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DOI： 10.1016/j.aspen.2020.05.001

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DOI： 10.1016/j.cbd.2015.07.003

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DOI： 10.1007/s11010-015-2529-5

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DOI： 10.1007/s12033-015-9866-1

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DOI： 10.1016/j.aspen.2015.03.006

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DOI： 10.1016/j.ibmb.2015.01.008

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DOI： 10.1016/j.ibmb.2014.10.001

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DOI： 10.1016/j.febslet.2014.09.009

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DOI： 10.1016/j.ibmb.2014.05.008

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DOI： 10.1007/s12010-014-0814-5

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DOI： 10.1007/s00253-013-4785-1

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DOI： 10.1111/j.1744-7917.2012.01569.x

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DOI： 10.1371/journal.pone.0052320

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DOI： 10.1007/s10529-012-0971-y

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DOI： 10.1016/j.cellbi.2005.07.007

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DOI： 10.1007/s11033-004-2474-y

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DOI： 10.1080/10425170400028871

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DOI： 10.1111/j.1365-2583.2004.00553.x

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DOI： 10.1016/j.bbagen.2005.02.016

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DOI： 10.1016/j.femsle.2005.01.032

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DOI： 10.1080/07924259.2004.9652579

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DOI： 10.1016/j.cbpc.2004.06.004

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DOI： 10.1016/j.bbrc.2003.10.169

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DOI： 10.1016/S1096-4959(02)00153-7

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DOI： 10.1016/S0965-1748(01)00074-1

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DOI： 10.1006/abio.2000.4745

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DOI： 10.1006/abbi.1995.9953

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Norovirus (NoV) is one of the major causes of acute viral gastroenteritis in humans. Genetic variation is abundant, and prevalent genotypes vary from year to year and region to region. Since NoVs are difficult to amplify in cultured cells, genome RNA-free virus-like particles (VLPs) that mimic the capsid structure of the virus are promising vaccine candidates for the prevention of NoVs infection, and the development of multivalent VLP vaccines is required to prevent NoV infection in a wide range of genotypes. In this study, we attempted to produce NoV VLPs of the top nine genotypes that have a history of epidemics in Japan using the silkworm-baculovirus expression vector system (silkworm-BEVS), which has a proven track record in the mass production of recombinant proteins. In silkworm pupae infected with recombinant baculoviruses constructed to express VP1s, the major protein that forms VLP, the NoV VP1 protein was expressed in large amounts. Most genotypes of VP1 accumulated in the cytoplasm as soluble proteins, but solubility was reduced for that of two genotypes. VP1s of five genotypes could be purified in large quantities (>0.9 mg per pupa) by a two-step purification process, and gel filtration chromatography analysis confirmed the formation of VLPs. This study demonstrates the utility of silkworm-BEVS in producing NoV VLPs of multiple genotypes and provides the basis for the development of a multivalent vaccine against genetically diverse NoV infections.

DOI： 10.1016/j.jviromet.2024.115038

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The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

DOI： 10.1016/j.jviromet.2024.115029

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Language：English   Publisher：Frontiers in Immunology  

A monovalent Omicron XBB.1.5 mRNA RBD analogue vaccine, MAFB-7256a (DS-5670d), was newly developed and approved in Japan in the Spring of 2024 for the prevention of COVID-19. However, clinical efficacy data for this vaccine are currently lacking. We previously established the Quantification of Antigen-specific Antibody Sequence (QASAS) method to assess the response to SARS-CoV-2 vaccination at the mRNA level using B-cell receptor (BCR) repertoire assays and the Coronavirus Antibody Database (CoV-AbDab). Here, we used this method to evaluate the immunogenicity of MAFB-7256a. We analyzed repeated blood samples using the QASAS method from three healthy volunteers before and after MAFB-7256a vaccination. BCR response increased rapidly one week post-vaccination and then decreased, as with conventional vaccine. Notably, the matched sequences after MAFB-7256a vaccination specifically bound to the receptor-binding domain (RBD), with no sequences binding to other epitopes. These results validate that MAFB-7256a is an effective vaccine that exclusively induces antibodies specific for the RBD, demonstrating its targeted immunogenic effect.

DOI： 10.3389/fimmu.2024.1468760

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Language：English   Publisher：International Journal of Molecular Sciences  

During the COVID-19 pandemic, diabetic and obese patients experienced higher rates of hospital admissions, severe illness, and mortality. However, vaccinations failed to provide those vulnerable populations the same level of protection against COVID-19 severity as those without diabetic and obese phenotypes. Our study aimed to investigate how diabetes mellitus (DM) impacts the immune response following vaccination including the artificially designed trimeric SARS-CoV-2 spike (S)-protein. By using two diabetic mouse models, ob/ob mice (obese, hyperglycemic, and insulin-resistant) and STZ-treated mice (insulin-deficient and hyperglycemic), we observed a significant reduction in S-protein-specific IgG antibody titer post-vaccination in both diabetic models compared to wild-type (WT) mice. Both diabetic mouse models exhibited significant abnormalities in spleen tissue, including marked reductions in splenic weight and the size of the white pulp regions. Furthermore, the splenic T-cell and B-cell zones were notably diminished, suggesting an underlying immune dysfunction that could contribute to impaired antibody production. Notably, vaccination with the S-protein, when paired with an optimal adjuvant, did not exacerbate diabetic cardiomyopathy, blood glucose levels, or liver function, providing reassurance about the vaccine′s safety. These findings offer valuable insights into potential mechanisms responsible for the decreased persistence of antibody production in diabetic patients.

DOI： 10.3390/ijms251910379

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DOI： 10.1002/jha2.932

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4–2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

DOI： 10.1016/j.pep.2024.106450

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Language：English   Publisher：Scientific Reports  

High pathogenicity avian influenza (HPAI) poses a significant threat to both domestic and wild birds globally. The avian influenza virus, known for environmental contamination and subsequent oral infection in birds, necessitates careful consideration of alternative introduction routes during HPAI outbreaks. This study focuses on blowflies (genus Calliphora), in particular Calliphora nigribarbis, attracted to decaying animals and feces, which migrate to lowland areas of Japan from northern or mountainous regions in early winter, coinciding with HPAI season. Our investigation aims to delineate the role of blowflies as HPAI vectors by conducting a virus prevalence survey in a wild bird HPAI-enzootic area. In December 2022, 648 Calliphora nigribarbis were collected. Influenza virus RT-PCR testing identified 14 virus-positive samples (2.2% prevalence), with the highest occurrence observed near the crane colony (14.9%). Subtyping revealed the presence of H5N1 and HxN1 in some samples. Subsequent collections in December 2023 identified one HPAI virus-positive specimen from 608 collected flies in total, underscoring the potential involvement of blowflies in HPAI transmission. Our observations suggest C. nigribarbis may acquire the HPAI virus from deceased wild birds directly or from fecal materials from infected birds, highlighting the need to add blowflies as a target of HPAI vector control.

DOI： 10.1038/s41598-024-61026-1

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Language：English   Publisher：Vaccine  

Background: SARS-CoV-2 Omicron breakthrough infection (Omicron-BTI) after vaccination has been frequently observed. A more detailed understanding of the humoral immunity against Omicron-BTI is required. Methods: We measured strain-specific live-virus based neutralizing activity, anti-spike IgG, and anti-receptor-binding domain (RBD) IgG titers in individuals with Omicron/BA.1-BTI and directly compared them with controls with diverse combinations of wild-type (WT) mRNA vaccination and infection history. Results: Omicron-BTI individuals showed markedly higher neutralizing titers against all the WT, Delta, and Omicron strains in convalescent sera, compared with unvaccinated Omicron-infection individuals with only Omicron neutralizing activity. Similar tendencies were found in strain-specific anti-spike and anti-RBD IgG titers. The Omicron-specificity (BA.1/WT neutralizing ratio), Omicron-neutralizing efficiency per antibody unit, and anti-Omicron RBD-directivity of anti-spike antibodies in Omicron-BTI individuals were all significantly lower than those in unvaccinated Omicron-infection individuals, but they were equivalent to or higher than those in uninfected vaccinees. The induction of Omicron-specific neutralizing activity after Omicron-BTI was not weakened for eight months from the last vaccination. Conclusions: These findings suggest that cross-reactive vaccine-induced immunity was intensively stimulated following Omicron breakthrough infection, which contributed to Omicron neutralization. Measuring SARS-CoV-2 variant-specific antibody levels as well as neutralizing activity is useful for evaluating humoral immunity after breakthrough infection in the current situation of antigenic gaps between vaccinated and epidemic (Omicron sub-lineages) strains.

DOI： 10.1016/j.vaccine.2023.10.035

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Tributyltin (TBT)-binding protein type 1 in Japanese medaka (Oryzias latipes) (O.latTBT-bp1) is a fish lipocalin implicated in TBT binding and detoxification. We purified recombinant O.latTBT-bp1 (rO.latTBT-bp1; ca. 30 kDa) by using a baculovirus expression system and His- and Strep-tag chromatography process. Then, we examined O.latTBT-bp1 binding to several endo/exogenous steroid hormones by means of competitive binding assay. The dissociation constants for the binding of rO.latTBT-bp1 to DAUDA and ANS, two fluorescent ligands of lipocalin, were 7.06 and 13.6 μM, respectively. Multiple model validations indicated that a single-binding-site model was the most appropriate for evaluating rO.latTBT-bp1 binding. In the competitive binding assay, testosterone, 11-ketotestosterone, and 17β-estradiol were each bound by rO.latTBT-bp1; rO.latTBT-bp1 showed the strongest affinity for testosterone (inhibition constant, Ki = 3.47 μM). Endocrine-disrupting chemical (synthetic steroid) also bound to rO.latTBT-bp1; the affinity for ethinylestradiol (Ki = 9.29 μM) was stronger than that for 17β-estradiol (Ki = 30.0 μM). To determine the function of O.latTBT-bp1, we produced TBT-bp1 knockout medaka (TBT-bp1 KO), which we exposed to ethinylestradiol for 28 days. After exposure, the number of papillary processes in TBT-bp1 KO genotypic male medaka was significantly fewer (3.5), compared to that in wild-type male medaka (22). Thus, TBT-bp1 KO medaka were more sensitive to the anti-androgenic effects of ethinylestradiol than wild-type medaka. These results indicate that O.latTBT-bp1 may bind to steroids and act as a gatekeeper of ethinylestradiol action by regulating the androgen–estrogen balance.

DOI： 10.1016/j.cbpc.2023.109660

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. In this study, we attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances our understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.

DOI： 10.3390/insects14080684

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：IOS Press  

BACKGROUND: A cost-effective and eco-friendly method is needed for the assessment of humoral immunity against SARS-CoV-2 in large populations. OBJECTIVE: We investigated the performance of an ELISA that uses silkworm-produced proteins to quantify the strain-specific anti-Spike IgG (anti-S IgG) titer. METHODS: The OD values for the anti-His-tag antibody, a standard material of ELISA quantification, were measured. Correlations between the ELISA for each strain and the Abbott SARS-CoV-2 IgG II Quant assay for the wild type were evaluated with serum samples from nine participants with various infection and vaccination statuses. RESULTS: Linear dose-responses were confirmed by high coefficients of determination: 0.994, 0.994, and 0.996 for the wild-type, Delta, and Omicron (BA.1) strain assays, respectively. The coefficient of determination for the wild-type and Delta strain assays was high at 0.959 and 0.892, respectively, while the Omicron strain assay had a relatively low value of 0.563. Booster vaccinees showed similar or higher titers against all strains compared to infected persons without vaccination. The Omicron-infected persons without vaccination had lower antibody titers against wild type than did the vaccinated persons. CONCLUSIONS: This study provides data indicating that the ELISA with silkworm-produced proteins makes it possible to discriminate and quantify the strain-specific anti-S IgG antibody induced by vaccination or infection.

DOI： 10.3233/hab-230006

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Language：English   Publisher：Journal of Pharmacological Sciences  

The outbreak of the SARS-2-CoV infection has become a global outbreak and continues to cause many deaths. In addition, the risk of pandemics continues to increase due to environmental changes and the globalization of human exchange and logistics. On the other hand, our preparedness for emerging infectious diseases caused by such unknown viruses is inadequate, and dealing with viral infections is one of the most important issues that need to be addressed immediately. Vaccine based disease control is considered an ideal countermeasure for infectious diseases, as it is expected to provide maximum efficacy at minimum cost. Although new nucleic acid-based vaccines are leading the way in the prevention of COVID-19, the mainstream of vaccines is still inactivated or live attenuated vaccines that use the pathogen virus itself. Subunit vaccines, in which specific virus-derived proteins are produced as recombinant proteins and used as vaccine antigens, have been developed, but production and development of many antigens that are difficult to mass-produce as recombinant proteins, such as the spike protein antigen of COVID-19 has not progressed. This paper describes the development of recombinant protein vaccines using the silkworm, which has an advantage in the production of such difficult-to-express vaccine antigens, especially virus-like particles.

DOI： 10.1016/j.jphs.2023.01.002

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Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Journal of Pharmaceutical Sciences  

Malaria is a mosquito-borne infectious disease that is widespread in developing countries. Malaria vaccines are important in efforts to eradicate malaria; however, vaccines are usually administered by injection, which requires medical personnel and has a risk of causing infection. Transdermal vaccines can be administered without damaging the skin and thus are ideal for the prevention of malaria. However, the stratum corneum forms a "brick and mortar" like structure in which stratum corneum cells are embedded in a hydrophobic matrix composed of lipids, which strongly inhibits the permeation of hydrophilic substances. In the present study, we designed a transdermal vaccine against vivax malaria using a solid-in-oil (S/O) dispersion. The S/O dispersion of a transmission blocking vaccine candidate, Pvs25 from Plasmodium vivax, showed higher skin penetration than that of the aqueous solution. Mice immunized with the S/O dispersion generated antibodies at similar titers as the mice immunized by injection, over the mid- to long-term. These results provide information for the development of transdermally administered malaria vaccines toward the eradication of malaria.

DOI： 10.1016/j.xphs.2022.10.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Molecular Sciences  

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of the key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells is initiated by binding with its receptor, angiotensin-converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we report that ibudilast, which we previously identified as a potent inhibitor of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2, attenuates the SARS-CoV-2 spike glycoprotein pseudovirus-evoked contractile and metabolic dysfunctions of neonatal rat cardiomyocytes (NRCMs). Epidemiologically reported risk factors of severe COVID-19, including cigarette sidestream smoke (CSS) and anti-cancer drug treatment, commonly upregulate ACE2 expression level, and these were suppressed by inhibiting TRPC3-Nox2 complex formation. Exposure of NRCMs to SARS-CoV-2 pseudovirus, as well as CSS and doxorubicin (Dox), induces ATP release through pannexin-1 hemi-channels, and this ATP release potentiates pseudovirus entry to NRCMs and human iPS cell-derived cardiomyocytes (hiPS-CMs). As the pseudovirus entry followed by production of reactive oxygen species was attenuated by inhibiting TRPC3-Nox2 complex in hiPS-CMs, we suggest that TRPC3-Nox2 complex formation triggered by panexin1-mediated ATP release participates in exacerbation of myocardial damage by amplifying ACE2-dependent SARS-CoV-2 entry.

DOI： 10.3390/ijms24010102

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Protein Expression and Purification  

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.

DOI： 10.1016/j.pep.2022.106096

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Heterochromatin protein 1 plays an important role in chromatin structure and gene expression regulation. Three HP1 genes have been found in Homo sapiens, and five HP1 genes have been reported in Drosophila melanogaster. On the other hand, in Bombyx mori, only two HP1 genes, BmHP1a and BmHP1b, were reported. In this research, we have reported the molecular and functional characterization of a novel Bombyx mori HP1 gene (BmHP1c), which had stronger transcriptional repression activity than BmHP1a. BmHP1a and BmHP1b is reported to form homo- and heterodimers, but in co-immunoprecipitation experiments, no homo- or hetero-dimer formation of BmHP1c with the other silkworm HP1s is detected. The intracellular localization of BmHP1c is not only in the nucleus but also in the cytoplasm like mammalian HP1γ. In contrast to human HP1a and b, all three BmHP1s were localized preferentially in the regions poorly stained with DAPI. Interestingly, the double knockdown of BmHP1a and b, but not BmHP1c with a or b, arrested the cell cycle at the G2/M phase. These results suggest that BmHP1c is not essential for cell progression and plays a different role than BmHP1a and BmHP1b.

DOI： 10.3390/insects13070631

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Pharmacological Sciences  

Coronavirus disease 2019 (COVID-19) remains prevalent worldwide since its onset was confirmed in Wuhan, China in 2019. Vaccines against the causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have shown a preventive effect against the onset and severity of COVID-19, and social and economic activities are gradually recovering. However, the presence of vaccine-resistant variants has been reported, and the development of therapeutic agents for patients with severe COVID-19 and related sequelae remains urgent. Drug repurposing, also called drug repositioning or eco-pharma, is the strategy of using previously approved and safe drugs for a therapeutic indication that is different from their original indication. The risk of severe COVID-19 and mortality increases with advancing age, cardiovascular disease, hypertension, diabetes, and cancer. We have reported three protein-protein interactions that are related to heart failure, and recently identified that one mechanism increases the risk of SARS-CoV-2 infection in mammalian cells. This review outlines the global efforts and outcomes of drug repurposing research for the treatment of severe COVID-19. It also discusses our recent finding of a new protein-protein interaction that is common to COVID-19 aggravation and heart failure.

DOI： 10.1016/j.jphs.2022.04.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Insect Biochemistry and Molecular Biology  

The ovary is an important organ in reproduction. In insects, especially lepidopteran insects, the oocytes and reproductive organs develop rapidly during the pupal stage. Despite their drastic morphological changes, the molecular mechanisms of ovary development are not fully understood. In this study, it is found that forkhead box transcription factor L2, member 1 (FoxL21), which is known to be involved in ovarian differentiation and maintenance in vertebrates, is required for the development of the ovary in the silkworm, Bombyx mori. FoxL21 was expressed in the ovary and ovariole during the larval and pupal stage, respectively. In silkworms in which FoxL21 was knocked out by genome editing, multiple ovarian dysfunctions, such as, abnormal egg formation, thinning of the ovariole sheaths, and defective connection of the oviductus geminus with the ovariole were observed. Finally, ovarian transplantation experiments using the knockout silkworms revealed that FoxL21 functions in the ovariole, but not in the oviductus geminus.

DOI： 10.1016/j.ibmb.2022.103737

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Language：English   Publishing type：Research paper (scientific journal)  

Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells. We produced recombinant leptin using silkworm pupae and investigated the effects of leptin on FSH and LH secretion and gene expression in the primary culture of pituitary cells from chub mackerel. The presence or absence of co-expression of lepr mRNA, FSH and LH b-subunit mRNA in gonadotropic cells was examined by double-labeled in situ hybridization. The addition of leptin significantly increased the secretion and gene expression of FSH and LH from male and female pituitary cells in primary culture. In contrast, gonadotropin-releasing hormone 1 affected neither FSH secretion in cells from females nor fshb and lhb expression in cells from males and females. The expression of lepr was observed in FSH- and LH-producing cells of both males and females. The results indicate that leptin directly regulates gonadotropin synthesis and secretion and plays an important role in the induction of puberty in teleost fish.

DOI： 10.3390/cells10123505

Language：English   Publishing type：Research paper (scientific journal)  

There are several known non-molting mutations of the silkworm, Bombyx mori, including non-molting dwarf (nm-d). Larvae with this mutation hatch normally and start eating leaves, but die before the completion of the first ecdysis. Genetic analysis of the nm-d mutation would contribute to the isolation of essential genes for the larval development of lepidopteran insects. To identify the causative gene of the nm-d locus, we conducted RNA-seq based rough mapping. Using two sets of RNA-seq data, one from a pooled sample of normal larvae, and one from a pooled sample of nm-d larvae, the nm-d locus was narrowed to a 500 kb region. Among the genes located in this region, an nm-d-specific exon loss was identified in the Bombyx homolog of the ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/Inosine 5'-monophosphate cyclohydrolase) (BmATIC) gene, which catalyzes the final two steps of the de novo purine biosynthetic pathway in mammals. PCR and subsequent sequencing analysis revealed that a region containing exon 9 of the BmATIC gene is deleted in the nm-d larvae. A knockout allele of the BmATIC gene (BmATICKO), that was generated using the CRISPR/Cas9 system, revealed that first instar knockout larvae died while exhibiting the dark brown larval body that is a typical feature of mutants that lack uric acid in the integument. Lethal larvae resulted from crosses between +/BmATICKO moths. The uric acid content in the whole-body of the first instar was drastically reduced in the nm-d larvae compared to normal larvae. These results indicated that the BmATIC gene is responsible for the nm-d phenotype, and that nm-d larvae have a defect in purine biosynthesis, including uric acid. We also discuss the possibility that the BmATIC mRNA is maternally transmitted to eggs. Our results indicated that RNA-seq based mapping using pooled samples is a practical method for the identification of the causative genes of lethal mutations.

DOI： 10.1016/j.ibmb.2021.103636

Language：English   Publishing type：Research paper (scientific journal)  

Parasitic helminths can reside in humans owing to their ability to disrupt host protective immunity. Receptor for advanced glycation end products (RAGE), which is highly expressed in host skin, mediates inflammatory responses by regulating the expression of pro-inflammatory cytokines and endothelial adhesion molecules. In this study, we evaluated the effects of venestatin, an EF-hand Ca2+-binding protein secreted by the parasitic helminth Strongyloides venezuelensis, on RAGE activity and immune responses. Our results demonstrated that venestatin bound to RAGE and downregulated the host immune response. Recombinant venestatin predominantly bound to the RAGE C1 domain in a Ca2+-dependent manner. Recombinant venestatin effectively alleviated RAGE-mediated inflammation, including footpad edema in mice, and pneumonia induced by an exogenous RAGE ligand. Infection experiments using S. venezuelensis larvae and venestatin silencing via RNA interference revealed that endogenous venestatin promoted larval migration from the skin to the lungs in a RAGE-dependent manner. Moreover, endogenous venestatin suppressed macrophage and neutrophil accumulation around larvae. Although the invasion of larvae upregulated the abundance of RAGE ligands in host skin tissues, mRNA expression levels of tumor necrosis factor-α, cyclooxygenase-2, endothelial adhesion molecules vascular cell adhesion protein-1, intracellular adhesion molecule-1, and E-selectin were suppressed by endogenous venestatin. Taken together, our results indicate that venestatin suppressed RAGE-mediated immune responses in host skin induced by helminthic infection, thereby promoting larval migration. The anti-inflammatory mechanism of venestatin may be targeted for the development of anthelminthics and immunosuppressive agents for the treatment of RAGE-mediated inflammatory diseases.

DOI： 10.1371/journal.ppat.1009649

Language：Others   Publishing type：Research paper (scientific journal)  

The tumor necrosis factor α (TNFα) has been employed as a promising reagent in treating autoimmunity and cancer diseases. To meet the substantial requirement of TNFα proteins, we report in this study that mature types of recombinant human and murine TNFα proteins are successfully expressed in the baculovirus expression system using silkworm larvae as hosts. The biological activities of purified products were verified in culture murine L929 cells, showing better performance over a commercial Escherichia coli-derived murine TNFα. By comparing the activity of purified TNFα with or without the tag removal, it is also concluded that the overall activity of purified TNFα cytokines could be further improved by the complete removal of C-terminal fusion tags. Collectively, our current attempt demonstrates an alternative platform for supplying high-quality TNFα products with excellent activities for further pharmaceutical and clinical trials.

DOI： 10.3390/insects12060517

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ibmb.2020.103458

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.biochem.0c00523

Language：English   Publishing type：Research paper (scientific journal)  

Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future.

DOI： 10.1016/j.aspen.2019.12.014

Language：English   Publishing type：Research paper (scientific journal)  

In insect cell culture, a fetal bovine serum is often used to maintain the cellular physiological conditions, irre-spective of its relatively high cost and unstable supply. Several serum-free media for insects were developed, but it is challenging to adopt some insect cells to these serum-free media. Silkworm serum was used as a re-placement of fetal bovine serum for a long time but not used widely due to the melanization of insect serum catalyzed by phenoloxidase (PO). PO inhibitors reported for insect serum preparation have a significantly nega-tive effect on the long-term culture of healthy insect cells. In this study, we evaluated several kinds of PO inhibi-tors and found the method to prepare silkworm serum, which is suitable for the maintenance of insect cells and the production of the recombinant proteins by the baculovirus expression system. When L-Glutathione (reduced form) or L-Cysteine is used as a PO inhibitor at the time of recovery of silkworm serum, it is possible to sup-press the browning of the silkworm serum. However, in this state of silkworm serum, browning is observed in long-term cell culture use. Therefore, it is preferable for cell culture to add 10% of silkworm serum treated at 50°C for 30 minutes to the medium with 2.7 mM L-Glutathione. Our results suggest that silkworm serum can be an alternative of mammalian serum for cell culture and used for producing the proteins for clinical application without any risk of contamination of zoonotic infectious disease viruses.

DOI： 10.11416/jibs.89.1_009

Language：English   Publishing type：Research paper (scientific journal)  

The centromere, in which kinetochore proteins are assembled, plays an important role in the accurate congression and segregation of chromosomes during cell mitosis. Although the function of the centromere and kinetochore is conserved from monocentric to holocentric, the DNA sequences of the centromere and components of the kinetochore are varied among different species. Given the lack of core centromere protein A (CENP-A) and CENP-C in the lepidopteran silkworm Bombyx mori, which possesses holocentric chromosomes, here we investigated the role of CENP-N, another important member of the centromere protein family essential for kinetochore assembly. For the first time, cellular localization and RNA interference against CENP-N have confirmed its kinetochore function in silkworms. To gain further insights into the regulation of CENP-N in the centromere, we analyzed the affinity-purified complex of CENP-N by mass spectrometry and identified 142 interacting proteins. Among these factors, we found that the chaperone protein heat shock cognate 70 (HSC70) is able to regulate the stability of CENP-N by prohibiting ubiquitin–proteasome pathway, indicating that HSC70 could control cell cycle-regulated degradation of CENP-N at centromeres. Altogether, the present work will provide a novel clue to understand the regulatory mechanism for the kinetochore activity of CENP-N during the cell cycle.

DOI： 10.3390/ijms20235823

Language：English   Publishing type：Research paper (scientific journal)  

The receptor for advanced glycation end products (RAGE) recognizes Ca⁺⁺-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca⁺⁺-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca⁺⁺ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca⁺⁺-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes.

DOI： 10.1016/j.meegid.2019.103964

Language：English   Publishing type：Research paper (scientific journal)  

Follitropin, an important gonadotropin hormone, participates in vitellogenesis and spermatogenesis. Equine chorionic gonadotropin (eCG) can induce gonadotropin hormone activity in non-equid species and exhibits a long biological half-life. Here, we report the production, using silkworm larval and pupal systems, of biologically active recombinant hybrid-type follitropins based on the coding sequence of the eCG C-terminal peptide (CTP) between the mature β- and α-chains of eel. The three constructs, rJeFSH, rJeFSH·eCG, and rJeFSH·2xeCG were produced and verified to be N- or O-glycosylated and secreted mature peptides. Although rJeFSH·eCG contains more elaborate O-linked carbohydrate chains than rJeFSH, it elicited no significant in vitro oocyte maturation, which may be a result of insufficient terminal sialylation of its N-and O-linked carbohydrate chains. Then, a hybrid of rJeFSH·2xeCG extended with two eCG CTP. Furthermore, the receptor binding assay revealed potency of rJeFSH and rJeFSH·2xeCG to be a few folds greater than that of rJeFSH·eCG. The findings of this study will be useful for the development of more efficient GTHs in teleosts, including eels, when various modifications with two or more extended eCG CTP produced by silkworm are included.

DOI： 10.1007/s12257-019-0045-2

Language：English   Publishing type：Research paper (scientific journal)  

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg²⁺ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm–baculovirus expression vector system (silkworm–BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg²⁺, Mn²⁺, Ca²⁺, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm–BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.

DOI： 10.1007/s12033-019-00184-4

Language：English   Publishing type：Research paper (scientific journal)  

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

DOI： 10.1016/j.pep.2019.03.010

Language：English   Publishing type：Research paper (scientific journal)  

Reverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that C- terminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase.

DOI： 10.1016/j.aspen.2019.02.008

Language：English   Publishing type：Research paper (scientific journal)  

The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.

DOI： 10.1016/j.aspen.2019.01.009

Language：English   Publishing type：Research paper (scientific journal)  

Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)
₄
–Stav or Stav–(HRP)
₄
, respectively) using a baculovirus-silkworm expression system. Both (HRP)
₄
–Stav and Stav–(HRP)
₄
were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)
₄
–Stav and Stav–(HRP)
₄
could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)
₄
–Stav was twofold higher than that of Stav–(HRP)
₄
, and the sensitivity of (HRP)
₄
-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)
₄
–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.

DOI： 10.1016/j.jbiotec.2019.03.007

Language：English   Publishing type：Research paper (scientific journal)  

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter.

DOI： 10.1093/femsle/fny295

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.11416/jibs.88.1_021

Language：English   Publishing type：Research paper (scientific journal)  

The polymerization of proteins can create newly active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N- and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O
₂
to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H
₂
O
₂
. The addition of H
₂
O
₂
in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.

DOI： 10.1002/biot.201800531

Language：English   Publishing type：Research paper (scientific journal)  

Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (β1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.

DOI： 10.1007/s00253-018-9309-6

Language：English   Publishing type：Research paper (scientific journal)  

As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses.

DOI： 10.1016/j.aspen.2018.05.002

Language：English   Publishing type：Research paper (scientific journal)  

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

DOI： 10.1002/biot.201700624

Language：English   Publishing type：Research paper (scientific journal)  

Baculovirus-host interactions are important models for studying the biological control of lepidopteran pests. Research on baculovirus-host interactions has focussed on baculovirus manipulation of cellular signalling pathways, including the extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3-kinases/protein kinase B (PI3K/Akt) signalling pathways. However, the mechanism underlying ERK and PI3K/Akt activation and function in response to baculovirus infection remains poorly understood. Here, we demonstrated that baculovirus activated the Bombyx mori ERK and PI3K/Akt signalling pathways via the B. mori epidermal growth factor receptor (BmEGFR). To further characterize the function of the BmEGFR/ERK signalling pathway in baculovirus replication, we calculated genome-wide changes in kinase-chromatin interactions for ERK after baculovirus infection using chromatin immunoprecipitation followed by high-throughput sequencing. A Gene Ontology analysis showed that virus infection had effects on the biological regulation, cellular process and metabolic process pathways. Moreover, ERK was shown to regulate the transcription of late viral genes. Taken together, our results suggest that baculoviruses manipulate components of the host cell machinery for replication via modulation of the BmEGFR signalling pathway.

DOI： 10.1111/imb.12386

Language：English   Publishing type：Research paper (scientific journal)  

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm–baculovirus expression vector system (silkworm–BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

DOI： 10.1099/jgv.0.001087

Language：English   Publishing type：Research paper (scientific journal)  

Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a ly-sine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines.

DOI： 10.11416/jibs.87.2_053

Language：English   Publishing type：Research paper (scientific journal)  

Human α
₁
-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α
₁
-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.

DOI： 10.1007/s12033-018-0127-y

Language：English   Publishing type：Research paper (scientific journal)  

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called “stereotypic RFs,” are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2–CH3 elbow in the canonical antigen-binding manner involving a large antigen–antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu⁴³²–His⁴³⁵ region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

DOI： 10.1074/jbc.M117.814475

Language：English   Publishing type：Research paper (scientific journal)  

We investigated the gene structure and predicted amino acid sequence of the antioxidant enzyme 2-Cys peroxiredoxin (2-Cys Prx) in the raphidophyte Chattonella marina, which is a harmful algal bloom (HAB) species. The open reading frame of 2-Cys Prx was 585 bp long and encoded a protein consisting of 195 amino acids. The putative amino acid sequence contained two cysteine residues located at the 49th and 170th amino acid positions from the N-terminal methionine residue. The sequence also possessed 2-Cys Prx characteristic motifs, F (FFYPLDFTFVCPTEI) and EVCP. The position of the 2-Cys Prx gene relative to several others (ycf59–2-CysPrx–rpl35–rpl20) was the same as that found in the chloroplast genome in the raphidophyte Heterosigma akashiwo. Upstream of the 2-Cys Prx gene, possible TATA and GGA motifs recognized by nuclear-encoded plastid RNA polymerase (NEP), and a possible -10 box and -35 box recognized by plastid-encoded plastid RNA polymerase (PEP) were observed. We measured the transcript levels of 2-Cys Prx in C. marina cells grown under three different light intensities (0, 100, 1000 µmol photons m^–2 s^–1, 14-h light/8-h dark photoperiod) by quantitative PCR. The 2-Cys Prx transcript level in cells grown under the highest light intensity on day 3 was threefold that on day 0 but two lower light intensities resulted in relatively stable transcription levels. The 2-Cys Prx transcript level was significantly positively related to the H₂O₂ concentration per cell and the H₂O₂ scavenging activity per cell. These results suggest that C. marina 2-Cys Prx functions in the chloroplast and its transcription could be regulated by both NEP and PEP. Moreover, the 2-Cys Prx transcript level might increase to remove excessive H₂O₂ produced under strong light conditions in order to maintain cell proliferation activity.

DOI： 10.1080/09670262.2017.1346206

Language：English   Publishing type：Research paper (scientific journal)  

Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator by its specific localization and activity during cell cycle. It has been shown that the phosphorylation and ubiquitylation of Plk1 are required for its own activation and localization. Here, we report that SUMOylation regulates the activity of Plk1 in the lepidopteran insect of Bombyx mori. In the absence of SUMOylation, it causes the lost localization of Plk1 on centrosomes and kinetochores, as well as an uneven distribution in midzone. We further identify that the putative SUMOylation site of Bombyx Plk1 at lysine 466 is required for its localization on centrosomes, and K466 mutation in Plk1 could influence its interaction with Smt3/Ubc9 complex. These findings are also confirmed by Drosophila Polo and human Plk1, which together reveals a conserved role of Plk1 SUMOylation in mammals. Moreover, conjugation of Smt3 to Plk1 SUMOylation mutant promotes its localization on centrosomes and kinetochores, and rescues functional defects of chromosome alignment in cells depleted of endogenous Plk1. Altogether, the present data indicate that the SUMOylation of Plk1 could participate in proper chromosome alignment and segregation during mitosis, and provides a novel layer for the regulation of Plk1 localization and activity throughout cell cycle.

DOI： 10.1038/s41598-017-15884-7

Language：English   Publishing type：Research paper (scientific journal)  

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to “nuage” in ovary-derived BmN4 cell.

DOI： 10.1016/j.bbrc.2017.09.107

Language：English   Publishing type：Research paper (scientific journal)  

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects.

DOI： 10.1016/j.ibmb.2017.08.006

Language：English   Publishing type：Research paper (scientific journal)  

PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24–32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell.

DOI： 10.1016/j.bbrc.2017.06.008

Language：English   Publishing type：Research paper (scientific journal)  

The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species.

DOI： 10.1016/j.ibmb.2017.04.005

Language：English   Publishing type：Research paper (scientific journal)  

The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

DOI： 10.1007/s12033-017-0008-9

Language：English   Publishing type：Research paper (scientific journal)  

Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.

DOI： 10.1007/s12033-017-0003-1

Language：English   Publishing type：Research paper (scientific journal)  

The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides.

DOI： 10.1093/femsle/fnx051

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

We investigated the ability of recombinant pufferfish saxitoxin and tetrodotoxin binding protein types 1 and 2 of Takifugu rubripes (rTrub.PSTBP1 and rTrub.PSTBP2) to bind to tetrodotoxin (TTX) and tributyltin. Both rTrub.PSTBPs bound to tributyltin in an ultrafiltration binding assay but lost this ability on heat denaturation. In contrast, only rTrub.PSTBP2 bound to TTX even heat denaturation. This result suggests that the amino acid sequence of PSTBP2 may be contributed for its affinity for TTX.

DOI： 10.1016/j.toxicon.2016.11.245

Language：English   Publishing type：Research paper (scientific journal)  

Recent advances in bacterial characterization methodologies have made taxonomic categorization significantly more accurate. Here, we re-evaluated the position of bacterial strains (532 and 652) belonging to the genus Hafnia, isolated from flacherie-diseased silkworms in 1965. Phylogenetic analysis based on the 16S rRNA gene sequences of these strains suggests that they belong to genus Enterobacter. Using multilocus sequence analysis (MLSA), these strains were further classified to MLSA group A, which is a “core” group of Enterobacter containing E. cloacae (the type species of the genus). Although these strains were closely related to E. mori, E. tabaci, and E. asburiae, they also had other MLSA characteristics that distinguished them from these neighboring bacterial species. These data were supported by further biochemical analysis. Thus, it appears that the 532 and 652 strains isolated almost half a century ago belong to genus Enterobacter, and their unique characteristics strongly suggest that they are a novel bacterial species.

DOI： 10.11416/jibs.86.2_035

Language：English   Publishing type：Research paper (scientific journal)  

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DOI： 10.1016/j.aspen.2016.07.007

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DOI： 10.1016/j.aspen.2016.03.014

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12257-016-0042-7

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DOI： 10.1007/s12033-016-9937-y

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.11416/jibs.85.1_007

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep18103

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DOI： 10.1016/j.jbiosc.2015.02.013

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12257-014-0806-x

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DOI： 10.1016/j.bbagen.2015.01.018

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0119429

Language：English   Publishing type：Research paper (scientific journal)  

Understanding the evolutionary mechanisms of toxin accumulation in pufferfishes has been long-standing problem in toxicology and evolutionary biology. Pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) is involved in the transport and accumulation of tetrodotoxin and is one of the most intriguing proteins related to the toxicity of pufferfishes. PSTBPs are fusion proteins consisting of two tandem repeated tributyltin-binding protein type 2 (TBT-bp2) domains. In this study, we examined the evolutionary dynamics of TBT-bp2 and PSTBP genes to understand the evolution of toxin accumulation in pufferfishes. Database searches and/or PCR-based cDNA cloning in nine pufferfish species (6 toxic and 3 nontoxic) revealed that all species possessed one or more TBT-bp2 genes, but PSTBP genes were found only in 5 toxic species belonging to genus Takifugu. These toxic Takifugu species possessed two or three copies of PSTBP genes. Phylogenetic analysis of TBT-bp2 and PSTBP genes suggested that PSTBPs evolved in the common ancestor of Takifugu species by repeated duplications and fusions of TBT-bp2 genes. In addition, a detailed comparison of Takifugu TBT-bp2 and PSTBP gene sequences detected a signature of positive selection under the pressure of gene conversion. The complicated evolutionary dynamics of TBT-bp2 and PSTBP genes may reflect the diversity of toxicity in pufferfishes.

DOI： 10.1111/jeb.12634

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12033-014-9767-8

Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s11033-014-3348-6

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DOI： 10.1371/journal.pone.0099200

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

DOI： 10.1111/imb.12072

Language：Japanese  

DOI： 10.1007/s00253-013-5437-1

Language：English  

DOI： 10.1111/1744-7917.12031

Language：English  

DOI： 10.1007/s12033-013-9694-0

Language：English  

DOI： 10.1016/j.aspen.2013.10.009

Language：English  

DOI： 10.1371/journal.pone.0092313

Language：English  

DOI： 10.1016/j.ibmb.2013.11.007

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DOI： 10.1007/s00253-013-5279-x

Language：English  

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DOI： 10.1016/j.ibmb.2013.04.004

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DOI： 10.1080/10826068.2012.762717

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DOI： 10.1007/s00253-012-4583-1

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DOI： 10.1007/s13355-013-0182-6

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DOI： 10.1007/s10529-013-1183-9

Language：English  

DOI： 10.1111/imb.12023

Language：English  

DOI： 10.1002/arch.21091

Language：English  

DOI： 10.1095/biolreprod.112.107292

Language：Japanese  

DOI： 10.1016/j.aspen.2012.09.004

Language：Japanese  

DOI： 10.1016/j.aspen.2012.06.004

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DOI： 10.2108/zsj.29.786

Language：Japanese  

DOI： 10.1007/s00438-012-0711-y

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DOI： 10.1016/j.pep.2004.07.010

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DOI： 10.1016/S0304-4165(01)00095-2

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DOI： 10.1016/S0378-1119(01)00631-X

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DOI： 10.1006/abbi.2001.2275

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DOI： 10.1303/aez.2000.427

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DOI： 10.1023/A:1007076511814

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DOI： 10.1271/bbb.64.1197

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DOI： 10.1074/jbc.274.16.11030

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DOI： 10.1016/0167-4781(94)90088-4

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DOI： 10.1016/0304-4165(89)90104-9

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市   Country：Japan  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市   Country：Japan  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌市   Country：Japan  

Event date： 2015.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Barcelona   Country：Spain  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Busan   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Busan   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Busan   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Busan   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Busan   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Busan   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2015.4

Language：English   Presentation type：Oral presentation (general)  

Venue：Daegu   Country：Korea, Republic of  

Event date： 2014.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2014.3

Language：English   Presentation type：Oral presentation (general)  

Venue：藤沢市   Country：Japan  

Event date： 2014.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：藤沢市   Country：Japan  

Event date： 2013.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：つくば   Country：Japan  

Event date： 2013.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：つくば   Country：Japan  

Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2013.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2013.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：平昌郡   Country：Korea, Republic of  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2012.3

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2011.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2011.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2011.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.12

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2011.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2011.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2011.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2011.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.12

Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2010.11

Presentation type：Oral presentation (general)  

Venue：tsukuba   Country：Japan  

Event date： 2010.8

Presentation type：Oral presentation (general)  

Venue：boston   Country：United States  

Event date： 2010.4

Presentation type：Oral presentation (general)  

Venue：長野   Country：Japan  

Event date： 2010.4

Presentation type：Oral presentation (general)  

Venue：長野   Country：Japan  

Event date： 2010.3

Presentation type：Oral presentation (general)  

Venue：沖縄   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.12

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Event date： 2009.10

Presentation type：Symposium, workshop panel (public)  

Venue：Chongching   Country：China  

Event date： 2009.9

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：札幌   Country：Japan  

Event date： 2009.9

Presentation type：Symposium, workshop panel (public)  

Venue：名古屋   Country：Japan  

Event date： 2009.9

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2009.9

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2009.9

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2009.9

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2009.9

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2009.9

Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：札幌   Country：Japan  

Event date： 2009.3

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Event date： 2009.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2009.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2009.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2009.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2009.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：nagoya   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：(Kakegawa, Japan)   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：(Kakegawa, Japan)   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：nagoya   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：nagoya   Country：Japan  

Event date： 2011.3

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Suwon   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：Suwon   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：福岡   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Suwon   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：横浜   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：茨城   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：茨城   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：岩手   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：岩手   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：岩手   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：岩手   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Presentation type：Symposium, workshop panel (public)  

Venue：Jeju   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：Jeju   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：Jeju   Country：Korea, Republic of  

Presentation type：Symposium, workshop panel (public)  

Venue：Jeju   Country：Korea, Republic of