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Abstract Background Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. Results In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. Conclusion The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.

Hepatitis viral infections are the most common cause of hepatitis liver disease, which eventually leads to cancer and fibrosis if not detected early. Therefore, early detection would allow for preventive and therapeutic actions. Here, a surface-enhanced Raman spectroscopy (SERS)-based biosensor was developed using plasmonic molybdenum trioxide quantum dots (MoO3-QDs) as the SERS substrates. The nanostructured substrate of MoO3-QDs was functionalized with a proteoglycan (syndecan-1) as a novel bioreceptor for the target hepatitis E virus (HEV). The innovative biodetection system achieved a detection limit of 1.05 fg/mL for the tested HEV target (ORF2), indicating superb clinically relevant sensitivity and performance. The designed biosensing system incorporating a glycan motif as a bioreceptor instead of the conventional antibodies or aptamers presents new insights for the ultrasensitive detection of HEV and other infectious viruses.

Highlights Dengue virus-like particles for serotype 1 and serotype 4 (DENV-LPs/1 and DENV-LPs/4) were produced in silkworm. Heparin-binding assay by ELISA and ITC showed that DENV-LPs/1 and DENV-LPs/4 contain Envelope Domain III. DENV-LPs/1 and DENV-LPs/4 showed affinity to sera from human dengue patients and immunized mice.

Recent progress has been made dramatically in decorating virus-like particles (VLPs) on the surface or inside with functional molecules, such as antigens or nucleic acids. However, it is still challenging to display multiple antigens on the surface of VLP to meet the requirement as a practical vaccine candidate. Herein this study, we focus on the expression and engineering of the capsid protein VP2 of canine parvovirus for VLP display in the silkworm-expression system. The chemistry of the SpyTag/SpyCatcher (SpT/SpC) and SnoopTag/SnoopCatcher (SnT/SnC) are efficient protein covalent ligation systems to modify VP2 genetically, where SpyTag/SnoopTag are inserted into the N-terminus or two distinct loop regions (Lx and L2) of VP2. The SpC-EGFP and SnC-mCherry are employed as model proteins to evaluate their binding and display on six SnT/SnC-modified VP2 variants. From a series of protein binding assays between indicated protein partners, we showed that the VP2 variant with SpT inserted at the L2 region significantly enhanced VLP display to 80% compared to 5.4% from N-terminal SpT-fused VP2-derived VLPs. In contrast, the VP2 variant with SpT at the Lx region failed to form VLPs. Moreover, the SpT (Lx)/SnT (L2) double-engineered chimeric VP2 variants showed covalent conjugation capacity to both SpC/SnC protein partners. The orthogonal ligations between those binding partners were confirmed by both mixing purified proteins and co-infecting cultured silkworm cells or larvae with desired recombinant viruses. Our results indicate that a convenient VLP display platform was successfully developed for multiple antigen displays on demand. Further verifications can be performed to assess its capacity for displaying desirable antigens and inducing a robust immune response to targeted pathogens.

Abstract White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1–25), VP15(26–57), VP15(58–80), and VP15(1–25,58–80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26–57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26–57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26–57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.

Abstract 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.

Abstract 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 × 102 to 1.45 × 105 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.

Abstract 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.

Abstract 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.

Nanomaterials have been explored in the sensing research field in the last decades. Mainly, 3D nanomaterials have played a vital role in advancing biomedical applications, and less attention was given to their application in the field of biosensors for pathogenic virus detection. The versatility and tunability of a wide range of nanomaterials contributed to the development of a rapid, portable biosensor platform. In this review, we discuss 3D nanospheres, one of the classes of nanostructured materials with a homogeneous and dense matrix wherein a guest substance is carried within the matrix or on its surface. This review is segmented based on the type of nanosphere and their elaborative application in various sensing techniques. We emphasize the concept of signal amplification strategies using different nanosphere structures constructed from a polymer, carbon, silica, and metal–organic framework (MOF) for rendering high-level sensitivity of virus detection. We also briefly elaborate on some challenges related to the further development of nanosphere-based biosensors, including the toxicity issue of the used nanomaterial and the commercialization hurdle.

Detection of viral biomarkers is important for disease treatment and prevention. Here, the authors report on a system that uses an electrical pulse-induced electrochemical sensor for the detection of hepatitis E virus, and demonstrate potential application of the device.

A Gram-positive strain, ADR1, was isolated from soil collected from the Algerian Sahara Desert. The ethyl acetate extract of the fermentation broth showed cytotoxic activity against the PANC-1 cell line (37.1 ± 1.3% viability when applied at a concentration of 100 µg/mL). Fractionation and NMR analysis of two peaks absorbing at 490 nm revealed that they represented β- and γ-rubromycin, anticancer antibiotic compounds. The ADR1 strain contained LL-diaminopimelic acid in the whole-cell hydrolysate, and the partial 16S ribosomal RNA gene sequence (1392 bp, Accession No. KF947515) showed 99% sequence similarity to Streptomyces species. Therefore, the name Streptomyces sp. ADR1 was proposed and deposited in the Wellness Industries Culture Collection (WICC) of the Institute of Bioproduct Development, UTM, Malaysia, under the number (WICC- B86). In a 16 L stirred-tank bioreactor, the stain was adapted to submerged culture conditions and produced rubromycins at a relatively high concentration, with maximums of 24.58 mg/L and 356 mg/L for β- and γ-rubromycins, respectively.

The treatment for mosquito-borne viral diseases such as dengue virus (DENV), zika virus (ZIKV), and chikungunya virus (CHIKV) has become difficult due to delayed diagnosis processes. In addition, sharing the same transmission media and similar symptoms at the early stage of infection of these diseases has become more critical for early diagnosis. To overcome this, a common platform that can identify the virus with high sensitivity and selectivity, even for the different serotypes, is in high demand. In this study, we have attempted an electrochemical impedimetric method to detect the ZIKV, DENV, and CHIKV using their corresponding antibody-conjugated sensor electrodes. The significance of this method is emphasized on the fabrication of a common matrix of gold–polyaniline and sulfur, nitrogen-doped graphene quantum dot nanocomposites (Au-PAni-N,S-GQDs), which have a strong impedimetric response based only on the conjugated antibody, resulting in minimum cross-reactivity for the detection of various mosquito-borne viruses, separately. As a result, four serotypes of DENV and ZIKV, and CHIKV have been detected successfully with an LOD of femtogram mL−1.

Abstract Recombinant proteins produced in insect cells and insects, unlike those produced in mammalian cells, have pauci-mannose-type N-glycans. In this study, we examined complex-type N-glycans on recombinant proteins via coexpression of human β-1,2-N-acetylglucosaminyltransferase II (hGnT II) and human β1,4-galactosyltransferase (hGalT I) in silkworm pupae, by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The actin A3 promoter from B. mori and the polyhedrin promoter from Autographa californica multiple nucleopolyhedroviruses (AcMNPVs) were used to coexpress hGnT II and hGalT I. These recombinant BmNPVs were coexpressed with human IgG (hIgG), hGnT II and hGalT I in silkworm pupae. When hIgG was coexpressed with hGnT II, approximately 15% of all N-glycans were biantennary, with both arms terminally modified with N-acetylglucosamine (GlcNAc). In contrast, when hIgG was coexpressed with both hGnT II and hGalT I under the control of the polyhedrin promoter, 27% of all N-glycans were biantennary and terminally modified with GlcNAc, with up to 5% carrying one galactose and 11% carrying two. The obtained N-glycan structure was dependent on the promoters used for coexpression of hGnT II or hGalT I. This is the first report of silkworm pupae producing a biantennary, terminally galactosylated N-glycan in a recombinant protein. These results suggest that silkworms can be used as alternatives to insect and mammalian hosts to produce recombinant glycoproteins with complex N-glycans.

Abstract An entomopathogenic fungus, Cordyceps sp. has been known to produce cordycepin which is a purine nucleoside antimetabolite and antibiotic with potential anticancer, antioxidant and anti‐inflammatory activities. Interestingly, Cordyceps militaris produces significantly higher amount in a liquid surface culture than in a submerged culture. The liquid surface culture consists of mycelia growing into the air (aerial mycelia) and mycelia growing toward the bottom into the medium (submerged mycelia). In this study, to clarify roles of aerial and submerged mycelia of C. militaris in the cordycepin production the difference in metabolism between these mycelia was investigated. From transcriptomic analyses of the aerial and submerged mycelia at the culture of 5, 12 and 19 days, the metabolism of the submerged mycelia switched from the oxidative phosphorylation to the fermentation pathway. This activated the pentose phosphate pathway to provide building block materials for the nucleotide biosynthetic pathway. Under hypoxic conditions, the 5‐aminolevulinic acid synthase (CCM_01504), delta‐aminolevulinic acid dehydratase (CCM_00935), coproporphyrinogen III oxidase (CCM_07483) and cytochrome c oxidase 15 (CCM_05057) genes of heme biosynthesis were significantly upregulated. In addition, the liquid surface culture revealed that metabolite coproporhyrinogen III and glycine, the product and precursor of heme, were increased at 12th day and decreased at 19th day, respectively. These results indicate that the submerged mycelia induce the activation of iron acquisition, the ergosterol biosynthetic pathway, and the iron cluster genes of cordycepin biosynthesis in a hypoxic condition. Even though, the expression of the cluster genes of cordycepin biosynthesis was not significantly different in both types of mycelia.

Cordyceps militaris 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 C. militaris during its infection were investigated. After the injection of conidia, C. militaris NBRC100741 killed silkworm larvae more rapidly than NBRC103752. At 96 and 120 h, Cmcns genes (Cmcns1–4), 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 Metarhizium anisopliae and Beauveria bassiana, 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 C. militaris in silkworm larvae by the influence of the expression of some genes in silkworm larvae.

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 15N-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-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori 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 13C-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.

Neosporosis, which is caused by Neospora caninum, is a well-known disease in the veterinary field. Infections in pregnant cattle lead to abortion via transplacental (congenitally from mother to fetus) transmission. In this study, a N. caninum profilin (NcPROF), was expressed in silkworm larvae by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid and was purified from the hemolymph. Three NcPROF constructs were investigated, native NcPROF fused with an N-terminal PA tag (PA-NcPROF), PA-NcPROF fused with the signal sequence of bombyxin from B. mori (bx-PA-NcPROF), and bx-PA-NcPROF with additional C-terminal transmembrane and cytoplasmic domains of GP64 from BmNPV (bx-PA-NcPROF-GP64TM). All recombinant proteins were observed extra- and intracellularly in cultured Bm5 cells and silkworm larvae. The bx-PA-NcPROF-GP64TM was partly abnormally secreted, even though it has the transmembrane domain, and only it was pelleted by ultracentrifugation, but PA-NcPROF and bx-PA-NcPROF were not. Additionally, bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph by anti-PA agarose beads while PA-NcPROF and bx-PA-NcPROF were not. The purified bx-PA-NcPROF-GP64TM protein bound to its receptor, mouse Toll-like receptor 11 (TLR-11), and formed unique nanoparticles. These results suggest that profilin fused with GP64TM was secreted as a nanoparticle with binding affinity to its receptor and this nanoparticle formation is advantageous for the development of vaccines to N. caninum.

Riboflavin production in a culture of Ashbya gossypii was enhanced by adding mineral support with adsorbed soybean oil. When the support in an amount of 1 % to the amount of medium was added into the culture at agitation intensity corresponding to impeller rotation rate of 600 rpm, the attained riboflavin concentration was 2.5 g/L at the culture time of 4 days, i.e. 1.6 times higher than that in the culture without adding mineral support. Riboflavin yield coefficient based on consumed substrate was also 1.6-fold higher than that in the culture without mineral support. In order to investigate the effect of mineral support on the riboflavin production and mycelial morphology variation, intracellular oil droplets were investigated by staining mycelia with Nile red. The A. gossypii cells, growing in submerged culture using soybean oil as a carbon source, were found to form intracellular micro-lipid bodies. The oil droplets became bigger as the culture time increased, and then the riboflavin leakage began, whereas lipid bodies gradually disappeared. When the soybean oil adsorbed on mineral support was added to the culture, the diameter of A. gossypii mycelia was much thicker and more riboflavin crystals were accumulated than in the pool of culture without mineral support; and what is more, mycelial autolysis was delayed for 2 days due to the presence of mineral support.

Plasmonic nanomaterials (P-NM) are receiving attention due to their excellent properties, which include surface-enhanced Raman scattering (SERS), localized surface plasmon resonance (LSPR) effects, plasmonic resonance energy transfer (PRET), and magneto optical (MO) effects. To obtain such plasmonic properties, many nanomaterials have been developed, including metal nanoparticles (MNP), bimetallic nanoparticles (bMNP), MNP-decorated carbon nanotubes, (MNP-CNT), and MNP-modified graphene (MNP-GRP). These P-NMs may eventually be applied to optical biosensing systems due to their unique properties. Here, probe biomolecules, such as antibodies (Ab), probe DNA, and probe aptamers, were modified on the surface of plasmonic materials by chemical conjugation and thiol chemistry. The optical property change in the plasmonic nanomaterials was monitored based on the interaction between the probe biomolecules and target virus. After bioconjugation, several optical properties, including fluorescence, plasmonic absorbance, and diffraction angle, were changed to detect the target biomolecules. This review describes several P-NMs as potential candidates of optical sensing platforms and introduces various applications in the optical biosensing field.

Background: Engineered multifunctional nanoparticles (NPs) have made a tremendous impact on the biomedical sciences, with advances in imaging, sensing and bioseparation. In particular, the combination of optical and magnetic responses through a single particle system allows us to serve as novel multimodal molecular imaging contrast agents in clinical settings. Despite of essential medical imaging modalities and of significant clinical application, only few nanocomposites have been developed with dual imaging contrast. A new method for preparing quantum dots (QDs) incorporated magnetic nanoparticles (MNPs) based on layer-by-layer (LbL) self-assembly techniques have developed and used for cancer cells imaging. Methods: Here, citrate - capped negatively charged Fe3O4 NPs were prepared and coated with positively - charged hexadecyltrimethyl ammonium bromide (CTAB). Then, thiol - capped negatively charged CdTe QDs were electrostatically bound with CTAB. Morphological, optical and magnetic properties of the fluorescent magnetic nanoparticles (FMNPs) were characterized. Prepared FMNPs were additionally conjugated with hCC49 antibodies fragment antigen binding (Fab) having binding affinity to sialylated sugar chain of TAG-72 region of LS174T cancer cells, which was prepared silkworm expression system, and then were used for imaging colon carcinoma cells. Results: The prepared nanocomposites were magnetically responsive and fluorescent, simultaneously that are useful for efficient cellular imaging, optical sensing and magnetic separation. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed that the particle size is around 50 nm in diameter with inner magnetic core and outer CdTe QDs core-shell structure. Cytotoxicity test of prepared FMNPs indicates high viability in Vero cells. NPs conjugated with anti cancer antibodies were successfully labeled on colon carcinoma cells (LS174) in vitro and showed significant specificity to target cells. Conclusion: The present report demonstrates a simple synthesis of CdTe QDs-Fe3O4 NPs. The surface of the prepared FMNPs was enabled simple conjugation to monoclonal antibodies by electrostatic interaction. This property further extended their in vitro applications as cellular imaging contrast agents. Such labeling of cells with new fluorescentmagneto nanoprobes for living detection is of interest to various biomedical applications and has demonstrated the potential for future medical use. [ABSTRACT FROM AUTHOR]

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity. [ABSTRACT FROM AUTHOR]

The circulating RA (renin–angiotensin) system is essential for the regulation of blood pressure and electrolyte balance. Recently, plasma prorenin has been reported to significantly increase its level in diabetes and to be possibly non-proteolytically activated by binding to the PRR [(pro)renin receptor] on the cell membrane reported in several tissues during circulation. Although many pathological aspects have been researched, there is a lack of sufficient information on the biochemical structure and biological function of this hPRR (human PRR) because of the difficulty in increasing hPRR expression. In the present study, GFPuv–hPRR (hPRR fused with green fluorescence protein when excited with long-wave UV light) was successfully expressed by using BmMNPV (Bombyx mori multiple nucleopolyhedrovirus) bacmid DNA in silkworm (Bombyx mori) larvae. Some of the hPRR was expressed in the haemolymph of silkworm larvae and some of the hPRR was located in the fat body of silkworm larvae. The binding ability of hPRR expressed in the haemolymph and fat body with renin or prorenin was analysed by ELISA and surface plasmon resonance using a biosensor respectively. These binding assays suggest that the expressed hPRR has a functional bioactivity. hPRR preparation in silkworm larvae would, therefore, be useful for biochemical and biomedical researches related to PRR. [ABSTRACT FROM AUTHOR]

A bacmid system of BmMNPV [Bombyx mori (silkworm) multiple nucleopolyhedrovirus] with both the cysteine-protease and chitinase genes deleted (BmMNPV-CP−-Chi−) was constructed. The viral protease and chitinase activities in the haemolymph of Bombyx mori larvae infected with this BmMNPV-CP−-Chi−bacmid were reduced by 95 and 50% respectively. By using this system, a GFPuv–β3GnT2 (green fluorescent protein excited by UV light–β1,3-N-acetylglucosaminyltransferase 2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; β3GnT2 activity improved 2.8-fold over that of unmodified bacmid. This BmMNPV-CP−-Chi−bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation. [ABSTRACT FROM AUTHOR]

Human β-1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) is indispensable for the conversion of lacto-N-triose II into lacto-N-tetraose and lacto-N-neotetraose. In this paper, we report multiple co-transfection in a single step using two different human cDNAs in an insect cell, β3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one-step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25–1500 μg/ml respectively during co-transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed β3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m-units/ml compared with 6.7 m-units/ml by a cell only carrying β3GnT2. In this study we correlated the activity of β3GnT2 with the amount of β3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and β3GnT2 cDNA were in a rough ratio of 1:1, the β3GnT2 activity was at a high level. In order to achieve better expression levels of β3GnT2 with less cost and time, efficient ways have to be devised. [ABSTRACT FROM AUTHOR]

Among the members of flaviviruses, the Zika virus (ZIKV) remains a potent infectious disease agent, with its associated pandemic prompting the World Health Organization (WHO) to declare it a global public health concern. Thus, rapid and accurate diagnosis of the ZIKV is needed. In this study, we report a new immunofluorescence biosensor for the detection of nonstructural protein 1 (NS1) of the ZIKV, which operates using the localized surface plasmon resonance (LSPR) signal from plasmonic gold nanoparticles (AuNPs) to amplify the fluorescence intensity signal of quantum dots (QDs) within an antigen-antibody detection process. The LSPR signal from the AuNPs was used to amplify the fluorescence intensity of the QDs. For ultrasensitive, rapid, and quantitative detection of NS1 of the ZIKV, four different thiol-capped AuNPs were investigated. Our biosensor could detect the ZIKV in a wide concentration range from 10-107 RNA copies/mL, and we found that the limit of detection (LOD) for the ZIKV followed the order Ab-L-cysteine-AuNPs (LOD = 8.2 copies/mL) > Ab-3-mercaptopropionic acid-AuNPs (LOD = 35.0 copies/mL). Immunofluorescence biosensor for NS1 exhibited excellent specificity against other negative control targets and could also detect the ZIKV in human serum.

Cordyceps militaris produces cordycepin, which is known to be a bioactive compound. Currently, cordycepin hyperproduction of C. militaris was carried out in a liquid surface culture because of its low productivity in a submerged culture, however the reason was not known. In this study, 4.92 g/L of cordycepin was produced at the 15th day of C. militaris NBRC 103752 liquid surface culture, but only 1 mg/L was produced in the submerged culture. RNA-Seq was used to clarify the gene expression profiles of the cordycepin biosynthetic pathways of the submerged culture and the liquid surface culture. From this analysis, 1036 genes were shown to be upregulated and 557 genes were downregulated in the liquid surface culture compared with the submerged culture. Specifically, adenylosuccinate synthetase and phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthase in purine nucleotide metabolism were significantly upregulated in the liquid surface culture. Thick mycelia formation in the liquid surface culture was found to induce the expression of hypoxia-related genes (GABA shunt, glutamate synthetase precursor, and succinate-semialdehyde dehydrogenase). Cytochrome P450 oxidoreductases containing heme were also found to be significantly enriched, suggesting that a hypoxic condition might be created in the liquid surface culture. These results suggest that hypoxic conditions are more suitable for cordycepin production in the liquid surface culture compared with the submerged culture. Our analysis paves the way for unraveling the cordycepin biosynthesis pathway and for improving cordycepin production in C. militaris.

Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.

Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

Neosporosis, caused by an intracellular parasite, Neospora caninum, is an infectious disease primarily of cattle and dogs. It occurs worldwide and causes huge damages to dairy farms. In this study, we immunized mice with recombinant surface-associated protein 1 of N. caninum (rNcSAG1) and developed two novel monoclonal antibodies, A10 and H3, against NcSAG1 using phage-display technology. Both clones bound to purified rNcSAG1 and the half maximal inhibitory concentrations of A10 and H3 are 50 and 72 nM of rNcSAG1, respectively. In immunofluorescence assays, both A10 and H3 Fabs bound to N. caninum parasites. Direct detection of N. caninum parasites was developed firstly using an enzyme-linked immunosorbent assay (ELISA) with A10 and H3. Binding of A10 and H3 antibodies to rNcSAG1 was also inhibited by some certain anti-N. caninum antibodies in the neosporosis-positive cattle sera, suggesting they might bind to the same epitopes of NcSAG1 with those anti-N. caninum antibodies of bovine. These antibodies were demonstrated to have a potential for monitoring the N. caninum parasites in a dairy farm, which may lead to protect livestock from parasite-infection.

A ligation independent cloning (LIC) system has been developed to facilitate the rapid and high-efficiency cloning of genes in a Bombyx mori expression system. This system was confirmed by the expression of human microsomal triglyceride transfer protein (hMTP) fused with EGFP in silkworm larvae and pupae. Moreover, hMTP and human protein disulfide isomerase (hPDI) genes were inserted into two LIC vectors harboring gcLINK sequences and were combined by using the LIC through gcLINK sequences. The constructed vector was incorporated into the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid, and injected into silkworm larvae. The expressed hMTP-hPDI complex was purified from the fat bodies of silkworm larvae. This LIC vector system was applied to express the E1, E2, and E3 subunits of human α-ketoglutarate dehydrogenase (KGDH) in silkworm larvae. The expressed proteins were purified easily from fat bodies using three different affinity chromatography steps. The LIC vectors constructed as described in this report allow for the rapid expression and purification of recombinant proteins or their complexes by using the BmNPV bacmid system.