Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields; however, its toxicological properties are not well documented. In our previous report, MGT showed genotoxicity in both in vitro and in vivo assay systems, and it was suggested that inflammatory responses exist behind the genotoxicity. To further clarify mechanisms underlying the genotoxicity, a comprehensive DNA adduct (DNA adductome) analysis was conducted using DNA samples derived from the lungs of mice exposed to MGT. In total, 30 and 42 types of DNA adducts were detected in the vehicle control and MGT-treated groups, respectively. Principal component analysis (PCA) against a subset of DNA adducts was applied and several adducts, which are deduced to be formed by inflammation or oxidative stress, as the case of etheno-deoxycytidine (εdC), revealed higher contributions to MGT exposure. By quantitative-LC-MS/MS analysis, εdC levels were significantly higher in MGT-treated mice than those of the vehicle control. Taken together with our previous data, it is suggested that inflammatory responses might be involved in the genotoxicity induced by MGT in the lungs of mice.

Neosporosis, which is caused by Neospora caninum, is a well-known disease in the veterinary field. Infections in pregnant cattle lead to abortion via transplacental (congenitally from mother to fetus) transmission. In this study, a N. caninum profilin (NcPROF), was expressed in silkworm larvae by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid and was purified from the hemolymph. Three NcPROF constructs were investigated, native NcPROF fused with an N-terminal PA tag (PA-NcPROF), PA-NcPROF fused with the signal sequence of bombyxin from B. mori (bx-PA-NcPROF), and bx-PA-NcPROF with additional C-terminal transmembrane and cytoplasmic domains of GP64 from BmNPV (bx-PA-NcPROF-GP64TM). All recombinant proteins were observed extra- and intracellularly in cultured Bm5 cells and silkworm larvae. The bx-PA-NcPROF-GP64TM was partly abnormally secreted, even though it has the transmembrane domain, and only it was pelleted by ultracentrifugation, but PA-NcPROF and bx-PA-NcPROF were not. Additionally, bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph by anti-PA agarose beads while PA-NcPROF and bx-PA-NcPROF were not. The purified bx-PA-NcPROF-GP64TM protein bound to its receptor, mouse Toll-like receptor 11 (TLR-11), and formed unique nanoparticles. These results suggest that profilin fused with GP64TM was secreted as a nanoparticle with binding affinity to its receptor and this nanoparticle formation is advantageous for the development of vaccines to N. caninum.

Photodynamic therapy (PDT) following lung-sparing extended pleurectomy for malignant pleural mesothelioma (MPM) has been investigated as a potential means to kill residual microscopic cells. High expression levels of folate receptor 1 (FOLR1) have been reported in MPM; therefore, targeting FOLR1 has been considered a novel potential strategy. The present study developed FOLR1‑targeting porphyrin-lipid nanoparticles (folate-porphysomes, FP) for the treatment of PDT. Furthermore, inhibition of activated epidermal growth factor (EGFR)-associated survival pathways enhance PDT efficacy. In the present study, these approaches were combined; FP-based PDT was used together with an EGFR-tyrosine kinase inhibitor (EGFR-TKI). The frequency of FOLR1 and EGFR expression in MPM was analyzed using tissue microarrays. Confocal microscopy and a cell viability assay were performed to confirm the specificity of FOLR1‑targeting cellular uptake and photocytotoxicity in vitro. In vivo fluorescence activation and therapeutic efficacy were subsequently examined. The effects of EGFR-TKI were also assessed in vitro. The in vivo combined antitumor effect of EGFR-TKI and FP-PDT was then evaluated. The results revealed that FOLR1 and EGFR were expressed in 79 and 89% of MPM samples, respectively. In addition, intracellular uptake of FP corresponded well with FOLR1 expression. When MPM cells were incubated with FP and then irradiated at 671 nm, there was significant in vitro cell death, which was inhibited in the presence of free folic acid, thus suggesting the specificity of FPs. FOLR1 targeting resulted in disassembly of the porphysomes and subsequent fluorescence activation in intrathoracic disseminated MPM tumors, as demonstrated by ex vivo tissue imaging. FP-PDT resulted in significant cellular damage and apoptosis in vivo. Furthermore, the combination of pretreatment with EGFR-TKI and FP-PDT induced a marked improvement of treatment responses. In conclusion, FP-based PDT induced selective destruction of MPM cells based on FOLR1 targeting, and pretreatment with EGFR-TKI further enhanced the therapeutic response.

Ovarian cancer is the most aggressive female reproductive tract tumours. Taxane (paclitaxel; TX) is widely used for ovarian cancer treatment. However, ovarian cancers often acquire chemoresistance. MicroRNAs (miR) have been reported to mediate many tumours'chemoresistance. We investigated the role of miR-363 in the chemoresistance of the ovarian cancer cell line, KF, and its TX-resistant derivative (KF-TX) cells. QRT-PCR indicated that miR-363 was upregulated in KF-TX cells, and introduction of miR-363 into sensitive ovarian cancer cells confers TX-resistance and significantly inhibited the expression of the Hippo member, LATS2, as indicated by viability, clonogenic assay and expression analysis. Furthermore, we validated the role of LATS2 in TX-response by sh-based silencing, which also confers TX-resistance to the ovarian cancer cells. On the other hand, specific inhibitor against miR-363 restored the response to TX in the resistant cells. In addition, miR-363 was found to bind to the 3'-UTR of LATS2 mRNA, confirming that miR-363 directly targets LATS2 as indicated by dual luciferase assay. RT-PCR-based evaluation of miR-363 in a panel of human ovarian tumours revealed its upregulation in most of the tumour tissues identified as resistant while it was downregulated in most of the tissues identified as sensitive ones. Moreover, higher levels of miR-363 in human ovarian cancer specimens were significantly correlated with TX chemoresistance. Taken together, our study reveals the involvement of miR-363 in chemoresistance by targeting LATS2 in ovarian cancers, raising the possibility that combination therapy with a miR-363 inhibitor and TX may increase TX efficacy and reduce the chance of TX-resistance.

Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55-56, 174-175, and 348-349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.

Insulin-like peptide 3 (INSL3) is a member of the relaxin/insulin superfamily and is expressed in testicular Leydig cells. Essential for fetal testis descent, INSL3 has been implicated in testicular and sperm function in adult males via interaction with relaxin/insulin-like family peptide receptor 2 (RXFP2). The INSL3 is typically prepared using chemical synthesis or overexpression in Escherichia coli followed by oxidative refolding and proteolysis. Here, we expressed and purified full-length porcine INSL3 (pINSL3) using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system. Biophysical measurements and proteomic analysis revealed that this recombinant pINSL3 exhibited the correct conformation, with the three critical disulfide bonds observed in native pINSL3, although partial cleavage occurred. In cAMP stimulation assays using RXFP2-expressing HEK293 cells, the recombinant pINSL3 possessed full biological activity. This is the first report concerning the production of fully active pINSL3 without post-expression treatments and provides an efficient production platform for expressing relaxin/insulin superfamily peptides.

Kaolin is white clay mineral with the chemical composition Al2Si2O5(OH)4, and many varieties of kaolins having different crystal structures are utilized in industrial, cosmetic and medical fields. To evaluate the effect of physicochemical character differences on the genotoxicity of kaolin, two types of kaolin, kaolin-S with smooth, sphere-shaped crystals, and kaolin-P with clusters of thin pseudohexagonal plates, were used in the study.

White spot syndrome virus (WSSV) is one of the most devastating pathogens in penaeid shrimp and can cause massive damage in shrimp aquaculture industries. Previously, the WSSV structural protein VP15 was identified as an antigenic reagent against WSSV infections. In this study, we truncated this protein into VP15(1-25), VP15(26-57), VP15(58-80), and VP15(1-25,58-80). The purified proteins from the E. coli expression system were assayed as potential protective agents in Kuruma shrimp (Marsupenaeus japonicus) using the prime-and-boost strategy. Among the four truncated constructs, VP15(26-57) provided a significant improvement in the shrimp survival rate after 20 days of viral infection. Subsequently, four peptides (KR11, SR11, SK10, and KK13) from VP15(26-57) were synthesized and applied in an in vivo assay. Our results showed that SR11 could significantly enhance the shrimp survival rate, as determined from the accumulated survival rate. Moreover, a multiligand binding protein with a role in the host immune response and a possible VP15-binding partner, MjgC1qR, from the host M. japonicus were employed to test its binding with the VP15 protein. GST pull-down assays revealed that MjgC1qR binds with VP15, VP15(26-57), and SR11. Taken together, we conclude that SR11 is a determinant antigenic peptide of VP15 conferring antiviral activity against WSSV.

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform.

Underwater walking is one of the most common hydrotherapeutic exercises. Therefore, understanding muscular control during underwater walking is important for optimizing training regimens. The effects of the water environment on walking are mainly related to the hydrostatic and hydrodynamic theories of buoyancy and drag force. To date, muscular control during underwater walking has been investigated at the individual muscle level. However, it is recognized that the human nervous system modularly controls multiple muscles through muscle synergies, which are sets of muscles that work together. We found that the same set of muscle synergies was shared between the two walking tasks. However, some task-dependent modulation was found in the activation combination across muscles and temporal activation patterns of the muscle synergies. The results suggest that the human nervous system modulates activation of lower-limb muscles during water walking by finely tuning basic locomotor muscle synergies that are used during land walking to meet the biomechanical requirements for walking in the water environment.

While the ethanol production from biomass by consolidated bioprocess (CBP) is considered to be the most ideal process, simultaneous saccharification and fermentation (SSF) is the most appropriate strategy in practice. In this study, one-pot bioethanol production, including cellulase production, saccharification of cellulose, and ethanol production, was investigated for the conversion of biomass to biofuel by co-culture of two different microorganisms such as a hyper cellulase producer, Acremonium cellulolyticus C-1 and an ethanol producer Saccharomyces cerevisiae. Furthermore, the operational conditions of the one-pot process were evaluated for maximizing ethanol concentration from cellulose in a single reactor.

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

Human parainfluenza virus type 1 (hPIV1) and type 3 (hPIV3) initiate infection by sialic acid binding. Here, we investigated sialic acid linkage specificities for binding and infection of hPIV1 and hPIV3 by using sialic acid linkage-modified cells treated with sialidases or sialyltransferases. The hPIV1 is bound to only α2,3-linked sialic acid residues, whereas hPIV3 is bound to α2,6-linked sialic acid residues in addition to α2,3-linked sialic acid residues in human red blood cells. α2,3 linkage-specific sialidase treatment of LLC-MK2 cells and A549 cells decreased the infectivity of hPIV1 but not that of hPIV3. Treatment of A549 cells with α2,3 linkage-specific sialyltransferase increased infectivities of both hPIV1 and hPIV3, whereas α2,6 linkage-specific sialyltransferase treatment increased only hPIV3 infectivity. Clinical isolates also showed similar sialic acid linkage specificities. We concluded that hPIV1 utilizes only α2,3 sialic acid linkages and that hPIV3 makes use of α2,6 sialic acid linkages in addition to α2,3 sialic acid linkages as viral receptors.

Lung tumors showing ground-glass opacities on high-resolution computed tomography indicate the presence of inflammation, atypical adenomatous hyperplasia, or localized bronchioloalveolar carcinoma. We adopted a two-staged video-assisted thoracoscopic lobectomy strategy involving completion lobectomy for localized bronchioloalveolar carcinoma with an invasive component according to postoperative pathological examination by permanent section after partial resection.

Nanomaterials are useful for their characteristic properties and are commonly used in various fields. Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields, whereas their toxicological properties are not well documented. A safety assessment is thus urgently required for MGT, and genotoxicity is one of the most serious concerns. In the present study, we examined genotoxic effects of MGT using mice and revealed that DNA damage analyzed by a comet assay in the lungs of imprinting control region (ICR) mice intratracheally instilled with a single dose of 0.05 or 0.2 mg/animal of MGT was approximately two- to three-fold higher than that of vehicle-control animals. Furthermore, in gpt delta transgenic mice, gpt mutant frequency (MF) in the lungs of the group exposed to four consecutive doses of 0.2 mg MGT was significantly higher than in the control group. Mutation spectrum analysis showed that base substitutions were predominantly induced by MGT, among which G:C to A:T transition and G:C to T:A transversion were the most significant. To clarify the mechanism of mutation caused by MGT, we analyzed the formation of DNA adducts in the lungs of mice exposed to MGT. DNA was extracted from lungs of mice 3, 24, 72 and 168 h after intratracheal instillation of 0.2 mg/body of MGT, and digested enzymatically. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and lipid peroxide-related DNA adducts were quantified by stable isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS). Compared with vehicle control, these DNA adduct levels were significantly increased in the MGT-treated mice. In addition to oxidative stress- and inflammation related-DNA adduct formations, inflammatory cell infiltration and focal granulomatous formations were also observed in the lungs of MGT-treated mice. Based on these findings, it is suggested that inflammatory responses are probably involved in the genotoxicity induced by MGT in the lungs of mice.

Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions.

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.

To develop monovalent dengue virus-like particle for serotype 3 (DENV-LP/3), we prepared and expressed two structural polyprotein constructs using silkworm and Bm5 cells: DENV-3 Capsid-premembrane-envelope (DENV-3CprME) and premembrane-envelope (DENV-3prME). The expressed PA-tagged 3CprME and 3prME polypeptides were partially purified by PA-tag affinity chromatography and had molecular weights of 85 and 75 kDa, respectively. Expressed proteins were separately verified using the following primary antibodies: the anti-PA tag antibody, DENV premembrane polyclonal antibody, and DENV envelope polyclonal antibody. Transmission electron microscopy revealed that these DENV-3CprME and 3prME formed rough, spherical DENV-LPs (DENV-LP/3CprME and DENV-LP/3prME), respectively, with a diameter of 30-55 nm. The heparin-binding assay demonstrated that these DENV-LPs contained the envelope protein domain III on their surfaces. Both DENV-LPs showed an affinity to sera from human dengue patients and immunized mice. Immunization of mice with DENV-LP/3prME significantly induced the level of antibodies compared with DENV-LP/3CprME. These results indicate that DENV-LP/3prME is suitable as a vaccine candidate compared with DENV-LP/3CprME.

Video-assisted thoracoscopic surgery (VATS) has become a standard approach for the treatment of lung cancer. However, its minimally invasive nature limits the field of view and reduces tactile feedback. These limitations make it vital that surgeons thoroughly familiarize themselves with the patient's anatomy preoperatively. We have developed a virtual reality (VR) surgical navigation system using head-mounted displays (HMD). The aim of this study was to investigate the potential utility of this VR simulation system in both preoperative planning and intraoperative assistance, including support during thoracoscopic sublobar resection.

Background. Robotic-assisted thoracic surgery (RATS) is now standard for lung cancer treatment, offering advantages over traditional methods. However, RATS's minimally invasive approach poses challenges like limited visibility and tactile feedback, affecting surgeons' navigation through com-plex anatomy. To enhance preoperative familiarization with patient-specific anatomy, we devel-oped a virtual reality (VR) surgical navigation system. Using head-mounted displays (HMDs), this system provides a comprehensive, interactive view of the patient's anatomy pre-surgery, aiming to improve preoperative simulation and intraoperative navigation. Methods. We integrated 3D data from preoperative CT scans into Perspectus VR Education software, displayed via HMDs for in-teractive 3D reconstruction of pulmonary structures. This detailed visualization aids in tailored preoperative resection simulations. During RATS, surgeons access these 3D images through Tile-ProTM multi-display for real-time guidance. Results. The VR system enabled precise visualization of pulmonary structures and lesion relations, enhancing surgical safety and accuracy. The HMDs offered true 3D interaction with patient data, facilitating surgical planning. Conclusions. VR sim-ulation with HMDs, akin to a robotic 3D viewer, offers a novel approach to developing robotic surgical skills. Integrated with routine imaging, it improves preoperative planning, safety, and accuracy of anatomical resections. This technology particularly aids in lesion identification in RATS, optimizing surgical outcomes.