A major endogenous glycoside hydrolase mediating quercetin uptake in Bombyx mori.

Quercetin is a common plant flavonoid which is involved in herbivore-plant interactions. Mulberry silkworms (domestic silkworm, Bombyx mori, and wild silkworm, Bombyx mandarina) take up quercetin from mulberry leaves and accumulate the metabolites in the cocoon, thereby improving its protective properties. Here we identified a glycoside hydrolase, named glycoside hydrolase family 1 group G 5 (GH1G5), which is expressed in the midgut and is involved in quercetin metabolism in the domestic silkworm. Our results suggest that this enzyme mediates quercetin uptake by deglycosylating the three primary quercetin glycosides present in mulberry leaf: rutin, quercetin-3-O-malonylglucoside, and quercetin-3-O-glucoside. Despite being located in an unstable genomic region that has undergone frequent structural changes in the evolution of Lepidoptera, GH1G5 has retained its hydrolytic activity, suggesting quercetin uptake has adaptive significance for mulberry silkworms. GH1G5 is also important in breeding: defective mutations which result in discoloration of the cocoon and increased silk yield are homozygously conserved in 27 of the 32 Japanese white-cocoon domestic silkworm strains and 12 of the 30 Chinese ones we investigated.

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

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 Bombyx mori, 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.

Role of a single odorant receptor in the chemotaxis behavior of Bombyx mori.

Silkworm (Bombyx mori), an insect herbivore, is attracted to cis-jasmone released from mulberry leaves. Its olfactory receptor, BmOr56, specifically responds to cis-jasmone. In this study, we constructed a BmOr56 deletion line and found that the attractive behavior of cis-jasmone was completely lost in the mutant, suggesting the involvement of a single receptor in this specific chemoattractive behavior.

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

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.

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

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)3 AS]2 RKLPDAS]8 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.

Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction.

Porcine zona pellucida proteins (ZPs) have been utilized as female immunocontraceptive antigens. The purpose of this study was to explore the potential use of silkworm recombinant bovine ZP4 as an alternative. When the protein was injected with monophosphoryl lipid A (MPL) - an immuno-stimulative agent - into two female goats, marked elevation of the anti-ZP4 titer was detected. Application of the purified specific IgG to a porcine in vitro fertilization system reduced the sperm penetration rate. In one goat, the cyclic profile of serum progesterone disappeared as the anti-ZP4 titer increased. Histological examination of the ovaries revealed degeneration of antral follicles with sparse infiltration of inflammatory cells in the theca, indicating that autoimmune oophoritis had been induced. Together, the present results suggest that recombinant ZP4 disturbs fertilization and exerts a pathogenic effect on follicle development in goats, thus indicating its potential as a female immunocontraceptive antigen.

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

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.

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

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.

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

We had previously reported a prostaglandin E synthase (bmPGES) in the silkworm Bombyx mori that catalyzes the isomerization of PGH2 to PGE2. 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 PGE2. 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 PGE2 in silkworms.

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

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 TRß1 that was fused with glutathione S-transferase. Using 160 larvae, the yield of the recombinant GST-TRß was approximately 4 mg, and the purified GST-TRß completely retained its physiological activity. Our results indicated that the recombinant TRß was secreted extracellularly using the silk fibroin signal peptide sequence. Moreover, we found that the expression system of silkworms was applicable to nuclear proteins.

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

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.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

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

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 (Dy3+), 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.

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori.

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori.

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

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.

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells.

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori.

Hoxgenes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hoxgenes can also function in terminally differentiated tissue of the lepidopteranBombyx mori In this species,Antennapedia(Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antpcan regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antpin the posterior silk gland induced ectopic expression of major silk protein genes such assericin-3,fhxh4, and fhxh5 These genes are normally expressed specifically in the middle silk gland as is Antp Therefore, the evidence strongly suggests that Antpactivates these silk protein genes in the middle silk gland. The putativesericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antpdirectly activates their expression. We also found that the pattern of gene expression was well conserved between B. moriand the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori We suggest that Hoxgenes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes.

Pheromone responsiveness threshold depends on temporal integration by antennal lobe projection neurons.

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs' responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.

Hox transcription factor Antp regulates sericin-1 gene expression in the terminal differentiated silk gland of Bombyx mori.

Hox genes are well-known master regulators in developmental morphogenesis along the anteroposterior axis of animals. However, the molecular mechanisms by which Hox proteins regulate their target genes and determine cell fates are not fully understood. The silk gland of Bombyx mori is a tubular tissue divided into several subparts along the anteroposterior axis, and the silk genes are expressed with specific patterns. The sericin-1 gene (ser1) is expressed in the middle silk gland (MSG) with sublocal specificity. Here we show that the Hox protein Antp is a component of the middle silk gland-specific complex, MIC (MSG-intermolt-specific complex), binds to the essential promoter element of ser1, and activates its expression. Ectopic expression of Antp in transgenic silkworms induced the expression of ser1 in the posterior silk gland (PSG), but not in the anterior part of MSG (MSG-A). Correspondingly, a MIC-like complex was formed by the addition of recombinant Antp in extracts from PSG with its cofactors Exd and Hth, but not in extracts from MSG-A. Splicing patterns of ser1 mRNA induced by the ectopic expression of Antp in PSG were almost the same as those in MSG at the fifth instar and altered depending on the induction timing of Antp. Other Hox genes were expressed with sublocal specificity in the silk gland. The Bombyx silk gland might provide a useful system for understanding how Hox proteins select and regulate their target genes.