Publications by authors named "Hideki Sezutsu"

121 Publications

Dynamic alterations of circulating T lymphocytes and the clinical response in patients with head and neck squamous cell carcinoma treated with nivolumab.

Cancer Immunol Immunother 2021 Aug 31. Epub 2021 Aug 31.

Department of Otolaryngology-Head and Neck Surgery, Gunma University Graduate School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma, 3718511, Japan.

Cancer immunotherapy using immune checkpoint inhibitors (ICIs) has been recognized as a novel therapeutic option for head and neck squamous cell carcinoma (HNSCC). However, only approximately 20-30% of patients with recurrent/metastatic (R/M) HNSCC benefit. Moreover, the mechanisms underlying the response to ICIs remain unclear. We investigated the proportion, activation status, and expression level of immune checkpoint molecules in circulating T cell subsets in R/M HNSCC patients treated with nivolumab using flow cytometry and mass cytometry, and then determined whether treatment response was associated with these values. We also assessed the changes in the frequency of tumor-associated antigens, MAGE-A4 and p53, -specific T cells prior to and after nivolumab treatment using the IFN-γ ELISPOT assay. The proportion of activated CD4+ and CD8+ TEMRA cells significantly increased in the disease-controlled patients but not in disease-progressed patients. As expected, the expression of PD-1 in T cells markedly decreased regardless of the therapeutic response. Meanwhile, T cell immunoglobulin mucin-3 expression on CD8+ T cells was significantly higher in patients with disease progression than in disease-controlled patients after treatment. The frequency of the tumor-associated antigens, MAGE-A4- and p53-specific T cells, was not correlated with clinical responses; however, in the disease-controlled patients, the frequency of MAGE-A4-specific T cells was significantly augmented. We concluded that in R/M HNSCC patients treated with nivolumab, circulating T cells show dynamic alterations depending on treatment efficacy. An analysis of the immunokinetics of circulating T cells could thus provide new insights into rational therapeutic strategies in cancer immunotherapy for HNSCC.
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http://dx.doi.org/10.1007/s00262-021-03042-yDOI Listing
August 2021

Reference Transcriptome Data in Silkworm .

Insects 2021 Jun 3;12(6). Epub 2021 Jun 3.

Database Center for Life Science (DBCLS), Joint Support-Center for Data Science Research, Research Organization of Information and Systems, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.

Herein, we performed RNA-seq analysis of ten major tissues/subparts of silkworm larvae. The sequences were mapped onto the reference genome assembly and the reference transcriptome data were successfully constructed. The reference data provided a nearly complete sequence for -, a major silk gene with a complex structure. We also markedly improved the gene model for other genes. The transcriptomic expression was investigated in each tissue and a number of transcripts were identified that were exclusively expressed in tissues such as the testis. Transcripts strongly expressed in the midgut formed tight genomic clusters, suggesting that they originated from tandem gene duplication. Transcriptional factor genes expressed in specific tissues or the silk gland subparts were also identified. We successfully constructed reference transcriptome data in the silkworm and found that a number of transcripts showed unique expression profiles. These results will facilitate basic studies on the silkworm and accelerate its applications, which will contribute to further advances in lepidopteran and entomological research as well as the practical use of these insects.
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http://dx.doi.org/10.3390/insects12060519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228281PMC
June 2021

Mutation in Bombyx mori fibrohexamerin (P25) gene causes reorganization of rough endoplasmic reticulum in posterior silk gland cells and alters morphology of fibroin secretory globules in the silk gland lumen.

Insect Biochem Mol Biol 2021 08 5;135:103607. Epub 2021 Jun 5.

Institute of Sericulture, Iikura 1053, 300-0324, Ami-machi, Ibaraki, Japan. Electronic address:

Larvae of many lepidopteran species produce a mixture of secretory proteins, known as silk, for building protective shelters and cocoons. Silk consists of a water-insoluble silk filament core produced in the posterior silk gland (PSG) and a sticky hydrophilic coating produced by the middle silk gland (MSG). In Bombyx mori, the fiber core comprises three proteins: heavy chain fibroin (Fib-H), light chain fibroin (Fib-L) and fibrohexamerin (Fhx, previously referred to as P25). To learn more about the role of Fhx, we used transcription activator-like effector nuclease (TALEN) mutagenesis and prepared a homozygous line with a null mutation in the Fhx gene. Our characterization of cocoon morphology and silk quality showed that the mutation had very little effect. However, a detailed inspection of the secretory cells in the posterior silk gland (PSG) of mid-last-instar mutant larvae revealed temporary changes in the morphology of the endoplasmic reticulum. We also observed a morphological difference in fibroin secretory globules stored in the PSG lumen of Fhx mutants, which suggests that their fibroin complexes have a slightly lower solubility. Finally, we performed an LC-MS-based quantitative proteomic analysis comparing mutant and wild-type (wt) cocoon proteins and found a high abundance of a 16 kDa secretory protein likely involved in fibroin solubility. Overall, our study shows that whilst Fhx is dispensable for silk formation, it contributes to the stability of fibroin complexes during intracellular transport and affects the morphology of fibroin secretory globules in the PSG lumen.
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http://dx.doi.org/10.1016/j.ibmb.2021.103607DOI Listing
August 2021

Production of cloned transgenic silkworms by breeding non-diapausing parthenogenetic strains.

J Insect Physiol 2021 Jul 4;132:104265. Epub 2021 Jun 4.

Institute of Sericulture, Iikura 1053, 300-0324 Ami-machi, Ibaraki, Japan. Electronic address:

Clonal transgenic silkworms are useful for the functional analysis of insect genes and for the production of recombinant proteins. Such silkworms have previously been created using an existing ameiotic parthenogenetic strain. However, the process was labor intensive, and the efficiency of producing transgenic silkworms was very low. To overcome this issue, we developed a more convenient and efficient method by breeding non-diapausing parthenogenetic strains. The strains produced non-diapausing eggs only when the embryogenesis of the parent eggs was performed at low temperatures, which could then be used for injecting vector plasmids. This demonstrated that transgenic silkworms could be produced with greater ease and efficiency. To breed the strains, we crossed the existing parthenogenetic strains with bivoltine strains and made F1 and F2 from each cross. Then we selected the silkworms whose eggs have a high ability of parthenogenesis and became non-diapausing. We also demonstrated that the germplasm could be cryopreserved in liquid nitrogen. Thus, this method increases the efficiency and ease of using genetically engineered silkworms to analyze gene function and produce recombinant proteins, potentially impacting various industries.
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http://dx.doi.org/10.1016/j.jinsphys.2021.104265DOI Listing
July 2021

In vitro assessment of antitumor immune responses using tumor antigen proteins produced by transgenic silkworms.

J Mater Sci Mater Med 2021 May 17;32(6):58. Epub 2021 May 17.

Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu, Gunma, 376-8515, Japan.

The evaluation of antitumor immune responses is essential for immune monitoring to predict clinical outcomes as well as treatment efficacies in cancer patients. In this study, we produced two tumor antigen (TA) proteins, melanoma antigen family A4 and wild type p53, using TG silkworm systems and evaluated anti-TA-specific immune responses by enzyme-linked immunosorbent spot assays in patients with head and neck cancer. Eleven (61.1%) of 18 patients showed significant IFN-γ production in response to at least one TA; however, the presence of TA-specific immune responses did not significantly contribute to better prognosis (overall survival, p = 0.1768; progression-free survival, p = 0.4507). Further studies will need to be performed on a larger scale to better assess the clinical significance of these systems. The production of multiple TA proteins may provide new avenues for the development of immunotherapeutic strategies to stimulate a potent and specific immune response against tumor cells as well as precise assessment of antitumor immune responses in cancer patients.
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http://dx.doi.org/10.1007/s10856-021-06526-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128804PMC
May 2021

Comparisons in temperature and photoperiodic-dependent diapause induction between domestic and wild mulberry silkworms.

Sci Rep 2021 Apr 13;11(1):8052. Epub 2021 Apr 13.

Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.

The bivoltine strain of the domestic silkworm, Bombyx mori, has two generations per year. It shows a facultative diapause phenotype determined by environmental conditions, including photoperiod and temperature, and nutrient conditions during embryonic and larval development of the mother. However, it remains unclear how the environmental signals received during development are selectively utilized as cues to determine alternative diapause phenotypes. We performed a comparative analysis between the Kosetsu strain of B. mori and a Japanese population of the wild mulberry silkworm B. mandarina concerning the hierarchical molecular mechanisms in diapause induction. Our results showed that for the Kosetsu, temperature signals during the mother's embryonic development predominantly affected diapause determination through the thermosensitive transient receptor potential ankyrin 1 (TRPA1) and diapause hormone (DH) signaling pathways. However, embryonic diapause in B. mandarina was photoperiod-dependent, although the DH signaling pathway and thermal sensitivity of TRPA1 were conserved within both species. Based on these findings, we hypothesize that TRPA1-activated signals are strongly linked to the signaling pathway participating in diapause induction in Kosetsu to selectively utilize the temperature information as the cue because temperature-dependent induction was replaced by photoperiodic induction in the TRPA1 knockout mutant.
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http://dx.doi.org/10.1038/s41598-021-87590-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044193PMC
April 2021

Bio-functionalized titanium surfaces with modified silk fibroin carrying titanium binding motif to enhance the ossific differentiation of MC3T3-E1.

Biotechnol Bioeng 2021 07 13;118(7):2585-2596. Epub 2021 Apr 13.

Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.

Silk fibroin (SF) from Bombyx mori has superior properties as both a textile and a biomaterial, and has been used to functionalize the surfaces of various medical inorganic materials including titanium (Ti). In this study, we endowed SF with reversible binding ability to Ti by embedding a titanium binding motif (minTBP-1 and RKLPDA). Artificial SF proteins were first created by conjugating gene cassettes for SF motif (AGSGAG) and minTBP-1 motif with different ratios, which have been shown to bind reversibly to Ti surfaces in quartz crystal microbalance analyses. Based on these results, the functionalized SF (TiBP-SF) containing the designed peptide [TS[(AGSGAG) AS] RKLPDAS] was prepared from the cocoon of transgenic B. mori, which accelerates the ossific differentiation of MC3T3-E1 cells when coated on titanium substrates. Thus, TiBP-SF presents an alternative for endowing the surfaces of titanium materials with osseointegration functionality, which would allow the exploration of potential applications in the medical field.
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http://dx.doi.org/10.1002/bit.27777DOI Listing
July 2021

Identification of Exploration and Exploitation Balance in the Silkmoth Olfactory Search Behavior by Information-Theoretic Modeling.

Front Comput Neurosci 2021 1;15:629380. Epub 2021 Feb 1.

Transgenic Silkworm Research Unit, National Agriculture and Food Research Organization, Tsukuba, Japan.

Insects search for and find odor sources as their basic behaviors, such as when looking for food or a mate. This has motivated research to describe how they achieve such behavior under turbulent odor plumes with a small number of neurons. Among different insects, the silk moth has been studied owing to its clear motor response to olfactory input. In past studies, the "programmed behavior" of the silk moth has been modeled as the average duration of a sequence of maneuvers based on the duration of periods without odor hits. However, this model does not fully represent the fine variations in their behavior. In this study, we used silk moth olfactory search trajectories from an experimental virtual reality device. We achieved an accurate input by using optogenetic silk moths that react to blue light. We then modeled such trajectories as a probabilistic learning agent with a belief of possible source locations. We found that maneuvers mismatching the programmed behavior are related to larger entropy decrease, that is, they are more likely to increase the certainty of the belief. This implies that silkmoths include some stochasticity in their search policy to balance the exploration and exploitation of olfactory information by matching or mismatching the programmed behavior model. We believe that this information-theoretic representation of insect behavior is important for the future implementation of olfactory searches in artificial agents such as robots.
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http://dx.doi.org/10.3389/fncom.2021.629380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7882484PMC
February 2021

Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm .

Proc Natl Acad Sci U S A 2021 01 21;118(1). Epub 2020 Dec 21.

Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan;

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm , embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.
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http://dx.doi.org/10.1073/pnas.2020028118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817158PMC
January 2021

Effects of transgenic silk materials that incorporate FGF-7 protein microcrystals on the proliferation and differentiation of human keratinocytes.

FASEB Bioadv 2020 Dec 20;2(12):734-744. Epub 2020 Oct 20.

Department of Applied Biology Kyoto Institute of Technology Kyoto Japan.

The silk glands of silkworms produce large quantities of fibroin, which is a protein that can be physically processed and used as a biodegradable carrier for cell growth factors in tissue engineering applications. Meanwhile, protein microcrystals known as polyhedra, which are derived from cypovirus 1, have been used as a vehicle to protect and release encapsulated cell growth factors. We report the generation of transgenic silkworms that express recombinant fibroblast growth factor-7 (FGF-7) fused with the polyhedron-encapsulating signal in polyhedra produced in the middle (MSG) and posterior (PSG) silk glands. Immunofluorescence showed that polyhedra from silk glands are associated with FGF-7. The MSG and PSG from transgenic silkworms were processed into fine powdery materials, from which FGF-7 activity was released to stimulate the proliferation of human keratinocyte epidermal cells. Powders from PSGs exhibited higher FGF-7 activity than those from MSGs. Moreover, PSG powder showed a gradual release of FGF-7 activity over a long period and induced keratinocyte proliferation and differentiation in 3D culture to promote the formation of stratified epidermis expressing positive differentiation marker proteins. Our results indicate that powdery materials incorporating the FGF-7-polyhedra microcrystals from silk glands are valuable for developing cell/tissue engineering applications in vivo and in vitro.
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http://dx.doi.org/10.1096/fba.2020-00078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734426PMC
December 2020

Functional Analysis of Adipokinetic Hormone Signaling in .

Cells 2020 12 11;9(12). Epub 2020 Dec 11.

Biology Centre of the Czech Academy of Sciences, Institute of Entomology, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic.

Insect adipokinetic hormones (AKHs) are short peptides produced in the corpora cardiaca and are responsible for mobilizing energy stores from the fat body to the hemolymph. Three related peptides, AKH1, AKH2, and AKH/corazonin-related peptide (ACP) as well as three AKH receptors have been reported in . AKH1 and AKH2 are specific for the AKHR1 receptor, whereas ACP interacts with the other two AKHRs. To assess the effect of the two silkworm AKHs and ACP in the regulation of energy homeostasis we examined the expression pattern of the three peptides and their receptors as well as their effect on the level of carbohydrates and lipids in the hemolymph. Our results support the hypothesis that only AKH1 and AKH2 peptides together with the AKHR1 receptor are involved in the maintenance of energy homeostasis. Because AKHR1 (BmAKHR1) seems to be a true AKHR we generated its mutation. The mutant larvae display significantly lower carbohydrate and lipid levels in the hemolymph and reduced sensitivity to starvation. Our study clarifies the role of BmAKHR1 in energy homeostasis.
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http://dx.doi.org/10.3390/cells9122667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764666PMC
December 2020

The Number of Larval Molts Is Controlled by Hox in Caterpillars.

Curr Biol 2021 02 11;31(4):884-891.e3. Epub 2020 Dec 11.

National Agricultural and Food Research Organization, Ibaraki 305-8634, Japan; Faculty of Food and Agricultural Sciences, Fukushima University, Fukushima 960-1296, Japan.

Animals with exoskeletons molt for further growth. In insects, the number of larval (or nymphal) molts varies inter- and intra-specifically, and it is widely accepted that the variation in the number of larval molts is an adaptive response to diverse environmental conditions. However, the molecular mechanism that underlies the variety and plasticity in the number of larval molts is largely unknown. In the silkworm, Bombyx mori, there are strains that molt three, four, or five times, and these numbers are determined by allelic variation at a single autosomal locus, Moltinism (M). Here, we demonstrate that the Hox gene Sex combs reduced (Scr) is responsible for the phenotypes of the M locus. Scr is selectively expressed in the larval prothoracic gland (PG), an endocrine organ that produces molting hormones.Scr represses the biosynthesis of molting hormones in the PG, thereby regulating the incremental increase in body size during each larval instar. Our experiments consistently suggest that the differential expression levels of Scr among the three M alleles result in different growth ratios that ultimately lead to the different number of larval molts. Although the role of Hox genes in conferring segmental identity along the body axis and in molding segment-specific structure later in development has been well established, the present study identifies an unexpected role of Hox gene in hormone biosynthesis. This new role means that, in addition to shaping segment-specific morphology, Hox genes also drive the evolution of life history traits by regulating animal physiology.
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http://dx.doi.org/10.1016/j.cub.2020.11.017DOI Listing
February 2021

Successful activation of rat T lymphocytes by sperm specific antigens in vitro.

J Reprod Dev 2020 Dec 3;66(6):599-605. Epub 2020 Oct 3.

Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Ibaraki 305-8634, Japan.

Autoimmune orchitis is a condition related to cellular immunity. A disease model involving transfer of T lymphocytes activated by known antigens would be useful for defining pathogenical molecules. Since no method for activating rat T cells using specific antigens is available, we started the study to develop the method. T cells were collected from draining lymph nodes of immunized rats, then co-cultured with syngeneic splenocytes as antigen-presenting cells (APC) in antigen-supplemented medium (= stimulation). The cells were then incubated in medium without antigens and APC (= resting). Repetitive stimulation and resting increased the number of the T cells more than 100-fold. The antigen-specific activation was demonstrated by cell proliferation assay and ELISA assay for interferon gamma. Flow cytometry revealed that > 95% of the cells expressed tumor necrosis factor alpha, a cytokine responsible for autoimmune orchitis. The present method will provide a new procedure to evaluate antigenicity of sperm molecules.
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http://dx.doi.org/10.1262/jrd.2020-106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768165PMC
December 2020

Transgenic and knockout analyses of Masculinizer and doublesex illuminated the unique functions of doublesex in germ cell sexual development of the silkworm, Bombyx mori.

BMC Dev Biol 2020 09 21;20(1):19. Epub 2020 Sep 21.

Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba, 277-8562, Japan.

Background: Masculinizer (Masc) plays a pivotal role in male sex determination in the silkworm, Bombyx mori. Masc is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. The male isoform of Bmdsx (BmdsxM) induces male differentiation in somatic cells, while females express the female isoform of Bmdsx (BmdsxF), which promotes female differentiation in somatic cells. Our previous findings suggest that Masc could direct the differentiation of genetically female (ZW) germ cells into sperms. However, it remains unclear whether Masc directly induces spermatogenesis or if it promotes male differentiation in germ cells indirectly by inducing the expression of BmdsxM.

Results: In this study, we performed genetic analyses using the transgenic line that expressed Masc, as well as various Bmdsx knockout lines. We found that Masc-expressing females with a homozygous mutation in BmdsxM showed normal development in ovaries. The formation of testis-like tissues was abolished in these females. On the other hand, Masc-expressing females carrying a homozygous mutation in BmdsxF exhibited almost complete male-specific development in gonads and germ cells. These results suggest that BmdsxM has an ability to induce male development in germ cells as well as internal genital organs, while BmdsxF inhibits BmdsxM activity and represses male differentiation. To investigate whether MASC directly controls male-specific splicing of Bmdsx and identify RNAs that form complexes with MASC in testes, we performed RNA immunoprecipitation (RIP) using an anti-MASC antibody. We found that MASC formed a complex with AS1 lncRNA, which is a testis-specific factor involved in the male-specific splicing of Bmdsx pre-mRNA.

Conclusions: Taken together, our findings suggest that Masc induces male differentiation in germ cells by enhancing the production of BmdsxM. Physical interaction between MASC and AS1 lncRNA may be important for the BmdsxM expression in the testis. Unlike in the Drosophila dsx, BmdsxM was able to induce spermatogenesis in genetically female (ZW) germ cells. To the best of our knowledge, this is the first report that the role of dsx in germ cell sexual development is different between insect species.
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http://dx.doi.org/10.1186/s12861-020-00224-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504827PMC
September 2020

Secretory expression of thyroid hormone receptor using transgenic silkworms and its DNA binding activity.

Protein Expr Purif 2020 12 5;176:105723. Epub 2020 Aug 5.

Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu, Gunma, 376-8515, Japan. Electronic address:

Silkworms are economically important insects that have the ability to produce large amounts of silk. They have mass breeding methods and silk glands, which are specialized tissues that secrete silk fibroin and sericin. Thus, the production of recombinant proteins in a transgenic silkworm system is a promising approach. We developed a silkworm, Bombyx mori, as a host expression insect for recombinant proteins and successfully produced different proteins including antibodies, glycoproteins, and membrane receptors. The thyroid hormone receptor (TR) is a regulatory factor for many physiological phenomena. It is a lipophilic protein that has DNA-binding and ligand-binding domains. Based on our previous experiences, it was inferred that the recombinant TR easily formed aggregates and precipitates which is potentially due to an unstructured hinge domain. We applied the silkworm expression system to produce mice TRβ1 that was fused with glutathione S-transferase. Using 160 larvae, the yield of the recombinant GST-TRβ was approximately 4 mg, and the purified GST-TRβ completely retained its physiological activity. Our results indicated that the recombinant TRβ was secreted extracellularly using the silk fibroin signal peptide sequence. Moreover, we found that the expression system of silkworms was applicable to nuclear proteins.
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http://dx.doi.org/10.1016/j.pep.2020.105723DOI Listing
December 2020

Structural insight and stability of TNFR-Fc fusion protein (Etanercept) produced by using transgenic silkworms.

J Biochem 2021 Feb;169(1):25-33

Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-9501, Japan.

Therapeutic proteins expressed using transgenic animals have been of great interest for several years. Especially, transgenic silkworm has been studied intensively because of its ease in handling, low-cost, high-yield and unique glycosylation patterns. However, the physicochemical property of the therapeutic protein expressed in transgenic silkworm remains elusive. Here, we constructed an expression system for the TNFR-Fc fusion protein (Etanercept) using transgenic silkworm. The TNFR-Fc fusion protein was employed to N-glycan analysis, which revealed an increased amount of afucosylated protein. Evidence from surface plasmon resonance analysis showed that the TNFR-Fc fusion protein exhibit increased binding affinity for Fcγ receptor IIIa and FcRn compared to the commercial Etanercept, emphasizing the profit of expression system using transgenic silkworm. We have further discussed the comparison of higher order structure, thermal stability and aggregation of the TNFR-Fc fusion protein.
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http://dx.doi.org/10.1093/jb/mvaa092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7868081PMC
February 2021

Transcriptomic analysis of the bagworm moth silk gland reveals a number of silk genes conserved within Lepidoptera.

Insect Sci 2021 Aug 25;28(4):885-900. Epub 2020 Jul 25.

Institute of Agrobiological Sciences, Transgenic Silkworm Research Unit, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.

Lepidopteran insects produce cocoons with unique properties. The cocoons are made of silk produced in the larval tissue silk gland and our understanding of the silk genes is still very limited. Here, we investigated silk genes in the bagworm moth Eumeta variegata, a species that has recently been found to produce extraordinarily strong and tough silk. Using short-read transcriptomic analysis, we identified a partial sequence of the fibroin heavy chain gene and its product was found to have a C-terminal structure that is conserved within nonsaturniid species. This is in accordance with the presence of fibroin light chain/fibrohexamerin genes and it is suggested that the bagworm moth is producing silk composed of fibroin ternary complex. This indicates that the fibroin structure has been evolutionarily conserved longer than previously thought. Other than fibroins we identified candidates for sericin genes, expressed strongly in the middle region of the silk gland and encoding serine-rich proteins, and other silk genes, that are structurally conserved with other lepidopteran homologues. The bagworm moth is thus considered to be producing conventional lepidopteran type of silk. We further found a number of genes expressed in a specific region of the silk gland and some genes showed conserved expression with Bombyx mori counterparts. This is the first study allowing comprehensive silk gene identification and expression analysis in the lepidopteran Psychidae family and should contribute to the understanding of silk gene evolution as well as to the development of novel types of silk.
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http://dx.doi.org/10.1111/1744-7917.12846DOI Listing
August 2021

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Molecules 2020 Feb 10;25(3). Epub 2020 Feb 10.

Silk Materials Research Unit, Division of Biotechnology, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 1-2, Owashi, Tsukuba, Ibaraki 305-8634, Japan.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.
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http://dx.doi.org/10.3390/molecules25030761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037070PMC
February 2020

A defective prostaglandin E synthase could affect egg formation in the silkworm Bombyx mori.

Biochem Biophys Res Commun 2020 01 24;521(2):347-352. Epub 2019 Oct 24.

State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Southwest University, Chongqing, 400716, China.

We had previously reported a prostaglandin E synthase (bmPGES) in the silkworm Bombyx mori that catalyzes the isomerization of PGH to PGE. The present study aimed to provide a genome-editing characterization of bmPGES in B. mori. Results showed bmPGES gene disruption to result in a reduced content of PGE. The change affected the expression of chorion genes and egg formation in silkworms. Collectively, the results indicated that bmPGES could be involved in reproduction of B. mori. Therefore, this study provides insights into the physiological role of bmPGES and PGE in silkworms.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.121DOI Listing
January 2020

Genome-wide SNP marker discovery and phylogenetic analysis of mulberry varieties using double-digest restriction site-associated DNA sequencing.

Gene 2020 Feb 19;726:144162. Epub 2019 Oct 19.

Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), Tsukuba 305-8634, Japan. Electronic address:

There has been long taxonomic debate on mulberry species (genus Morus) because the classification of mulberry species has relied on morphological characteristics. Although attempts for classifying mulberry species using molecular markers have been performed, phylogenetic relationships among diploid mulberry species remain unclear. In this study, we aim to investigate the genetic relationship between 54 diploid mulberry varieties belonging to seven different Morus species (M. alba, M. indica, M. bombycis, M. acidosa, M. latifolia, M. kagayamae, and M. rotundiloba) and one unspecified Morus species ('Enbu') using genome-wide SNP discovery and phylogenetic analysis via double-digest restriction site-associated DNA sequencing (ddRAD-seq). Genome-wide 2229 homozygous SNPs of 54 mulberry varieties in the eight species were identified by ddRAD-seq. Results of the phylogenetic analysis identified only three clear monophyletic clades in two Japanese native species, M. acidosa and M. kagayamae, which are found on different geographically isolated islands and a Thai species, M. rotundiloba, whereas the other species were non-monophyletic. Varieties of M. bombycis, another Japanese native species, were roughly classified into three groups. Of these, two M. bombycis groups were monophyletic with M. acidosa and M. kagayamae, respectively, while another M. bombycis group was not monophyletic. Varieties of M. indica, an Indian native species, were classified into two different monophyletic clades. Of these, one clade was clearly monophyletic with an indigenous variety in Kenya, 'Enbu', while another clade was monophyletic with M. rotundiloba and one M. latifolia variety. There were no clear monophyletic clades within M. alba and M. latifolia varieties, which could be a result of several hybridization events after their introductions from China to Japan. Our results suggested that it was difficult to clearly classify the hybridized mulberry varieties even with genome-wide DNA markers. In addition to phylogenetic analysis, we also evaluated morphological characteristics of mulberry leaves for each variety. The results of morphological evaluation indicated that leaf tip ratio may correlate to genetic difference among the two M. bombycis groups in monophyletic clades and another M. bombycis group in non-monophyletic clades. These results suggested that leaf tip ratio might be used for evaluating hybridization of M. bombycis varieties. Over all, our results may provide new insights into taxonomic debate of mulberry species.
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http://dx.doi.org/10.1016/j.gene.2019.144162DOI Listing
February 2020

A synthetic male-specific sterilization system using the mammalian pro-apoptotic factor in a malaria vector mosquito.

Sci Rep 2019 06 3;9(1):8160. Epub 2019 Jun 3.

Division of Medical Zoology, Department of Infection and Immunity, Jichi Medical University, Yakushiji, Shimotsuke, Tochigi, Japan.

Conditional cell death systems are useful for various aspects of basic science with a wide range of applications, including genetic pest control. We recently demonstrated that expression of the mammalian pro-apoptotic factor, B-cell leukaemia/lymphoma 2-associated X protein (Bax), can induce apoptosis in specific tissues by using tissue specific promoters in silkworm and mosquito. Here, we newly identified a functional promoter in the Asian malaria vector, Anopheles stephensi, which enables gene expression specifically in the testis. We produced a transgenic mosquito line that expresses mouse Bax under the control of this testis-specific promoter. Transgenic mosquito males exhibited aberrant testes without functional sperm and complete sterility, whereas transgenic females maintained normal fecundity. Despite their abnormal testes, the transgenic males maintained normal function of male accessory glands and typical mating behaviour. As a result of mating with these males, females showed refractoriness to further mating. These results suggest that transgenic males induce female sterility via mating. The mosquito is one of the most important disease vectors, and the control of their population benefits global public health. Thus, this Bax-mediated synthetic male-specific sterilization system could be applied to population control of mosquitoes.
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http://dx.doi.org/10.1038/s41598-019-44480-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547752PMC
June 2019

Analysis of the role of wind information for efficient chemical plume tracing based on optogenetic silkworm moth behavior.

Bioinspir Biomim 2019 05 31;14(4):046006. Epub 2019 May 31.

Department of System Innovation, Osaka University, 1-2 Machikaneyama-cho, Toyonaka, Osaka, 560-8531, Japan. Author to whom correspondence may be addressed.

Many animals use olfactory information to search for feeding areas and other individuals in real time and with high efficiency. We focus on the chemical plume tracing (CPT) ability of male silkworm moths and investigate an efficient CPT strategy for an autonomous robot. In the case of flying insects, the wind direction is an important factor in CPT, because the wind carries odors amongst other environmental information. However, whether the same phenomenon occurs in the walking silkworm moth has not been investigated. Therefore, we examine how the silkworm moth uses wind information during CPT. To accurately investigate the response to the wind direction, we introduce an optogenetic approach that replaces the odor stimulation with light stimulation, allowing us to separate the 'wind stimulus' from the 'odor stimulus'. We examine how the moth uses wind direction information in a biological experiment, and find that the movement speed is significantly reduced when the wind speed is relatively fast (1.0 m s). By implementing this phenomenon in an autonomous robot, we can improve the successful search rate over that of the conventional moth-inspired algorithm. Regarding the search time, the proposed algorithm finds the odor source faster in a low-frequency odorant emission environment, whereas the search is slower than the conventional method when the odor frequency is higher. Therefore, switching from the use of wind direction information to odor information according to the frequency with which the odor is encountered leads to efficient CPT performance.
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http://dx.doi.org/10.1088/1748-3190/ab1d34DOI Listing
May 2019

Involvement of the Clock Gene period in the Circadian Rhythm of the Silkmoth Bombyx mori.

J Biol Rhythms 2019 06 5;34(3):283-292. Epub 2019 Apr 5.

Graduate School of Science, Kyoto University, Kyoto, Japan.

In Lepidoptera, the roles of period ( per) and the negative feedback involving this gene in circadian rhythm are controversial. In the present study, we established a per knockout strain using TALEN in Bombyx mori, and compared eclosion and hatching rhythms between the per-knockout and wild-type strains to examine whether per is actually involved in these rhythms. The generated per knockout allele was considered null, because it encoded an extensively truncated form of PERIOD (198 aa due to a 64-bp deletion in exon 7, in contrast to 1113 aa in the wild-type protein). In this per knockout strain, circadian rhythms in eclosion and hatching were disrupted. Under LD cycles, however, a steep peak existed at 1 h after lights-on in both eclosion and hatching, and was considered to be produced by a masking effect-a direct response to light. In the per-knockout strain, temporal expression changes of per and timeless ( tim) were also lost. The expression levels of tim were continuously high, probably due to the loss of negative feedback by per and tim. In contrast, the expression levels of per were much lower in the per knockout strain than in the wild type at every time point. From these results, we concluded that per is indispensable for circadian rhythms, and we suggest that the negative feedback loop of the circadian rhythm involving per functions for the production of behavioral rhythms in B. mori.
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http://dx.doi.org/10.1177/0748730419841185DOI Listing
June 2019

Multicomponent structures in camouflage and mimicry in butterfly wing patterns.

J Morphol 2019 01;280(1):149-166

Transgenic Silkworm Research Unit, Division of Biotechnology, Institute of Agrobiological Sciences, NARO, Ibaraki, Japan.

Understanding how morphological structures are built is essential for appreciating the morphological complexity and divergence of organisms. One representative case of morphological structures is the camouflage and mimicry of butterfly wing patterns. Some previous studies have questioned whether camouflage and mimicry are truly structures, considering that they rely on coloration. Nevertheless, our recent study revealed that the leaf pattern of Kallima inachus butterfly wings evolved through the combination of changes in several pigment components in a block-wise manner; it remains unclear whether such block-wise structures are common in other cases of camouflage and mimicry in butterflies and how they come about. Previous studies focused solely on a set of homologous components, termed the nymphalid ground plan. In the present study, we extended the scope of the description of components by including not only the nymphalid ground plan but also other common components (i.e., ripple patterns, dependent patterns, and color fields). This extension allowed us to analyze the combinatorial building logic of structures and examine multicomponent structures of camouflage and mimicry in butterfly wing patterns. We investigated various patterns of camouflage and mimicry (e.g., masquerade, crypsis, Müllerian mimicry, Batesian mimicry) in nine species and decomposed them into an assembly of multiple components. These structural component analyses suggested that camouflage and mimicry in butterfly wing patterns are built up by combining multiple types of components. We also investigated associations between components and the kinds of camouflage and mimicry. Several components are statistically more often used to produce specific types of camouflage or mimicry. Thus, our work provides empirical evidence that camouflage and mimicry patterns of butterfly wings are mosaic structures, opening up a new avenue of studying camouflage, and mimicry from a structural perspective.
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http://dx.doi.org/10.1002/jmor.20927DOI Listing
January 2019

Characterisation of a diazinon-metabolising glutathione S-transferase in the silkworm Bombyx mori by X-ray crystallography and genome editing analysis.

Sci Rep 2018 11 15;8(1):16835. Epub 2018 Nov 15.

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Previously, we found an unclassified glutathione S-transferase 2 (bmGSTu2) in the silkworm Bombyx mori that conjugates glutathione to 1-chloro-2,4-dinitrobenzene and also metabolises diazinon, an organophosphate insecticide. Here, we provide a structural and genome-editing characterisation of the diazinon-metabolising glutathione S-transferase in B. mori. The structure of bmGSTu2 was determined at 1.68 Å by X-ray crystallography. Mutation of putative amino acid residues in the substrate-binding site showed that Pro13, Tyr107, Ile118, Phe119, and Phe211 are crucial for enzymatic function. bmGSTu2 gene disruption resulted in a decrease in median lethal dose values to an organophosphate insecticide and a decrease in acetylcholine levels in silkworms. Taken together, these results indicate that bmGSTu2 could metabolise an organophosphate insecticide. Thus, this study provides insights into the physiological role of bmGSTu2 in silkworms, detoxification of organophosphate insecticides, and drug targets for the development of a novel insecticide.
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http://dx.doi.org/10.1038/s41598-018-35207-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237972PMC
November 2018

In vivo functional characterisation of pheromone binding protein-1 in the silkmoth, Bombyx mori.

Sci Rep 2018 09 10;8(1):13529. Epub 2018 Sep 10.

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.

Male moths detect sex pheromones emitted by conspecific females with high sensitivity and specificity by the olfactory sensilla on their antennae. Pheromone binding proteins (PBPs) are highly enriched in the sensillum lymph of pheromone sensitive olfactory sensilla and are supposed to contribute to the sensitivity and selectivity of pheromone detection in moths. However, the functional role of PBPs in moth sex pheromone detection in vivo remains obscure. In the silkmoth, Bombyx mori, female moths emit bombykol as a single attractive sex pheromone component along with a small amount of bombykal that negatively modulates the behavioural responses to bombykol. A pair of olfactory receptor neurons, specifically tuned to bombykol or bombykal, co-localise in the trichodeum sensilla, the sensillum lymph of which contains a single PBP, namely, BmPBP1. We analysed the roles of BmPBP1 using BmPBP1-knockout silkmoth lines generated by transcription activator-like effector nuclease-mediated gene targeting. Electroantennogram analysis revealed that the peak response amplitudes of BmPBP1-knockout male antennae to bombykol and bombykal were significantly reduced by a similar percentage when compared with those of the wild-type males. Our results indicate that BmPBP1 plays a crucial role in enhancing the sensitivity, but not the selectivity, of sex pheromone detection in silkmoths.
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http://dx.doi.org/10.1038/s41598-018-31978-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131395PMC
September 2018

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

Biochem Biophys Res Commun 2018 09 14;503(4):2633-2638. Epub 2018 Aug 14.

Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 3-25-26 Tonomachi Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan.

Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.
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http://dx.doi.org/10.1016/j.bbrc.2018.08.015DOI Listing
September 2018

[Construction of a Platform for the Development of Pharmaceutical and Medical Applications Using Transgenic Silkworms].

Yakugaku Zasshi 2018 ;138(7):863-874

Institute of Agrobiological Sciences, National Agriculture and Food Research Organization.

 We have been constructing a platform for the development of pharmaceutical and medical applications using the domesticated silkworm, Bombyx mori, as a new animal model for drug development and evaluation. Because silkworm larvae originally have the capacity to synthesize up to 0.5 g of silk proteins, genetically modified silkworms (transgenic silkworms) are expected to have high potential in the production of recombinant silks/proteins. An innovative method for generating transgenic silkworms was established in 2000, and ever since this epoch-defining technological development, longstanding efforts have succeeded in developing novel silks that enable the manufacture of new textile materials for regenerative medical uses. Furthermore, we have succeeded in developing a new system of recombinant protein production. This recombinant protein production system is currently capable of producing a maximum of approximately 15 mg recombinant protein per silkworm larva. Transgenic silkworms have also been shown to produce a wide variety of useful proteins, including antibodies and membrane proteins. Some of these recombinant proteins have been in commercial use since 2011. In addition, we have been developing transgenic silkworms as a novel animal model for testing medicines based on metabolic similarities between silkworms and mammals. These applications show the suitability and potential of transgenic silkworms for medical use. Here, we will describe the challenges faced in creating a transgenic silkworm-based platform for pharmaceutical and medical applications.
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http://dx.doi.org/10.1248/yakushi.17-00202-1DOI Listing
August 2018

A single amino acid substitution in the Bombyx-specific mucin-like membrane protein causes resistance to Bombyx mori densovirus.

Sci Rep 2018 05 9;8(1):7430. Epub 2018 May 9.

Division of Insect Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634, Japan.

Bombyx mori densovirus type 1 (BmDV) is a pathogen that causes flacherie disease in the silkworm. The absolute nonsusceptibility to BmDV among certain silkworm strains is determined independently by two genes, nsd-1 and Nid-1. However, neither of these genes has been molecularly identified to date. Here, we isolated the nsd-1 gene by positional cloning and characterized the properties of its product, NSD-1. Sequence and biochemical analyses revealed that this gene encodes a Bombyx-specific mucin-like glycoprotein with a single transmembrane domain. The NSD-1 protein was specifically expressed in the larval midgut epithelium, the known infection site of BmDV. Sequence analysis of the nsd-1 gene from 13 resistant and 12 susceptible strains suggested that a specific arginine residue in the extracellular tail of the NSD-1 protein was common among susceptible strains. Germline transformation of the susceptible-type nsd-1 (with a single nucleotide substitution) conferred partial susceptibility to resistant larvae, indicating that the +  gene is required for the susceptibility of B. mori larvae to BmDV and the susceptibility is solely a result of the substitution of a single amino acid with arginine. Taken together, our results provide striking evidence that a novel membrane-bound mucin-like protein functions as a cell-surface receptor for a densovirus.
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http://dx.doi.org/10.1038/s41598-018-25388-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943349PMC
May 2018

Melanoma antigen family A4 protein produced by transgenic silkworms induces antitumor immune responses.

Exp Ther Med 2018 Mar 4;15(3):2512-2518. Epub 2018 Jan 4.

Faculty of Science and Technology, Division of Molecular Science, Gunma University, Kiryu, Gunma 376-8515, Japan.

Recent clinical trials with the aim of developing tumor antigen (TA)-specific cancer vaccines against a number of malignancies have focused on the identification of TAs presented by tumor cells and recognized by T cells. In the present study, the TA melanoma antigen family A4 (MAGE-A4) protein was produced using a transgenic (TG) silkworm system. Using stimulation, it was subsequently determined whether MAGE-A4 protein induced MAGE-A4-specific T cells from peripheral blood mononuclear cells of healthy donors. TG silkworm lines expressing a MAGE-A4 gene under an upstream activating sequence (UAS) were mated with those expressing a yeast transcription activator protein (GAL4) at the middle silk glands (MSGs) and embryos that harbored both the GAL4 and UAS constructs were selected. Recombinant MAGE-A4 protein was extracted from the MSGs of TG silkworms and evaluated using SDS-PAGE and western blot analysis. It was observed that MAGE-A4 produced by the TG silkworm system successfully induced MAGE-A4-specific CD4 T cell responses. Furthermore, MAGE-A4-specific CD4 T cells recognized antigen-presenting cells when pulsed with a MAGE-A4 tumor cell lysate. The present data suggests that recombinant tumor antigen production using the TG silkworm system may be a novel tool in the preparation of cancer vaccines.
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http://dx.doi.org/10.3892/etm.2018.5703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854940PMC
March 2018
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