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In general, it is difficult to raise novel monoclonal antibodies against relatively low-molecular weight antigen, and particularly those with high homology for the mouse protein. The optimized B-cell targeting (BCT) technique can overcome this limitation. The point of this advanced technology is the selection of sensitized B lymphocytes by the antigen through B-cell receptors (BCRs). This strict selection by specific and strong interaction between antigen and antibody enables the efficient production of monoclonal antibodies with high specificity and affinity. It also offers the condensation of sensitized target B lymphocytes to selectively generate hybridoma cells secreting desired monoclonal antibodies. In this study, several kinds of biotinylated human myoglobin (hMyo) were prepared to select sensitized B lymphocytes via BCRs. Biotinylated hMyo prepared by a 3.75- and 7.5-fold molar excess of N-hydroxysuccinimide (NHS)-biotin provided high antigenicity of 68-88%. B lymphocytes selected by these biotinylated antigens had an ELISA-positive rate17 times higher than that with usual biotinylated antigen. Monoclonal antibodies generated by the optimized BCT technology by preselecting sensitized B lymphocytes with the target antigen were identified to specifically recognize lower antigenic epitopes in hMyo with high affinity, while this would be impossible by the polyethylene glycol (PEG) method. Furthermore, combination of these high-affinity monoclonal antibodies gave the best binding rate in an epitope binning assay. These outcomes could be attributed to the unique characteristic that BCRs on sensitized B lymphocytes themselves can select the target epitopes in the antigen. The BCRs may act as a strict sensor of B lymphocytes to precisely select the target epitopes, even though the number of immunized B lymphocytes is low.

This paper reports a superiority of the asymmetric electric field formed in the rectangle microwell array for the electrofusion of splenocytes and myeloma cells with different diameters. The upper substrate with microband electrodes was mounted on the lower substrate with the microwell array. Two electrodes were arranged at the both sides of the microwells on the bottom surface. An attractive force of positive dielectrophoresis was employed to capture splenocytes with smaller diameter and myeloma cells with larger diameter at the right and left of microwells by applying AC electric field. The splenocytes and myeloma cells were fused by the asymmetric electric field that was generated in the microwells by applying DC electric pulse to the bottom electrode at the right side. The asymmetric field could allow to the formation of small openings on the membrane for the fusion of smaller splenocytes by experiencing higher field and the suppression for the disruption of larger myeloma cells by experiencing lower field.

Until now, various kinds of monoclonal antibodies have been raised against many antigens. Nevertheless, the production of these monoclonal antibodies was usually limited to only one antigen. If simultaneous generation of monoclonal antibodies against multiple antigens were available at one time, we could reduce not only laborious work, but also experimental animals. Here, we developed a multitargeting (MT) method that enables simultaneous production of monoclonal antibodies against multiple antigens on the basis of strict selection of sensitized B lymphocytes by the target antigens via B-cell receptors. After immunization using multiple antigens, monoclonal antibodies against four different antigens containing lower antigenic one were successfully generated only in one experiment. At maximum, more than 90 % of ELISA-positive wells to hybridoma-positive ones was obtained by this advanced technology. This must be attributed to strict selection of sensitized B lymphocytes by different antigens. In the MT method, sensitized B lymphocytes were selected by means of each desired antigen regardless of their antigenic differences. Selective fusion of B cell-myeloma cell complexes by electrical pulses was also of critical importance for efficient generation of hybridoma cells secreting desired monoclonal antibodies. This study strongly suggests that simultaneous production of novel monoclonal antibodies directed against multiple antigens of interest by the MT method can be feasible.

To develop efficient applications of monoclonal antibodies for therapeutic purposes, stereospecific recognition of the target antigens is needed. DNA immunization is one of the best methods for sensitizing B lymphocytes that can produce conformation-specific antibodies. Here we verified the class-switching of monoclonal antibodies by DNA immunization followed by cell immunization for the generation of stereospecific monoclonal antibodies against native G protein-coupled receptor (GPCR) using the optimized stereospecific targeting (SST) technique. This technology facilitates the efficient selection of sensitized B lymphocytes through specific interaction with the intact antigen via B-cell receptors (BCRs). We demonstrate that multiple DNA immunizations followed by a single cell immunization in combination with a longer sensitization period (three to four months) are an appropriate sensitizing strategy for the generation of IgG-type stereospecific monoclonal antibodies by class-switching, and the characteristics of antibody production could be transferred to hybridoma cells provided by the optimized SST technique.

We have developed a simple and rapid formation of a cell-based array on microwell array electrodes by an attractive force of positive dielectrophoresis (p-DEP), even after removing an upper disk electrode stick that was used as a counter electrode to the microwell array electrodes. The attractive force of p-DEP generated by the scanning of the disk electrode allows the formation of a cell-based array on all microwell arrays. We demonstrated an exploration of target cells spiked with a low ratio after removing the disk electrode.

It is quite difficult to generate monoclonal antibodies that recognize the three-dimensional structures of the antigens of interest. To address this limitation, we developed a new hybridoma technology termed "optimized stereospecific targeting (SST)". Here we aimed at generating stereospecific monoclonal antibodies against a G protein-coupled receptor (GPCR). The optimized SST technique enabled the efficient production of conformation-specific monoclonal antibodies against human corticotropin-releasing hormone receptor 1 (huCRHR1). Hybridoma cells secreting stereospecific monoclonal antibodies were selectively cloned by a limiting dilution method and the target monoclonal antibodies were purified by protein A column chromatography. They specifically cross-reacted with native huCRHR1 expressed on the surface of CHO cells, whereas they showed no affinity for MDA-MB-231 cancer cells, which abundantly express EphA2 on the cell surface. Furthermore, immunofluorescence analysis revealed that treatment of huCRHR1-expressing CHO cells with 4% paraformaldehyde led to a decrease in the affinity of purified monoclonal antibodies for intact huCRHR1 on the cell surface. In addition, purified monoclonal antibodies showed no cross-reactivity with huCRHR1 expressed on Sf9 insect cells. These results strongly suggest that monoclonal antibodies generated by the optimized SST technique feature specific binding to the intact form of the target GPCR on mammalian cells.

High-performance affinity column chromatography is considered to be an important analytical tool for further advancements and popularisation of antibody–drug remedies. In this context, there is a great need for the design and development of a new protein A column system with desired functionalities at low cost. Here, we developed mesoporous silica (MPS) with pore diameters in the range of 2.3–31.2 nm, which was synthesised by a sol–gel method using organic templates, as a carrier for staphylococcal protein A. The surface of the MPS was organo-functionalised using γ-glycidoxypropyltrimethoxysilane (Gly), phenyltriethoxysilane (Ph) and 3-mercaptopropyltrimethoxysilane (SH). The specific binding of Immunoglobulin G (IgG) to the prepared protein A–MPS composites has been analysed as a function of pore size and surface properties. MPS composites with a smaller pore size (under 12.7 nm) demonstrate higher IgG binding activity than non-porous carrier composites. Of all the composites, the MPS-2.7–Gly–protein A composite, having mesochannels of size 2.7 nm and covalently bound to protein A, exhibits the highest binding efficiency. The molecular ratio of immobilised protein A to bound IgG is found to be approximately 1 : 1. These results imply that the composite MPS-2.7–Gly–protein A is at least 15 times more efficient than commercial carrier composites such as acrylic polymer–protein A. In addition, this composite shows remarkable stability towards low pH and chaotropic agents. Furthermore, the possibility of reusing the MPS-2.7–Gly–protein A composite is established by cycling it 5 times, with no loss in IgG extraction capacity. We expect that development of such MPS will have a significant impact on the industrial-scale advancement of high-efficiency and low-cost protein A affinity column chromatography.

Fibrinogen from human blood is used as a main component of coagulants, including surgical tissue sealants. The development of a recombinant human fibrinogen (rFib) is anticipated to eliminate the risks of blood-borne infections. Here, we report the efficient production of rFib in a transgenic silkworm system. A silkworm line carrying cDNAs of the fibrinogen Aα and γ chains (Aα/γ-silkworm) produced Aα and γ chains in its cocoons, however, the Bβ chains were not detected from cocoons of another silkworm line carrying the cDNA of fibrinogen Bβ chains (Bβ-silkworm). A silkworm line for all three fibrinogen chains was generated by crossing Aα/γ-silkworms with Bβ-silkworms, which secreted Aα2Bβ2γ2 fibrinogen (rFib) into cocoons at high contents. The N-terminal amino acid sequences of the three rFib chains were identical to those of the corresponding chains of native fibrinogen (nFib). The N-glycan profile of the rFib comprised oligomannose-type (53%), complex-type (34%), and paucimannose-type (13%); neither high-mannose-type (six or more mannose residues) nor core-fucosylated glycans were observed. The coagulation activity of the rFib was evaluated for the amount of thrombin-released fibrinopeptide A (FpA) and the kinetics for turbidity increase (non-covalent network formation) in the solution. FpA release rates were equivalent between rFib and nFib; by contrast, the kinetics of the turbidity increase for rFib were accelerated nearly two-fold, for both the rate and maximum value, compared to those of nFib. These results demonstrate that the rFib produced in the transgenic silkworm system is comparable to nFib in both physical and coagulative properties. This rFib is a promising candidate component for safe hemostatic pharmaceuticals.

Background The generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. An alternative method of production may yield recombinant glycoproteins with reduced antigenicity. Results A cDNA clone encoding the fifteenth subtype of FeIFN-α (FeIFN-α15) was isolated from a Japanese domestic cat. This clone encoded a protein of 189 amino acids with a molecular mass of 21.1 kDa. The rFeIFN-α15 was expressed using a transgenic silkworm system, which was expected to yield an N-glycan structure with reduced antigenicity compared with the protein produced by the baculovirus system. The resulting rFeIFN-α15 accumulated in the sericin layer of silk fibres and was easily extracted and purified by column chromatography. The N-terminal amino acid sequence of purified rFeIFN-α15 was identical to the mature form of natural sequence. Moreover, its N-glycans did not include detectable core α 1,3 fucosyl residues. Its anti-vesicular stomatitis virus activity (2.6 × 108 units/mg protein) was comparable to that of the baculovirus-expressed rFeIFN. Conclusions The lower allergy risk of rFeIFN produced by the transgenic silkworm system than by the baculovirus expression system is due to the former lacking core α 1,3 fucosyl residues in its N-glycans. The rFeIFN-α15 produced by the transgenic silkworm system may be a prospective candidate for the next generation of rFeIFN in veterinary medicine.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

There exists an increasing need to produce useful proteins in recombinant technologies. In particular, most biologics for medical purposes are produced as recombinant proteins. Various host cell/vector systems have been developed, but it remains difficult to efficiently produce large molecular weight proteins with complex structures. As a result of breeding for several thousand years, the silkworm has acquired the ability to synthesize bulk amounts of silk proteins. To utilize this capacity for the mass production of useful proteins, transgenic silkworms have been generated that synthesize recombinant proteins in the silk gland and secrete them into the silk cocoon. Using this transgenic silkworm system, various proteins, including antibodies, collagen, and fibrinogen, have been successfully produced and are being developed as materials for diagnostic or research-use reagents, as well as for cosmetics. Moreover, several silkworm-produced proteins are being developed as biologics for therapeutic use. Transgenic silkworms need to be reared under good manufacturing practices (GMP)-compliant conditions to produce biologics. Therefore, we have constructed a GMP-compliant pilot plant for producing biologics using transgenic silkworm, and are now developing silkworm-rearing technology under GMP-compliant conditions.

Giant vesicles (GVs) are commonly used as artificial membranes and microcompartments in biochemical and biophysical studies. The efficiency and/or yield of their production can be strongly affected by the conditions and procedures used, and thus many protocols have been developed. A reverse-phase protocol has been used to prepare liposomes for decades, and is reported to be suitable for preparing GVs. By revisiting a reverse-phase (W/O/W emulsification) mechanism, in this study we verified how simply and efficiently GVs could be generated using the following alternative method. First, phospholipid/diethyl ether was mixed with an aqueous solution containing sucrose to form an emulsion, and the mixture was centrifuged. The resulting GVs together with tubular and sheet aggregates in the lower water phases could be purified by a second centrifugation after the addition of a glucose aqueous solution. This “reverse-phase/centrifugation” method provided a large amount of GVs of ∼20 μm in diameter from egg phosphatidylcholine as well as multiple lipid components. Spherical GVs were obtained in the presence of physiological salt concentrations (phosphate-buffered saline, PBS) when phosphatidylglycerol was included. The appearance of microdomains on the vesicle membranes may indicate that giant unilamellar vesicles (GUVs) can form through this process.

Close homolog of L1 (CHL1) and its truncated form mainly play crucial roles in mouse brain development and neural functions. Herein, we newly identified that truncated form of CHL1 is produced and released from lung tumor tissue in a mouse model expressing human EML4-ALK fusion gene. Both western blot and direct ELISA analysis revealed that mouse CHL1 level in serum (including serum extracellular vesicles) was significantly elevated in EML4-ALK transgenic mice. The correlation between the tumor size and the amount of CHL1 secretion could be examined in this study, and showed a significant positive correlation in a tumor size-dependent manner. Considering these results, the measurement of circulating CHL1 level may contribute to assess a tumor progression in human lung tumor patients.

Deoxyribonucleic acid (DNA) adsorption onto particles has applications in biosensors, separation methods, and gene delivery. Mesoporous silica (MPS), which exhibits a high surface area and large pore volume, is used in these applications because its pore size is easily controlled and its surface functional groups are easily exchanged. In this study, three types of MPSs with different pore sizes (2.4, 5.6, and 11.8 nm) were functionalized with different aminosilane coupling reagents and the effects of the MPS pore size and surface functional groups on DNA adsorption were evaluated. As the pore size of MPS increased, MPSs with diethylenetriamine (–3NH2) adsorbed higher amounts of DNA, whereas MPSs with hexylenediamine groups (–2HNH2) adsorbed lower amounts of DNA. Moreover, the fitting of DNA adsorption equilibrium data to Langmuir and Freundlich isotherm models was investigated.

Attention to therapeutic monoclonal antibodies has been dramatically increasing year by year. Their highly specific targeting of antigens can provide very effective medical treatment, and the advent of molecular-targeting medicine is allowing development of a new generation of therapeutic agents. However, there is one critical obstacle to overcome. Most of the established therapeutic monoclonal antibodies have specificity for the primary structures of target antigens, although all proteins harbor original native intact structures for their own specific functions. Stereo-specific monoclonal antibodies recognizing conformational structures of target antigens may thus offer a markedly more versatile approach. Their application may change the very concepts underlying use of therapeutic antibodies.

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.

Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen–antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

The spherical aberration corrector for high-resolution electron microscopes has been newly designed and constructed. This aberration correction device is formed by an electrostatic lens consisted of only two electrodes having annular and circular holes and a constant-voltage supply. We call it ‘annular and circular electrodes (ACE) corrector’. The ACE corrector has big advantages in easy alignment and very low-cost over the conventional Cs-correctors. In this study, we have constructed the corrector for the scanning transmission electron microsope (STEM) probe-forming optical system. The developed device is very small (less than several tens millimeter) so that this can be assembled at the tip of the conventional aperture holder and installed in a 200kV-STEM column. In experiments using specimens of nanocrystals, we have successfully controlled the spatial resolution of the dark-field STEM images by changing the voltage applied to the corrector and also have imaged lattice fringes. These results clearly demonstrate that our developed device can correct effectively the inherent aberration of the objective lens of the STEM. Copyright © 2016 John Wiley & Sons, Ltd.

A highly sensitive biosensor electrode was designed using a novel mesoporous carbon (MPC)-sheet. In this study, the MPC-sheet was prepared by the hard-template method using a mesoporous silica (MPS) sheet. The MPS sheet has unique characteristics such as thin shape, porous structure, and highly capacity as an absorbing material. The shape of synthesized carbon materials was similar with the template MPS sheet which was approximately 1–2 μm width and 100 nm a folded thickness. Surprisingly, the physicochemical properties of MPC-sheet were demonstrated different characteristics depending on the template materials. The MPC-sheet-modified electrode demonstrated excellent electrocatalytic activity for the simultaneous determination of biological compounds.

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

Recently, proteoglycan was purified from the nasal cartilage of salmon. Although several physiological effects have been reported, the effect of salmon nasal cartilage proteoglycan (salmon PG) on glucose metabolism remains unclear. We studied the effect of salmon PG on rat plasma glucose levels. Oral administration of 1% salmon PG significantly attenuated the increase in portal plasma glucose levels following an oral glucose tolerance test (OGTT). Additionally 1% salmon PG delayed the increase in peripheral glucose concentration induced by the OGTT. Mucosal administration of 1% salmon PG significantly decreased active glucose transport using the everted jejunal sac method. Furthermore, transmural potential difference (ΔPD) measurements using the everted jejunum revealed that 1% salmon PG significantly decreased glucose-dependent and phlorhizin (inhibitor of sodium-glucose co-transporter 1; SGLT1)-sensitive ΔPD. These results suggest that salmon PG decreases glucose absorption via SGLT1 in the jejunum, thereby attenuating the increase in portal and peripheral plasma glucose levels in rats.

A notably high-capacity antibody adsorbent was prepared using dispersed gold nanoparticles immobilised on a mesoporous silica sheet. The mesoporous silica sheet (MPS sheet) was prepared via a dual-templating method using tetraethoxysilane and 3-aminopropyltriethoxysilane as the silica framework materials. Gold nanoparticles were loaded onto the amino-functionalised surface of the MPS sheet via deposition–precipitation using HAuCl4 as the Au colloid precursor and then crystallised via calcination. The properties of the gold nanoparticle–silica composite and the dispersion of the nanoparticles were characterised using transmission electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction analysis. The surface characteristics of gold nanoparticle composite materials calcined at different temperatures were also evaluated using Fourier transform-infrared spectroscopy, X-ray photoelectron spectroscopy and thermogravimetry data. The silica–gold nanoparticle composite sheet crystallised at 550 °C (sheet-Au-550) was mesoporous in character (pore diameter = 3.8 nm) with a BET surface area of 258.0 m2 g−1. With respect to antibody adsorption, the sheet materials had a relatively higher capacity than the typical cylindrical mesoporous silica, and sheet-Au-550 exhibited a strong performance (maximum adsorption quantity QMax = 0.26 mg IgG per mg carrier). In addition, the IgG adsorption equilibrium for sheet-Au-550 fits the Langmuir isotherm model better than the Freundlich plot model. These results will be very useful for the design of adsorption materials for biomolecules, such as antibodies.

Bisphenol A (BPA) is of global concern because of its disruption of endocrine systems and ubiquity in aquatic environment. In this study, BPA antibody was successfully immobilised on novel mesoporous silica (MPS) carriers that display unique properties such as high surface area, highly uniform pore distribution and high adsorption capacity. Mobil Crystalline Material MCM-41 (2.7nm), Santa Barbara Amorphous SBA-15-1 (12.3nm) and SBA-15-2 (24.0nm) materials were used as supports for these antibodies. On these carriers, the BPA antibody immobilisation reached 40μgmg−1. For each MPS, 15ng of BPA antigen was adsorbed on 1mg of MPS–antibody composite, which resulted in an antibody activity of 30%. The highest recovery rate of BPA antigen was observed for 80% acetonitrile in 10mM phosphate buffer (pH 7). After six repeated runs, BPA antibodies immobilised on SBA-15-1 and SBA-15-2 retained about 30% of their initial activity. In contrast, these antibodies showed 13% lower residual activity on MCM-41 than on SBA-15-1 and SBA-15-2. This result indicated that entire antibody molecules were adsorbed inside SBA-15-1 and SBA-15-2 pores, stabilising their structural conformation.

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Pythium species, isolated from seedlings of Glycyrrhiza uralensis with blight, were identified as P. myriotylum, P. aphanidermatum, and P. spinosum on the basis of morphological characteristics and sequences of the internal transcribed spacer regions of rDNA. In pathogenicity tests, the isolates of the three Pythium species caused blight, producing the original disease symptoms. The primary inoculum source was determined using a multiplex PCR to detect the pathogen. All the Pythium species were detected in the soils of fields with the diseased plants and in soils of adjacent field soils.

Here, we introduce a new synthetic approach to mesoporous silica (MPS) sheets (thickness ≤ 100 nm). By using the carboxylate surfactant (N-palmitoyl-L-alanine) and Pluronic P123 as dual-templating agents, an MPS sheet was synthesised under similar conditions to SBA-15. Cytochrome c immobilised on an MPS sheet showed extremely high relative activity.

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases.

Quadrupole Magnetic Field-Flow Fractionation (QMgFFF) is a technique for characterization of sub-micrometer magnetic particles based on their retention in the magnetic field from flowing suspensions. Different magnetic field strengths and volumetric flow rates were tested using on-off field application and two commercial nanoparticle preparations that significantly differed in their retention parameter, λ (by nearly 8-fold). The fractograms showed a regular pattern of higher retention (98.6% v. 53.3%) for the larger particle (200 nm v. 90 nm) at the higher flow rate (0.05 mL/min v. 0.01 mL/min) at the highest magnetic field (0.52 T), as expected because of its lower retention parameter. The significance of this approach is a demonstration of a system that is simpler in operation than a programmed field QMgFFF in applications to particle mixtures consisting of two distinct particle fractions. This approach could be useful for detection of unwanted particulate contaminants, especially important in industrial and biomedical applications.