Publications by authors named "Enoch Y Park"

197 Publications

Cargo encapsulated hepatitis E virus-like particles for anti-HEV antibody detection.

Biosens Bioelectron 2021 Apr 22;185:113261. Epub 2021 Apr 22.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan; Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. Electronic address:

Viral capsid-nanoparticle hybrid structures incorporating quantum dots (QDs) into virus-like particles (VLPs) constitute an emerging bioinspired type of nanoarchitecture paradigm used for various applications. In the present study, we packed inorganic QDs in vitro into the hepatitis E virus-like particle (HEV-LP) and developed a fluorometric biosensor for HEV antibody detection. Firstly, for the preparation of QDs-encapsulated HEV-LPs (QDs@HEV-LP), the HEV-LPs produced by a recombinant baculovirus expression system were disassembled and reassembled in the presence of QDs using the self-assembly approach. Thus, the prepared QDs@HEV-LP exhibited excellent fluorescence properties similar to QDs. Further, in the presence of HEV antibodies in the serum samples, when mixed with QDs@HEV-LP, bind together and further bind to anti-IgG-conjugated magnetic nanoparticles (MNPs). The target-specific anti-IgG-MNPs and QDs@HEV-LP enrich the HEV antibodies by magnetic separation, and the separated QDs@HEV-LP-bound HEV antibodies are quantified by fluorescence measurement. This developed method was applied to detect the HEV antibody from sera of HEV-infected monkey from 0 to 68 days-post-infection and successfully diagnosed for HEV antibodies. The viral RNA copies number from monkey fecal samples by RT-qPCR was compared to the HEV antibody generation. This study first used QDs-encapsulated VLPs as useful fluorescence emitters for biosensing platform construction. It provides an efficient route for highly sensitive and specific antibody detection in clinical diagnosis research.
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http://dx.doi.org/10.1016/j.bios.2021.113261DOI Listing
April 2021

Effects of Cordycepin in during Its Infection to Silkworm Larvae.

Microorganisms 2021 Mar 25;9(4). Epub 2021 Mar 25.

Laboratory of Biotechnology, Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.

produces cordycepin, a secondary metabolite that exhibits numerous bioactive properties. However, cordycepin pharmacology in vivo is not yet understood. In this study, the roles of cordycepin in during its infection were investigated. After the injection of conidia, NBRC100741 killed silkworm larvae more rapidly than NBRC103752. At 96 and 120 h, genes (-), which are part of the cordycepin biosynthesis gene cluster, were expressed in fat bodies and cuticles. Thus, cordycepin may be produced in the infection of silkworm larvae. Further, cordycepin enhanced pathogenicity toward silkworm larvae of and , that are also entomopathogenic fungi and do not produce cordycepin. In addition, by RNA-seq analysis, the increased expression of the gene encoding a lipoprotein 30K-8 (Bmlp20, KWMTBOMO11934) and decreased expression of genes encoding cuticular proteins (KWMTBOMO13140, KWMTBOMO13167) and a serine protease inhibitor (serpin29, KWMTBOMO08927) were observed when cordycepin was injected into silkworm larvae. This result suggests that cordycepin may aid the in vivo growth of in silkworm larvae by the influence of the expression of some genes in silkworm larvae.
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http://dx.doi.org/10.3390/microorganisms9040681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065576PMC
March 2021

Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

J Biol Chem 2021 Jan 15:100299. Epub 2021 Jan 15.

Laboratory of Glycobiology, Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland. Electronic address:

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.
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http://dx.doi.org/10.1016/j.jbc.2021.100299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949097PMC
January 2021

Self-assembled chromogen-loaded polymeric cocoon for respiratory virus detection.

Nanoscale 2021 Jan 22;13(1):388-396. Epub 2020 Dec 22.

Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan.

Inspired by the self-assembly approach, in this work, the chromogen, 3,3',5,5'-tetramethylbenzidine (TMB), was successfully co-precipitated in aqueous solution to form collective nanoparticles (NPs) of signal molecules (TMB-NPs). Utilizing poly(lactide-co-glycolide) (PLGA) in the molecular delivery approach, the formed emulsion nanovesicle (TMB-NPs@PLGA) exhibits an enrichment of the collective signal molecules in a single antibody-antigen conjugation. A specific antibody-conjugated TMB-NPs@PLGA forms an immunocomplex sandwich structure upon the addition of influenza virus (IV)/A. The addition of dimethyl sulfoxide (DMSO) dissolves the PLGA nanovesicles, releasing the encapsulated TMB-NPs. Sequentially, the TMB-NPs release TMB molecules upon the addition of DMSO. The released TMB is catalytically oxidized by HO with self-assembled protein-inorganic nanoflowers, where copper nanoflowers (CuNFs) acted as the nanozyme. The developed immunoassay demonstrates high sensitivity for IV/A with a limit of detection (LOD) as low as 32.37 fg mL and 54.97 fg mL in buffer and serum, respectively. For practical needs, a clinically isolated IV/A/H3N2 and spike protein of SARS-CoV-2 were detected with the LODs of 17 pfu mL and 143 fg mL, respectively. These results show the applicability of the advanced TMB-NPs@PLGA-based colorimetric sensor for the highly sensitive detection of airborne respiratory viruses.
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http://dx.doi.org/10.1039/d0nr06893dDOI Listing
January 2021

Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

Mikrochim Acta 2020 Nov 25;187(12):674. Epub 2020 Nov 25.

Laboratory of Biotechnology, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 protein (CHIK-VP) as an example. The hydrophobic quantum dots (QDs) embedded in the lipid bilayer of liposome and methylene blue (MB) encapsulated in the inner core of liposomes played a role of dual-signaling modulator. After CHIK-VP addition, the nanocomposites and APTES-coated FeO nanoparticles (FeO NPs) were conjugated with antibodies to form a sandwich structure and separated from the medium magnetically. The nanoconjugates have been burst out by chloroform as surfactant, and both the QDs and MB are released from the liposome and were then monitored through changes in the fluorescence and electrochemical signals, respectively. These two fluorometric and electrochemical signals alteration quantified the CHIK-VP in the range of femtogram to nanogram per milliliter level with a LOD of 32 fg mL, making this liposomal system a potential matrix in a virus detection platform. Graphical abstract.
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http://dx.doi.org/10.1007/s00604-020-04656-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688297PMC
November 2020

Ni-modified magnetic nanoparticles for affinity purification of His-tagged proteins from the complex matrix of the silkworm fat body.

J Nanobiotechnology 2020 Nov 6;18(1):159. Epub 2020 Nov 6.

Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

Purification of recombinant proteins is often a challenging matter because high purity and high recovery are desired. If the expressed recombinant protein is also in a complex matrix, such as from the silkworm expression system, purification becomes more challenging. Even if purification from the silkworm expression system is troublesome, it benefits from a high capacity for the production of recombinant proteins. In this study, magnetic nanoparticles (MNPs) were investigated as a suitable tool for the purification of proteins from the complex matrix of the silkworm fat body. The MNPs were modified with nickel so that they have an affinity for His-tagged proteins, as the MNP purification protocol itself does not need special equipment except for a magnet. Among the three different kinds of investigated MNPs, MNPs with sizes of 100 nm to 200 nm and approximately 20 nm-thick nickel shells were the most suitable for our purpose. With them, the total protein amount was reduced by up to at least approximately 77.7%, with a protein recovery of around 50.8% from the silkworm fat body. The minimum binding capacity was estimated to be 83.3 µg protein/mg MNP. Therefore, these MNPs are a promising tool as a purification pretreatment of complex sample matrices.
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http://dx.doi.org/10.1186/s12951-020-00715-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648358PMC
November 2020

Structural insight into the substrate specificity of Bombyx mori β-fructofuranosidase belonging to the glycoside hydrolase family 32.

Insect Biochem Mol Biol 2020 12 24;127:103494. Epub 2020 Oct 24.

Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; Department of Applied Life Sciences, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

Sucrose-hydrolyzing enzymes are largely divided into β-fructofuranosidase and sucrose α-glucosidase. The domestic silkworm Bombyx mori possesses both enzymes, BmSUC1 and BmSUH, belonging to the glycoside hydrolase family 32 (GH32) and GH13, respectively. BmSUC1 was presumed to be acquired by horizontal gene transfer from bacteria based on phylogenetic analysis and related to tolerance to sugar-mimic alkaloids contained in mulberry latex. Here we investigated the substrate specificity of recombinant BmSUC1 that can hydrolyze not only sucrose but also fructooligosaccharides and fructans, and revealed that the enzyme was competitively inhibited by 1,4-dideoxy-1,4-imino-D-arabinitol, one of the alkaloids. Moreover, the crystal structures of BmSUC1 in apo form and complex with sucrose were determined, and the active site pocket was shallow and suitable for shorter substrates but was related to more relaxed substrate specificity than the strict sucrose α-glucosidase BmSUH. Considering together with the distribution of BmSUC1-orthologous genes in many lepidopterans, our results suggest that BmSUC1 contributes to the digestion of fructooligosaccharides and fructans derived from feed plants.
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http://dx.doi.org/10.1016/j.ibmb.2020.103494DOI Listing
December 2020

Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling.

Biomolecules 2020 10 26;10(11). Epub 2020 Oct 26.

Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan.

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.
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http://dx.doi.org/10.3390/biom10111482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692867PMC
October 2020

Electrochemical detection of white spot syndrome virus with a silicone rubber disposable electrode composed of graphene quantum dots and gold nanoparticle-embedded polyaniline nanowires.

J Nanobiotechnology 2020 Oct 27;18(1):152. Epub 2020 Oct 27.

Laboratory of Biotechnology, Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

Background: With the enormous increment of globalization and global warming, it is expected that the number of newly evolved infectious diseases will continue to increase. To prevent damage due to these infections, the development of a diagnostic method for detecting a virus with high sensitivity in a short time is highly desired. In this study, we have developed a disposable electrode with high-sensitivity and accuracy to evaluate its performances for several target viruses.

Results: Conductive silicon rubber (CSR) was used to fabricate a disposable sensing matrix composed of nitrogen and sulfur-co-doped graphene quantum dots (N,S-GQDs) and a gold-polyaniline nanocomposite (AuNP-PAni). A specific anti-white spot syndrome virus (WSSV) antibody was conjugated to the surface of this nanocomposite, which was successfully applied for the detection of WSSV over a wide linear range of concentration from 1.45 × 10 to 1.45 × 10 DNA copies/ml, with a detection limit as low as 48.4 DNA copies/ml.

Conclusion: The engineered sensor electrode can retain the detection activity up to 5 weeks, to confirm its long-term stability, required for disposable sensing applications. This is the first demonstration of the detection of WSSV by a nanofabricated sensing electrode with high sensitivity, selectivity, and stability, providing as a potential diagnostic tool to monitor WSSV in the aquaculture industry.
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http://dx.doi.org/10.1186/s12951-020-00712-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590724PMC
October 2020

Hollow magnetic-fluorescent nanoparticles for dual-modality virus detection.

Biosens Bioelectron 2020 Dec 2;170:112680. Epub 2020 Oct 2.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan; Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. Electronic address:

Combination of magnetic nanomaterials with multifunctionality is an emerging class of materials that exhibit tremendous potential in advanced applications. Synthesizing such novel nanocomposites without compromising magnetic behavior and introducing added functional properties is proven challenging. In this study, an optically active quantum dot (QD) (core) encapsulated inside iron oxide (hollow shell) is prepared as the first electrochemical/fluorescence dual-modality probe. Presence of magnetic layer on the surface enables excellent magnetic property and the encapsulating of QDs on the hollow shell structure maintains the fluorescence with minimal quenching effect, endowing for potential application with fluorescence modality readout. We successfully demonstrate dual-modality sensing utilizing of QD-encapsulated magnetic hollow sphere nanoparticles (QD@MHS NPs) with magnetic separation ability and highly integrated multimodal sensing for the detection of various viruses including hepatitis E virus (HEV), HEV-like particles (HEV-LPs), norovirus-like particles (NoV-LPs), and norovirus (NoV) from clinical specimens. Most importantly, fecal samples of HEV-infected monkey are successfully diagnosed with sensitivity similar to gold standard real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). This well-defined QD@MHS NPs-based nanoplatform intelligently integrates dual-modality sensing and magnetic bio-separation, which open a gateway to provide an efficient point-of care testing for virus diagnostics.
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http://dx.doi.org/10.1016/j.bios.2020.112680DOI Listing
December 2020

Controlling distance, size and concentration of nanoconjugates for optimized LSPR based biosensors.

Biosens Bioelectron 2020 Dec 26;170:112657. Epub 2020 Sep 26.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan; Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan. Electronic address:

In this report, we have examined the distance- and size-dependent localized surface plasmon resonance (LSPR) between fluorescent quantum dots (QDs) and adjacent gold nanoparticles (AuNPs) to provide a comprehensive evaluation, aiming for practical application in biosensing platform. A series of peptides with different chain lengths, connected between QDs and AuNPs is initially applied to prepare various CdSe QDs-peptide-AuNP systems to optimize LSPR signal. Separation distance between two nanoparticles of these systems before and after conjugation is also confirmed by quantum mechanical modeling and corroborated with their LSPR influenced fluorescence variations. After detailed optimizations, it can be noted that larger sized AuNPs make strong quenching of QDs, which gradually shows enhancement of fluorescence with the increment of distance and the smaller sized AuNPs. Depending on the requirement, it is possible to tune the optimized structure of the CdSe QD-peptide-AuNP nanostructures for the application. In this work, two different structural designs with different peptide chain length are chosen to construct two biosensor systems, observing their fluorescence enhancement and quenching effects, respectively. Using different structural orientation of these biosensors, two nanoconjugates has applied for detection of norovirus and influenza virus, respectively to confirm their application in sensing.
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http://dx.doi.org/10.1016/j.bios.2020.112657DOI Listing
December 2020

Ultrasensitive Detection of the Hepatitis E Virus by Electrocatalytic Water Oxidation Using Pt-CoO Hollow Cages.

ACS Appl Mater Interfaces 2020 Nov 6;12(45):50212-50221. Epub 2020 Oct 6.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka 422-8529, Japan.

A sensitive virus detection method applicable for an early stage increases the probability of survival. Here, we develop a simple and rapid detection strategy for the detection of the hepatitis E virus (HEV) by an electrocatalytic water oxidation reaction (WOR) using a platinum (Pt)-incorporated cobalt (Co)-based zeolite imidazole framework (ZIF-67). The surface cavity of ZIF-67 enables the rich loading of Pt NPs, and subsequent calcination etches the cavity, promoting the electrocatalytic activity of Pt-CoO HCs. The Pt-CoO HCs show excellent behavior for the WOR due to the synergistic interaction of Pt and CoO, evaluated by voltammetry and chronoamperometry. The synthesized Pt-CoO HCs are conjugated with anti-HEV antibody (Ab@Pt-CoO HCs); the electrocatalytic activity of Ab@Pt-CoO HCs is combined with that of antibody-conjugated magnetic nanoparticles (MNPs) for HEV detection by a magneto-and-nanocomposite sandwich immunoassay. The sensor is challenged to detect the HEV in spiked serum samples and HEV G7 genotypes collected from the cell culture supernatant, reaching a low limit of detection down to 61 RNA copies mL. This work establishes a free-indicator one-step approach with the controlled design of Pt-CoO HCs, which presents an effective WOR technique for virus detection in a neutral pH solution, which can be extended to electrocatalytic studies in the future integrated biosensing systems.
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http://dx.doi.org/10.1021/acsami.0c13247DOI Listing
November 2020

Molybdenum Trioxide Nanocubes Aligned on a Graphene Oxide Substrate for the Detection of Norovirus by Surface-Enhanced Raman Scattering.

ACS Appl Mater Interfaces 2020 Sep 21;12(39):43522-43534. Epub 2020 Sep 21.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.

A novel biosensing system based on graphene-mediated surface-enhanced Raman scattering (G-SERS) using plasmonic/magnetic molybdenum trioxide nanocubes (mag-MoO NCs) has been designed to detect norovirus (NoV) via a dual SERS nanotag/substrate platform. A novel magnetic derivative of MoO NCs served as the SERS nanotag and the immunomagnetic separation material of the biosensor. Single-layer graphene oxide (SLGO) was adopted as the 2D SERS substrate/capture platform and acted as the signal reporter, with the ability to accommodate an additional Raman molecule as a coreporter. The developed SERS-based immunoassay achieved a signal amplification of up to ∼10-fold resulting from the combined electromagnetic and chemical mechanisms of the dual SERS nanotag/substrate system. The developed biosensor was employed for the detection of NoV in human fecal samples collected from infected patients by capturing the virus with the aid of NoV-specific antibody-functionalized magnetic MoO NCs. This approach enabled rapid signal amplification for NoV detection with this biosensing technology. The biosensor was tested and optimized using NoV-like particles within a broad linear range from 10 fg/mL to 100 ng/mL and a limit of detection (LOD) of ∼5.2 fg/mL. The practical applicability of the developed biosensor to detect clinical NoV subtypes in human fecal samples was demonstrated by effective detection with an LOD of ∼60 RNA copies/mL, which is ∼10-fold lower than that of a commercial enzyme-linked immunosorbent assay kit for NoV.
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http://dx.doi.org/10.1021/acsami.0c14729DOI Listing
September 2020

Agglutination of Human Polyomaviruses by Using a Tetravalent Glycocluster as a Cross-Linker.

ACS Omega 2020 Sep 18;5(34):21940-21947. Epub 2020 Aug 18.

Department of Virology and Parasitology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192, Japan.

Two kinds of tetravalent double-headed sialo-glycosides with short/long spacers between the Neu5Acα2,6Galβ1,4GlcNAc unit and ethylene glycol tetraacetic acid (EGTA) scaffold were found to be capable of binding to virus-like particles of Merkel cell polyomavirus (MCPyV-LP). The binding process and time course of interaction between the tetravalent ligand and MCPyV-LP were assessed by dynamic light scattering (DLS). On the addition of increasing concentrations of ligand to MCPyV-LP, larger cross-linked aggregates formed until a maximum size was reached. The binding was stronger for the tetravalent ligand with a short spacer than for that with a long spacer. The binding of the former ligand to the virus was observed to proceed in two stages during agglutination. The first step was the spontaneous formation of small aggregates comprising the cross-linked ligand-virus complex. In the second step, the aggregates grew successively larger by cooperative binding among the initially produced small aggregates. In transmission electron microscopy, the resulting complex was observed to form aggregates in which the ligands were closely packed with the virus particles. The cross-linked interaction was further confirmed by a simple membrane filtration assay in which the virus-like particles were retained on the membrane when complexed with a ligand. The assay also showed the effective capture of particles of pathogenic, infectious human polyomavirus JCPyV when complexed with a ligand, suggesting its possible application as a method for trapping viruses by filtration under conditions of virus aggregation. Collectively, these results show that the tetravalent glycocluster serves as a ligand not only for agglutinating MCPyV-LP but also for trapping the pathogenic virus.
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http://dx.doi.org/10.1021/acsomega.0c03269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469642PMC
September 2020

Plasmonic/magnetic molybdenum trioxide and graphitic carbon nitride quantum dots-based fluoroimmunosensing system for influenza virus.

Sens Actuators B Chem 2020 Oct 22;321:128494. Epub 2020 Jun 22.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan.

A novel magnetic/plasmonic-assisted fluoro-immunoassay system is developed for the detection of influenza virus using magnetic-derivatized plasmonic molybdenum trioxide quantum dots (MP-MoO QDs) as the plasmonic/magnetic agent and fluorescent graphitic carbon nitride quantum dots (gCNQDs) as the monitoring probe. Specific antibody against influenza A virus was conjugated onto the surface of MP-MoO QDs and gCNQDs, respectively. In the presence of influenza A virus (as the test virus), a core-satellite immunocomplex is formed between the antibody-conjugated nanomaterials (Ab-MP-MoO QDs and Ab-gCNQDs) and their interaction resulted in the modulation and gradual enhancement of the fluorescence intensity of the detection probe with the influenza virus concentration-dependent increase. In addition, PL change without influenza A virus was not observed. Limits of detection of 0.25 and 0.9 pg/mL were achieved for Influenza virus A/New Caledonia (20/99/IVR/116) (H1N1) detection in deionized water and human serum, respectively. Clinically isolated influenza virus A/Yokohama (110/2009) (H3N2) was detected in the range of 45 - 25,000 PFU/mL, with a limit of detection ca 45 PFU/mL (as opposed to a minimum of 5000 PFU/mL for a commercial test kit). This developed biosensor provides a robust, sensitive as well as a selective platform for influenza virus detection.
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http://dx.doi.org/10.1016/j.snb.2020.128494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313904PMC
October 2020

Production of dengue virus-like particles serotype-3 in silkworm larvae and their ability to elicit a humoral immune response in mice.

AMB Express 2020 Aug 17;10(1):147. Epub 2020 Aug 17.

Laboratory of Biotechnology, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

To develop monovalent dengue virus-like particle for serotype 3 (DENV-LP/3), we prepared and expressed two structural polyprotein constructs using silkworm and Bm5 cells: DENV-3 Capsid-premembrane-envelope (DENV-3CprME) and premembrane-envelope (DENV-3prME). The expressed PA-tagged 3CprME and 3prME polypeptides were partially purified by PA-tag affinity chromatography and had molecular weights of 85 and 75 kDa, respectively. Expressed proteins were separately verified using the following primary antibodies: the anti-PA tag antibody, DENV premembrane polyclonal antibody, and DENV envelope polyclonal antibody. Transmission electron microscopy revealed that these DENV-3CprME and 3prME formed rough, spherical DENV-LPs (DENV-LP/3CprME and DENV-LP/3prME), respectively, with a diameter of 30-55 nm. The heparin-binding assay demonstrated that these DENV-LPs contained the envelope protein domain III on their surfaces. Both DENV-LPs showed an affinity to sera from human dengue patients and immunized mice. Immunization of mice with DENV-LP/3prME significantly induced the level of antibodies compared with DENV-LP/3CprME. These results indicate that DENV-LP/3prME is suitable as a vaccine candidate compared with DENV-LP/3CprME.
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http://dx.doi.org/10.1186/s13568-020-01087-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431508PMC
August 2020

Boosting the energy storage performance of VO nanosheets by intercalating conductive graphene quantum dots.

Nanoscale 2020 Aug;12(32):16944-16955

Institute of Analytical and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013, Taiwan. and Faculty of Science and Technology, Airlangga University, Sarabaya, 60115, Indonesia.

2-Dimensional (2D) transition metal oxides are an emerging class of energy materials that offer a wide spectrum of potential applications in electrochemical energy storage. In this study, V2O5 nanosheets have been nano-engineered with 0D graphene quantum dots (GQDs) via a solvothermal treatment process, and they serve as an anode material to boost electrochemical energy storage properties. The interlayer embedded GQD endows V2O5 (VNS-GQD) with structural and compositional advantages for high-performance energy storage, including expanded interlayer distances between layers, fast electrochemical kinetics, and additional stability to buffer the volume variation. Moreover, the strong coupling effect between GQDs and VNS, an ultra-large interfacial area and enhanced electrical conductivity promote the intercalation pseudocapacitance. VNS-GQD exhibits the specific capacitance of 572 F g-1 at a current density of 1 A g-1 and retains 92% of the initial capacitance after 10 000 charge-discharge cycles. The asymmetric supercapacitor exhibits superior electrochemical performance at a voltage window of 1.5 V. The energy density is 31.25 W h kg-1 at the power density of 2.25 kW kg-1, and maintains a superior energy density of 20.62 W h kg-1 at the high power density of 14.86 kW kg-1. The results of this study can provide an avenue for fabricating nano-sandwiched composites by embedding GQDs into interlayers of 2D transition metal oxide for ultra-high performance applications of energy storage devices.
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http://dx.doi.org/10.1039/d0nr04362aDOI Listing
August 2020

Preparation of divalent antigen-displaying enveloped virus-like particles using a single recombinant Bombyx mori nucleopolyhedrovirus bacmid in silkworms.

J Biotechnol 2020 Nov 7;323:92-97. Epub 2020 Aug 7.

Laboratory of Biotechnology, Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan; Laboratory of Biotechnology, Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Suruga-ku, Shizuoka, Japan; Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan. Electronic address:

Silkworms have been used as a host for the production of recombinant proteins in a baculovirus expression system using Bombyx mori nucleopolyhedrovirus (BmNPV). To coexpress several recombinant proteins, a silkworm must be coinfected with several recombinant BmNPVs, which requires a difficult DNA manipulation procedure. In this study, we constructed recombinant BmNPVs containing three expression cassettes, Rous sarcoma virus (RSV) Gag protein, surface antigen 1 of Neospora caninum (NcSAG1) and SAG1-related sequence 2 of N. caninum (NcSRS2), by Gibson assembly and the Bac-to-Bac system, designated BmNPV/SAG-SRS-Gag and BmNPV/SAG-Gag-SRS. BmNPV/SAG-SRS-Gag was expressed in silkworms and characterized. NcSAG1 and NcSRS2 were purified with RSV Gag proteins using sucrose density gradient centrifugation and affinity chromatography. RSV Gag formed virus-like particles (RSV-LPs) at a diameter of 20-30 nm based on transmission electron microscopy (TEM). Immuno-TEM analysis showed that both NcSAG1 and NcSRS2 were displayed on the surface of the RSV-LPs. These results indicate that RSV-LPs displaying two different kinds of proteins were produced in the hemolymph of silkworm larvae by the single polycistronic strategy. This expression platform is efficient for generating multiantigen-displaying VLPs and facilitates the development of vaccines against infectious diseases.
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http://dx.doi.org/10.1016/j.jbiotec.2020.08.002DOI Listing
November 2020

Fluoroimmunoassay of influenza virus using sulfur-doped graphitic carbon nitride quantum dots coupled with AgS nanocrystals.

Mikrochim Acta 2020 07 21;187(8):466. Epub 2020 Jul 21.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

Novel sulfur-doped graphitic carbon nitride quantum dots (S-gCNQDs) are synthesized using a single-source precursor in a one-step solvothermal process. The S-gCNQDs with a size of ~ 5-nm displayed a strong green intrinsic fluorescence at 512 nm when excited at 400 nm, with a quantum yield of ~ 33% in aqueous solution. The prepared S-gCNQDs and AgS nanocrystals were applied as innovative functional materials to fabricate a biosensor for virus detection based on the conjugation of specific anti-human influenza A monoclonal antibody to the S-gCNQDs and AgS NCs, respectively. In the presence of the influenza A virus, an interaction between the S-gCNQDs/AgS-labeled antibody resulted in the formation of a nanosandwich structure, which is accompanied by the fluorescence enhancement of the S-gCNQDs. The change in fluorescence intensity linearly correlats with the concentration of the influenza A virus (H1N1) in the 10 fg/mL to 1.0 ng/mL range, with a limit of detection of 5.5 fg/mL. The assay was applied to the assay of clinically isolated influenza A virus (H3N2/Yokohama) mixed with human serum. The obtained limit of detection was 100 PFU/mL within the detection range of 10- 5 × 10 PFU/mL for the H3N2 virus. Graphical abstract.
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http://dx.doi.org/10.1007/s00604-020-04433-1DOI Listing
July 2020

α-L-Fucosidase from Bombyx mori has broad substrate specificity and hydrolyzes core fucosylated N-glycans.

Insect Biochem Mol Biol 2020 09 17;124:103427. Epub 2020 Jun 17.

Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.

N-glycans play a role in physiological functions, including glycoprotein conformation, signal transduction, and antigenicity. Insects display both α-1,6- and α-1,3-linked fucose residues bound to the innermost N-acetylglucosamine of N-glycans whereas core α-1,3-fucosylated N-glycans are not found in mammals. Functions of insect core-fucosylated glycans are not clear, and no α-L-fucosidase related to the N-glycan degradation has been identified. In the genome of the domestic silkworm, Bombyx mori, a gene for a protein, BmFucA, belonging to the glycoside hydrolase family 29 is a candidate for an α-L-fucosidase gene. In this study, BmFucA was cloned and recombinantly expressed as a glutathione-S-transferase tagged protein (GST-BmFucA). Recombinant GST-BmFucA exhibited broad substrate specificity and hydrolyzed p-nitrophenyl α-L-fucopyranoside, 2'-fucosyllactose, 3-fucosyllactose, 3-fucosyl-N,N'-diacetylchitobiose, and 6-fucosyl-N,N'-diacetylchitobiose. Further, GST-BmFucA released fucose from both pyridylaminated complex-type and paucimannose-type glycans that were core-α-1,6-fucosylated. GST-BmFucA also shows hydrolysis activity for core-fucosylated glycans attached to phospholipase A from bee venom. BmFucA may be involved in the catabolism of core-fucosylated N-glycans in B. mori.
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http://dx.doi.org/10.1016/j.ibmb.2020.103427DOI Listing
September 2020

Structure-function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 -α-glucosidases.

J Biol Chem 2020 06 7;295(26):8784-8797. Epub 2020 May 7.

Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka, Japan.

The domestic silkworm expresses two sucrose-hydrolyzing enzymes, BmSUH and BmSUC1, belonging to glycoside hydrolase family 13 subfamily 17 (GH13_17) and GH32, respectively. BmSUH has little activity on maltooligosaccharides, whereas other insect GH13_17 α-glucosidases are active on sucrose and maltooligosaccharides. Little is currently known about the structural mechanisms and substrate specificity of GH13_17 enzymes. In this study, we examined the crystal structures of BmSUH without ligands; in complexes with substrates, products, and inhibitors; and complexed with its covalent intermediate at 1.60-1.85 Å resolutions. These structures revealed that the conformations of amino acid residues around subsite -1 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including - and -acting hydrolases, such as α-glucosidases and α-amylases. Amino acid residues at subsite +1 are not conserved in BmSUH and other GH13_17 α-glucosidases, but subsite -1 residues are absolutely conserved. Substitutions in three subsite +1 residues, Gln, Tyr, and Glu, decreased sucrose hydrolysis and increased maltase activity of BmSUH, indicating that these residues are key for determining its substrate specificity. These results provide detailed insights into structure-function relationships in GH13 enzymes and into the molecular evolution of insect GH13_17 α-glucosidases.
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http://dx.doi.org/10.1074/jbc.RA120.013595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324511PMC
June 2020

Crystal structure of the Enterococcus faecalis α-N-acetylgalactosaminidase, a member of the glycoside hydrolase family 31.

FEBS Lett 2020 07 22;594(14):2282-2293. Epub 2020 May 22.

Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Japan.

Glycoside hydrolases catalyze the hydrolysis of glycosidic linkages in carbohydrates. The glycoside hydrolase family 31 (GH31) contains α-glucosidase, α-xylosidase, α-galactosidase, and α-transglycosylase. Recent work has expanded the diversity of substrate specificity of GH31 enzymes, and α-N-acetylgalactosaminidases (αGalNAcases) belonging to GH31 have been identified in human gut bacteria. Here, we determined the first crystal structure of a truncated form of GH31 αGalNAcase from the human gut bacterium Enterococcus faecalis. The enzyme has a similar fold to other reported GH31 enzymes and an additional fibronectin type 3-like domain. Additionally, the structure in complex with N-acetylgalactosamine reveals that conformations of the active site residues, including its catalytic nucleophile, change to recognize the ligand. Our structural analysis provides insight into the substrate recognition and catalytic mechanism of GH31 αGalNAcases.
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http://dx.doi.org/10.1002/1873-3468.13804DOI Listing
July 2020

Genomic analysis of a riboflavin-overproducing Ashbya gossypii mutant isolated by disparity mutagenesis.

BMC Genomics 2020 Apr 23;21(1):319. Epub 2020 Apr 23.

Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan.

Background: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out.

Results: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process.

Conclusion: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.
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http://dx.doi.org/10.1186/s12864-020-6709-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181572PMC
April 2020

Draft Genome Sequence of the Aspergillus terreus High-Itaconic-Acid-Productivity Strain IFO6365.

Microbiol Resour Announc 2020 Apr 16;9(16). Epub 2020 Apr 16.

Graduate School of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi, Japan

Itaconic acid is an important organic acid used in the chemical industry. strain IFO6365 is one of the highest-yielding itaconic acid-producing wild-type strains. Here, we report the draft genome sequence of IFO6365, enhancing the understanding of the role and biosynthesis of itaconic acid in this fungus.
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http://dx.doi.org/10.1128/MRA.00080-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163008PMC
April 2020

Advancement of capture immunoassay for real-time monitoring of hepatitis E virus-infected monkey.

Anal Chim Acta 2020 May 12;1110:64-71. Epub 2020 Feb 12.

Department of Applied Biological Chemistry, College of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan; Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. Electronic address:

Rapid increasing outbreak of Hepatitis E virus (HEV) shows an urgent need of HEV detection. Instead of time consuming and expensive RT-qPCR, an efficient and quick monitoring system is in utmost demand which can be comparable with the RT-qPCR in term of reliability and detection limit. An advanced platform for immunoassay has been constructed in this study by a nanozyme that constitutes anti-HEV IgG antibody-conjugated gold nanoparticles (Ab-AuNPs) as core and in situ silver deposition on the surface of Ab-AuNPs as outer shell. The virus has been entrapped on the nanocomposites while the silver-shell has decomposed back to the silver ions (Ag) by adding a tetramethylbenzidine (TMBZ) and hydrogen peroxide (HO) which indirectly quantifies the target virus concentration. Counterpart to only applying nanozyme, by incorporation of the enhanced effect of Ag shell on the AuNP-based nanozyme, the advance deposition has been confirmed to prove the signal amplification mechanism in the proposed immunoassay. Most importantly, the sensor performances have examined on the HEV, collected from the HEV-infected monkey over a period of 45 days. It was successfully correlated with the standard RT-qPCR data, showing the applicability of this immunoassay as a real-time monitoring on the HEV infection. The in situ formation of AuNPs@Ag as nanozyme in this capture immunoassay leads to a promising advancement over the conventional methods and nanozyme-based immunoassay in real application which can be a good substitute of RT-qPCR in near future.
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http://dx.doi.org/10.1016/j.aca.2020.02.020DOI Listing
May 2020

Fluorometric virus detection platform using quantum dots-gold nanocomposites optimizing the linker length variation.

Anal Chim Acta 2020 May 22;1109:148-157. Epub 2020 Feb 22.

Laboratory of Biotechnology, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. Electronic address:

In this study, a tunable biosensor using the localized surface plasmon resonance (LSPR), controlling the distance between fluorescent CdZnSeS/ZnSeS quantum dots (QDs) and gold nanoparticles (AuNPs) has been developed for the detection of virus. The distance between the AuNPs and QDs has been controlled by a linkage with a peptide chain of 18 amino acids. In the optimized condition, the fluorescent properties of the QDs have been enhanced due to the surface plasmon effect of the adjacent AuNPs. Successive virus binding on the peptide chain induces steric hindrance on the LSPR behavior and the fluorescence of QDs has been quenched. After analyzing all the possible aspect of the CdZnSeS/ZnSeS QD-peptide-AuNP nanocomposites, we have detected different concentration of influenza virus in a linear range of 10 to 10 g mL with detection limit of 17.02 fg mL. On the basis of the obtained results, this proposed biosensor can be a good alternative for the detection of infectious viruses in the various range of sensing application.
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http://dx.doi.org/10.1016/j.aca.2020.02.039DOI Listing
May 2020

Dual modality sensor using liposome-based signal amplification technique for ultrasensitive norovirus detection.

Biosens Bioelectron 2020 Jun 26;157:112169. Epub 2020 Mar 26.

Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan; Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku, Shizuoka, 422-8529, Japan. Electronic address:

Sensitive and accurate detection methods for infectious viruses are the pressing need for effective disease diagnosis and treatment. Herein, based on VO nanoparticles-encapsulated liposomes (VONP-LPs) we demonstrate a dual-modality sensing platform for ultrasensitive detection of the virus. The sensing performance relies on intrinsic peroxidase and electrochemical redox property of VO nanoparticles (VO NPs). The target-specific antibody-conjugated VONP-LPs and magnetic nanoparticles (MNPs) enrich the virus by magnetic separation and the separated VONP-LPs bound viruses are hydrolyzed to release the encapsulated VO NPs. These released nanoparticles from captured liposomes act as peroxidase mimics and electrochemical redox indicator resulting in noticeable colorimetric and robust electrochemical dual-signal. Utilizing the superiority of dual-modality sensor with two quantitative analysis forms, norovirus like particles (NoV-LPs) can be detected by electrochemical signals with a wide linear range and low detection limit. To verify the applicability in real samples, norovirus (NoV) collected from actual clinical samples are effectively-identified with excellent accuracy. This proposed detection method can be a promising next-generation bioassay platform for early-stage diagnosis of virus disease and surveillance for public health.
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http://dx.doi.org/10.1016/j.bios.2020.112169DOI Listing
June 2020

Antigenic properties of VP15 from white spot syndrome virus in kuruma shrimp Marsupenaeus japonicus.

Fish Shellfish Immunol 2020 Jun 29;101:152-158. Epub 2020 Mar 29.

Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan; Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan. Electronic address:

White spot syndrome virus (WSSV) is known as one of the most lethal pathogenic viruses in shrimp causing massive damage to shrimp aquaculture industries. To date, no effective treatment or prevention has been found. In this study, five recombinant viral proteins VP15, VP19, VP24, VP26, and VP28 were expressed and purified in E. coli, which were employed as candidates against WSSV in Kuruma shrimp Marsupenaeus japonicus. In vivo antiviral assay in this study newly revealed that VP15 of major nucleocapsid protein, being known as a DNA-binding protein provided the substantial protection against the viral infection when pre-injected into shrimps. Furthermore, we also verified the immunogenic effects of purified VP15 and VP19 proteins produced in a silkworm-bacmid expression system. Taken together, our study identified VP15 as an effective candidate against WSSV infection in the Kuruma shrimp. It is interesting to uncover why and how VP15 is involved in the immune memory in shrimp in the future study.
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http://dx.doi.org/10.1016/j.fsi.2020.03.061DOI Listing
June 2020

A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Feb 31;1138:121964. Epub 2019 Dec 31.

Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan; Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. Electronic address:

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.
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http://dx.doi.org/10.1016/j.jchromb.2019.121964DOI Listing
February 2020

Draft Genome Sequence of Aspergillus terreus High-Itaconic-Acid-Productivity Mutant TN-484.

Microbiol Resour Announc 2019 Dec 5;8(49). Epub 2019 Dec 5.

Graduate School of Horticulture, Chiba University, Matsudo, Japan

Itaconic acid is an important organic acid used in the chemical industry. strain TN-484 is a high-itaconic-acid-productivity mutant derived from strain IFO6365. Here, we report the draft genome sequence of strain TN-484, advancing the understanding of the biosynthesis of itaconic acid in filamentous fungi.
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http://dx.doi.org/10.1128/MRA.01170-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895305PMC
December 2019