The biological role of core 1β1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori.

O-glycosylation of secreted and membrane-bound proteins is an important post-translational modification that affects recognition of cell surface receptors, protein folding, and stability. However, despite the importance of O-linked glycans, their biological functions have not yet been fully elucidated and the synthetic pathway of O-glycosylation has not been investigated in detail, especially in the silkworm. In this study, we aimed to investigate O-glycosylation in silkworms by analyzing the overall structural profiles of mucin-type O-glycans using LC-MS.

Construction of gene co-expression networks in cultured silkworm cells and identification of previously uncharacterized lepidopteran-specific genes required for chromosome dynamics.

Advances in sequencing technology and bioinformatics have accelerated gene discovery and homology-based functional annotation in many species, and numerous targeted gene studies have greatly expanded the understanding of gene functions. Nevertheless, there are still many genes that lack homology with genes in other evolutionary lineages and are left as genes with unknown functions. We constructed a gene co-expression network from the Bombyx mori ovary-derived cell line, BmN4, and attempted to infer the biological roles of uncharacterized genes based on the correlation between the function-known and unknown genes.

Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development.

The ovary is an important organ in reproduction. In insects, especially lepidopteran insects, the oocytes and reproductive organs develop rapidly during the pupal stage. Despite their drastic morphological changes, the molecular mechanisms of ovary development are not fully understood.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells.

Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel.

Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2.

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development.

Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System.

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 -derived murine TNFα.

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR.

Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm-Baculovirus Expression System.

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 Mg and ATP. Although vTopIB has been effectively produced in E.

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system.

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.

Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms.

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter.

Expression, Purification, and Characterization of Recombinant Human α-Antitrypsin Produced Using Silkworm-Baculovirus Expression System.

Human α-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT).

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

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.

Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae.

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.

Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells.

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters.

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori.

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.