Search

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.

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.

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.

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.

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, a 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 (BmATIC KO ), 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 +/ BmATIC KO 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 .

Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases. Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

Simple Summary Baculovirus expression vector system (BEVS) is widely employed to produce eukaryotic recombinant proteins with desired post-translational modifications. The tumor necrosis factor α (TNFα) is a promising reagent in treating autoimmunity and cancer diseases. In the current study, we designed to express and purify human and murine TNFα proteins in a silkworm larva-based baculovirus expression vector system (silkworm-BEVS). The results demonstrated that the desirable productivity of proteins with similar biological activity was experimentally confirmed. It was revealed that the C-terminal fusion tags negatively impacted their biological activity, as confirmed in the cytotoxicity assay. Taken together, silkworm-BEVS is an alternative platform for supplying high-quality TNFα products for various purposes. Abstract 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.

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are 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 found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript™ III Platinum™ One-step RT-qPCR and TaqPath™ 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility.

Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-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.

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.

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.

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.

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 Leu(432)–His(435) 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.

The compressive stress distribution of concrete was assessed to serve as a basis for evaluation of the flexural performance of structural members using electric arc furnace (EAF) oxidising slag aggregates. Eccentric loading tests were conducted on C-type specimens with aggregate type and compressive strength as variables. Compared with concrete made using natural aggregates, concrete made with EAF oxidising slag aggregates was found to result in a 20% increase in compressive strength for the same concrete mix and required a lower proportion of cement to attain the same strength. Compared with the specimens made with natural aggregates, specimens made with EAF oxidising slag aggregates had larger areas under the ascending curves because of their higher elastic moduli and superior compressive strain, arising from a more gradual decrease in the descending curves representing stress–strain relationships. In addition, specimens made with EAF oxidising slag aggregates exhibited a more gradual rate of decrease in ultimate strain with an increase in compressive strength.

This study investigated the structural characteristics of reinforced concrete column comprised of EAF (Electric Arc Furnace) oxidizing slag aggregates, which are less detrimental to the environment, because they are generated during the steel-making process and are considered a form of recycling. Six column specimens were prepared with the axial force ratio and the amount of EAF oxidizing slag aggregates as the variables. Static loading tests were performed to analyze the load versus strain relationship, failure mode, distribution of the axial and transverse strain, plastic hinge length, and energy dissipation capacity. The analysis showed that reinforced concrete columns made from EAF oxidizing slag aggregates had a superior ductile capacity after they reached their ultimate strength compared to those made from natural aggregates. The former also experienced significantly less crushing at the column ends up to the limit state. The use of EAF oxidizing slag aggregates in the columns delayed the initiation of plastic hinges at the column ends and enabled the plastic hinges to occur first in the beams, thereby effectively establishing a strong-column weak-beam mechanism (if the slag is used in the structural design).

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.

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 transcrip...

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.

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.

We report an improved 3-stacked white organic light emitting diode (WOLED) structure comprised of layers emitting red and yellow-green phosphorescence in the 2nd stacked device. We focused on developing the WOLED devices to realize 4K premium OLED TV displaying very high brightness and excellent color characteristics, with the optimized color layer. The WOLED has a high current efficiency of 83 cd/A, and cool white color whose color coordinate is (0.297, 0.317). 65-inch 4K premium OLED TV employing the new WOLED shows 800 nit of peak brightness, and 99 percent color gamut in DCI color space.

Neutrophils are innate immune cells that constitute the first line of defense against invading pathogens. Due to this characteristic, they are exposed to diverse immunological environments wherein sources for nutrients are often limited. Recent advances in the field of immunometabolism revealed that neutrophils utilize diverse metabolic pathways in response to immunological challenges. In particular, neutrophils adopt specific metabolic pathways for modulating their effector functions in contrast to other immune cells, which undergo metabolic reprogramming to ensure differentiation into distinct cell subtypes. Therefore, neutrophils utilize different metabolic pathways not only to fulfill their energy requirements, but also to support specialized effector functions, such as neutrophil extracellular trap formation, ROS generation, chemotaxis, and degranulation. In this review, we discuss the basic metabolic pathways used by neutrophils and how these metabolic alterations play a critical role in their effector functions.

Autophagy is a highly conserved intracellular degradation and energy-recycling mechanism that contributes to the maintenance of cellular homeostasis. Extensive researches over the past decades have defined the role of autophagy innate immune cells. In this review, we describe the current state of knowledge regarding the role of autophagy in neutrophil biology and a picture of molecular mechanism underlying autophagy in neutrophils. Neutrophils are professional phagocytes that comprise the first line of defense against pathogen. Autophagy machineries are highly conserved in neutrophils. Autophagy is not only involved in generalized function of neutrophils such as differentiation in bone marrow but also plays crucial role effector functions of neutrophils such as granule formation, degranulation, neutrophil extracellular traps release, cytokine production, bactericidal activity and controlling inflammation. This review outlines the current understanding of autophagy in neutrophils and provides insight towards identification of novel therapeutics targeting autophagy in neutrophils.

Background & Aims Cholestasis comprises a spectrum of liver diseases characterized by the accumulation of bile acids. Bile acids and activation of the farnesoid X receptor (FXR) can inhibit autophagy, a cellular self-digestion process necessary for cellular homeostasis and regeneration. In mice, autophagy appears to be impaired in cholestasis and induction of autophagy may reduce liver injury. Methods Herein, we explored autophagy in human cholestasis in vivo and investigated the underlying molecular mechanisms in vitro. FXR chromatin immunoprecipitation-sequencing and qPCR were performed in combination with luciferase promoter studies to identify functional FXR binding targets in a human cholestatic liver sample. Results Autophagic processing appeared to be impaired in patients with cholestasis and in individuals treated with the FXR ligand obeticholic acid (OCA). In vitro, chenodeoxycholic acid and OCA inhibited autophagy at the level of autophagosome to lysosome fusion in an FXR-dependent manner. Rubicon, which inhibits autophago-lysosomal maturation, was identified as a direct FXR target that is induced in cholestasis and by FXR-agonistic bile acids. Genetic inhibition of Rubicon reversed the bile acid-induced impairment of autophagic flux. In contrast to OCA, ursodeoxycholic acid (UDCA), which is a non-FXR-agonistic bile acid, induced autophagolysosome formation independently of FXR, enhanced autophagic flux and was associated with reduced Rubicon levels. Conclusion In models of human cholestasis, autophagic processing is impaired in an FXR-dependent manner, partly resulting from the induction of Rubicon. UDCA is a potent inducer of hepatic autophagy. Manipulating autophagy and Rubicon may represent a novel treatment concept for cholestatic liver diseases. Lay summary Autophagy, a cellular self-cleansing process, is impaired in various forms of human cholestasis. Bile acids, which accumulate in cholestatic liver disease, induce Rubicon, a protein that inhibits proper execution of autophagy. Ursodeoxycholic acid, which is the first-line treatment option for many cholestatic liver diseases, induces hepatic autophagy along with reducing Rubicon.

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.

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.

MG132 has been used as a proteasome inhibitor on Bombyx cells, but its physiological effects on autophagy still have not been elucidated. In this study, we find that the lipidated BmAtg8, BmAtg8-PE as an autophagosomal marker protein, is only localized to membranes. Then we established systems to monitor autophagic flux in Bombyx cells: Induction of autophagy reduces exogenous BmAtg8 and exogenous BmAtg8-PE, facilitates formation of autophagosomes indicated by green EGFP-BmAtg8 puncta after cotreatment by Rapamycin and Bafilomycin A1, and causes accumulation of free EGFP from EGFP-BmAtg8 cleavage in autolysosomes. Using these established systems, we find that exposure of MG132 inhibits both basal and Rapamycin-induced autophagy when polyubiquitinated proteins are accumulated markedly in Bombyx cells. Interestingly, we reveal that attenuation of autophagy in these cells is ascribed as distinct suppression of formation of autophagosomes after MG132 treatment.

G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantities with outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eight G proteins (Gs, G12, Gα4, Gq, Gβ2, Gβ3, Gβ5 and Gγ) and subsequently monitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system.

Structural performance of rectangular concrete section confined by squared spirals was investigated. Through the uni-axial compression test using concrete prisms with squared spirals and without longitudinal bars, the enhancement of strength and deformation capacity of rectangular concrete section was scrutinized. By the experimental observations on the properties of lateral expansion of concrete prism, a moment of reaching the axial peak stress in section was predicted in a good accuracy. Qualitative relationships between the structural performance (the enhancement of strength and deformation capacity) of concrete prism and confinement efficiency were studied. Also, for the effective use of normal- and high-strength lateral reinforcement, a design implication using the qualitative relationship of the tensile stress in lateral reinforcement at failure of concrete prism with parameters was proposed.

Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GRLysM) or HSC (GRhGFAP): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GRLysM mice, whereas the suppression of fibrotic gene expression was diminished in GRhGFAP mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.

Interleukin 2 (IL-2) is a pharmacologically vital cytokine secreted mainly by activated CD4 + T lymphocyte. Recombinant human IL-2 (rhIL-2) protein has already been globally applied as an immune-therapeutic reagent for various diseases. Therefore, there is great interest in developing an active form of rhIL-2 in very large amounts and with excellent purity for clinical use. In this study, we successfully mass-expressed and purified N- or C-terminal tandem tag-fused rhIL-2 in a baculovirus expression vector system (BEVS) using silkworm larvae as factories. We confirmed that the intrinsic instability of hIL-2 causes the loss and low recovery of N-tagged rhIL-2 and that C-tagged rhIL-2 is more suitable for mass production. Furthermore, the activity of purified rhIL-2s was further validated by a cell proliferation assay of a human natural killer cell line, and both rhIL-2 proteins produced in the silkworm-BEVS exhibited comparable activity to that of the commercial E.coli -derived rhIL-2. Taken together, our results and strategies could contribute greatly to the mass production of active rhIL-2.

Luteinizing hormone (LH), a gonadotropin hormone (GTH) of the pituitary glycoprotein family, is important in oocyte maturation, ovulation, and spermiation. In this study, we generated a Japanese eel LH (JeLH) with and without the equine chorionic gonadotropin (eCG) carboxyl-terminal peptides under the control of the polyhedron in the silkworm pupae BES to determine their expression levels, glycosylation profile, and in vitro bioactivity. The target proteins were highly expressed in the pupae hemolymph. Recombinant JeLH·eCG and JeLH were N- or O-glycosylated, as shown by periodic-acid Schiff staining, deglycosidase enzyme treatment, and lectin blot analyses, and showed no significant difference in the in vitro bioactivity. Both single-chain hormones and salmon pituitary extract induced maturation of Japanese eel oocytes in vitro. Recombinant LHs produced in silkworm pupae might be suitable candidates for in vivo experiments, because they can be produced in sufficient amount and can undergo N-glycosylation.

A new reinforced concrete (RC) structural system suitable for long-span structures was developed. The system consists of a slab, a column, double beams and a drop panel. To investigate the structur...

2 The most important factor in the structural design of ships and offshore structures operating in arctic region is ice load, which results from ice-structure interaction during the ice collision process. The mechanical properties of ice related to strength and failure, however, show very complicated aspect varying with temperature, volume fraction of brine, grain size, strain rate and etc. So it is nearly impossible to establish a perfect material model of ice satisfying all the mechanical characteristics completely. Therefore, in general, ice collision analysis was carried out by relatively simple material models considering only specific aspects of mechanical characteristics of ice and it would be the most significant cause of inevitable errors in the analysis. Especially, it is well-known that the most distinctive mechanical property of ice is high dependency on strain rate. Ice shows brittle attribute in higher strain rate while it becomes ductile in lower strain rate range. In this study, the simulation method of ice collision to ship hull using the nonlinear dynamic FE analysis was dealt with. To consider the strain rate effects of ice during ice-structural interaction, strain rate dependent constitutive model in which yield stress and hardening behaviors vary with strain rate was adopted. To reduce the huge amount of computing time, the modeling range of ice and ship structure were restricted to the confined region of interest. Under the various scenario of ice-ship hull collision, the structural behavior of hull panels and failure modes of ice were examined by nonlinear FE analysis technique.

Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.

Pseudomonas aeruginosa PAO1 organisms harbouring different plasmids were cultured in broths containing appropriate antibiotic(s). Extracellular proteins were more abundant in the presence of tetracycline or kanamycin than in the presence of other antibiotics. Zymography revealed that alkaline protease (AprA) production was interfered by these antibiotics. Extracellular proteins were not observed at the same level when AprA-deficient EG03 strains were cultured in the presence of different antibiotics. The extracellular protein levels were dependent on the antibiotics and plasmid derivative groups. Levels of extracellular protein were not significantly different between PAO1 (pBBR1MCS-5) and EG03 (pAprcomp-MCS5), and profiles of the extracellular proteome were comparable. In contrast, the level of EG03 (pBBR1MCS-MCS5) extracellular protein was higher than those observed in the other two strains. These results suggested that although AprA partially contributes to the alteration of extracellular protein level, the effect is limited.

p-oily (op) is a novel mutant of Bombyx mori exhibiting translucent larval integument and male infertility. Elucidation of the causative gene of the op mutant will help understand the genetic mechanism underlying larval integument coloration and male fertility. Using polymorphisms between B. mori and B. mandarina, the op locus was narrowed down to a 375-kb region. Using RNA-seq analysis, we found that op mutants have a frameshift mutation in the KWMTBOMO13770 gene located in the 375-kb region. A database search indicated that this gene is the human cytosolic 5'-nucleotidase II gene (cN-II) homolog in Bombyx, which mediates the conversion of inosine monophosphate (IMP) to inosine, a precursor of uric acid. CRISPR/Cas9-mediated knockout mutants of the Bm-cN-II gene showed translucent integuments, and there appeared translucent larvae in the crosses between knockout moths and +/op moths. Moreover, the translucent phenotype of, and decreased uric acid content in the larval integument caused by the mutations in the Bm-cN-II gene were rescued by oral administration of inosine. These results indicated that the Bm-cN-II gene is responsible for the op phenotype and that the molecular function of the Bm-cN-II gene is the conversion of IMP to inosine. We also discuss the genetic relationship between the Bm-cN-II gene and male fertility.

This study describes a new assessment technique for evaluating marine coating performance on ice. The current evaluation methods related to the ice friction performance are investigated and their limitations are analyzed. To ensure the accuracy and validity of the study, ice abrasion specimens were prepared using paints applied to actual ice-class vessels. Instead of the commonly used rubber material roller, the cold model ice was used as the corresponding abrasion materials on the coating. To assess ice friction and abrasion performance, a new test procedures are developed. Abrasion was generated in the unit of abrasion distance of about 3 km per coating, and abrasion was finally generated up to about 12 km. As the ice abrasion progressed, scratches and peelings occurred on the surface of the IPC, therefore, it was found that the surface roughness and frictional coefficient of the IPC gradually increased. AFC, on the other hand, tended to separate like dust as the surface coatings were ground on ice, and the surface roughness but also frictional coefficient did not change significantly. It was confirmed that the ice abrasion phenomena were different according to the type of paints and the ice friction performance effect of the marine coating could be varied.

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.

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.

Human α

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson’s disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.

This study proposes a model to predict bond stress-slip behavior by conducting tests on RC members that experience splitting bond failure when subject to cyclic loading such as seismic load...

A new composite structural system that is able to enhance constructability and is suitable for realization of long-span structures was developed. Steel beams, steel reinforced concrete columns, reinforced concrete drop panels, and slabs are placed in the system. To determine the structural performance of the system, the gravity and lateral load resisting properties of the system were investigated. It was found that the presence of a reinforced concrete drop panel at the top of a column brings about a reduction of the negative moment at the top of the column. Also, it was observed that a reinforced concrete drop panel on the top of the column significantly affected the deflection-control of the beam. In a lateral load resisting test, cracks in slabs of the new structural system disperse in a better manner than is the case for crack patterns in the conventional composite system. Also, via push-over analysis using a package of structural analysis software, it was found that the developed structural system is effective in the prevention of stress concentration in the column zone.

This paper proposes a new analytical model for the relationship between the axial and transverse strains of confined concrete in uniaxial compression. The proposed model was developed based on empirical data for a circular section confined by spirals. A column with a circular section and subjected to uniaxial compression was considered. The proposed model predicts the stress in the transverse confinement reinforcement to estimate the relationship between the axial strain of the column and tensile strain of the transverse confinement reinforcement. The model can also produce the axial stress–strain relationship of the confined concrete of the column. The predicted axial and transverse strains of the confined concrete section corresponded to the observed axial peak stress of a column evaluated in previous research with good accuracy. This model will be an effective tool for investigating the effect of circular spirals on the structural behavior of confined concrete and enhancing the accuracy of structural behavior analysis for a column under uniaxial compression.

We reported previously that baculovirus AcMNPV host-ranges in silkworm strains are controlled by a novel third chromosomal locus. To further isolate the potential host factor and uncover the functional pathway involved, in this study we analyzed hemolymph proteins from AcMNPV-resistant or -sensitive silkworm strains infected with baculoviruses. All the protein spots from 2D electrophoresis were characterized by MALDI-TOF MS and further systematically assessed for differentially regulated proteins at different stages of infection. Subsequently, six candidates were selected for functional analysis using Bm5 cells, where the candidates were knocked-down or overexpressed. We observed that mRNA expression levels of beta-N-acetylglucosaminidase and prophenoloxidase subunit 2 are significantly upregulated during AcMNPV infections in Bm5 cells. Ultimately, we found that RNA interference of ribosomal protein RpL34 causes serious damages to cell viability as well as abortive infection, indicating that ribosomal components are essential for productive baculovirus infection.