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α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O -mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O -mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O -mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity. Fukutin-related protein (FKRP) catalyses the addition of ribitol-phosphate (RboP) to the O-mannosyl glycan of α-dystroglycan and mutations in FKRP cause dystroglycanopathy. Here the authors provide insights into its oligomerization and recognition of the substrates, CDP-Rbo and the RboP-(phospho-)core M3 glycan, by determining the crystal structures of human FKRP.

A cytocompatible porous scaffold mimicking the properties of extracellular matrices (ECMs) has great potential in promoting cellular attachment and proliferation for tissue regeneration. A biomimetic scaffold was prepared using silk fibroin (SF)/sodium alginate (SA) in which regular and uniform pore morphology can be formed through a facile freeze-dried method. The scanning electron microscopy (SEM) studies showed the presence of interconnected pores, mostly spread over the entire scaffold with pore diameter around 54~532 μm and porosity 66~94%. With significantly better water stability and high swelling ratios, the blend scaffolds crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) provided sufficient time for the formation of neo-tissue and ECMs during tissue regeneration. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) results confirmed random coil structure and silk I conformation were maintained in the blend scaffolds. What’s more, FI-TR spectra demonstrated crosslinking reactions occurred actually among EDC, SF and SA macromolecules, which kept integrity of the scaffolds under physiological environment. The suitable pore structure and improved equilibrium swelling capacity of this scaffold could imitate biochemical cues of natural skin ECMs for guiding spatial organization and proliferation of cells in vitro , indicating its potential candidate material for soft tissue engineering.

The mechanical properties of continuous fiber-reinforced thermoplastic (C-FRTP) composites are commonly lower than those of continuous fiber-reinforced thermosetting plastic (C-FRP) composites. We have developed a new molding method for C-FRTP. In this study, pre-impregnated materials were successfully prepared by polymer solution impregnation method and, finally, C-FRTP was fabricated. The viscosity of the thermoplastic matrix was decreased to approximately 3dPa×s, the same level of epoxy, and the fiber volume fraction was increased from approximately 45 to 60%. The cross-section of specimens were polished by an ion milling system and impregnation condition was investigated by scanning electron microscopy (SEM). The micrographs suggested that thermoplastic polymer was impregnated to every corner of the fiber, and no void was found on the cross-section. It revealed that void-free composites with perfect mechanical properties can be manufactured with this new molding method. All specimens were submitted to a mechanical measuring equipment, and the mechanical properties of the composite specimens were investigated. Mechanical analysis revealed that tensile property and flexural property of C-FRTP were enhanced up to the same level with C-FRP.

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

In the present paper, damage development within Carbon fiber reinforced plastic (CFRP) laminates and CFRP/Aluminum (Al) honeycomb core sandwich panels by impact loading was evaluated, and change in material properties due to the damage development was investigated. Falling weight impact tests, 3-point bending tests and cross-sectional observation were carried out. As results, it is found that falling rate of bending elastic modulus due to internal damage in the laminate only is lower than that in the upper face-sheet of the sandwich panel, and that difference in the falling rate between them becomes maximum at the impact energy of 5.1 J. As a result of investigating the relationship between reduction in bending elastic modulus and internal damage development, the reduction is caused by delamination within CFRP laminates mainly. Since total length of cracks in CFRP/Al honeycomb core sandwich panels is smaller than that in the laminates only, the sandwich panels have high impact tolerance because of absorption of impact energy by damaging Al honeycomb core.

Sialyl glycans have several biological functions. We have previously reported on the preparation and bifidogenic activity of milk-derived sialylglycopeptide (MSGP) concentrate containing sialyl O-glycans. The current study qualitatively and quantitatively analyzed the sialyl O-glycans present in the MSGP concentrate. Notably, our quantitative analysis indicated that a majority of O-glycopeptides in the MSGP concentrate were derived from glycomacropeptides. The concentrate was found to contain mainly three types of sialyl core 1 O-glycans, with the disialyl core 1 O-glycan being the most abundant. We successfully quantified three types of sialyl core 1 O-glycans using a meticulous method that used homogeneous O-glycopeptides as calibration standards. Our results provide valuable insights into assessment strategies for the quality control of O-glycans in dietary products and underscore the potential applications of MSGP concentrate in the food industry and other industries.

To better understand the material properties of cast aluminum alloys, the mechanical properties of microscopic structures (the eutectic structure and the α-Al phase) have been systematically examined using a special nano-indentation hardness test machine. In this approach, a triangular indentation is applied directly to the eutectic structure or α-Al phase, and the mechanical properties evaluated through hardness and load-strain relationships. The hardness of the eutectic phase varied, depending on the structural characteristics of the different intermetallic compounds. High values of hardness were obtained structures of the DO 3 type, e.g., Al 5 FeSi, but low hardness for CuAl 2 and Mg 2 Si. The hardness of CuAl 2 and Mg 2 Si had almost the same values as that of the α-Al matrix. In addition, from the nano-indentation hardness test, the effects of α-Al grain characteristics on the mechanical properties were clarified. The hardness of the α-Al grain was linearly related to the grain size and the distance from the grain boundary, where the higher the hardness, the smaller the grain size and the closer to the grain boundary. The hardness level was attributed to the different severity of slip resistance of the atoms during the indentation loading.

To elucidate a biological role of the methylated mannose residues found in N-glycans of terrestrial worm Enchytraeus japonensis , we first synthesized 3- O -methyl mannose- and 4- O -methyl mannose-derivatives and immobilized them to Sepharose 4B beads in order to isolate the sugar-binding protein. When whole protein extracts from the worms was applied to a series of the columns immobilized with the modified and unmodified mannose-derivatives, respectively, a protein with a molecular weight of 25,000 was isolated by 4- O -methyl mannose-immobilized column chromatography, and termed as a methylated mannose-binding protein (mMBP). mMBP bound weakly to a mannose-immobilized column and moderately to a 3- O -methyl mannose-immobilized column. The N-terminal amino acid sequences of mMBP and its endoprotease-digested peptides were determined. Using the degenerate first primers synthesized based on the primary sequence, a genomic DNA fragment was isolated. Then, the second primers were synthesized based on the genomic DNA fragment, and with use of them two cDNA fragments were obtained by the 3′- and 5′-RACE methods. Finally, the third primers were synthesized based on the sequences of the two cDNA fragments and one genomic DNA fragment, and with use of them a full-length cDNA of mMBP was isolated and shown to comprise a putative 633 bp open reading frame encoding 210 amino acid residues. BLAST analysis revealed that mMBP has identities by 26 ~ 55% to several proteins including the regeneration-upregulated protein 3 from the same species. Whether mMBP is involved in the regeneration of the worm is under investigation.

Gastric cancer with peritoneal dissemination is difficult to treat surgically, and frequently recurs and metastasizes. Currently, there is no effective treatment for this disease, and there is an urgent need to elucidate the molecular mechanisms underlying peritoneal dissemination and metastasis. Our previous study demonstrated that galectin-4 participates in the peritoneal dissemination of poorly differentiated gastric cancer cells. In this study, the glycan profiles of cell surface proteins and glycosphingolipids (GSLs) of the original (wild), galectin-4 knockout (KO), and rescue cells were investigated to understand the precise mechanisms involved in the galectin-4-mediated regulation of associated molecules, especially with respect to glycosylation. Glycan analysis of the NUGC4 wild type and galectin-4 KO clones with and without peritoneal metastasis revealed a marked structural change in the glycans of neutral GSLs, but not in N-glycan. Furthermore, mass spectrometry (MS) combined with glycosidase digestion revealed that this structural change was due to the presence of the lacto-type (β1-3Galactosyl) glycan of GSL, in addition to the neolacto-type (β1-4Galactosyl) glycan of GSL. Our results demonstrate that galectin-4 is an important regulator of glycosylation in cancer cells and galectin-4 expression affects the glycan profile of GSLs in malignant cancer cells with a high potential for peritoneal dissemination.

A new etching technique for revealing the plastic deformation zone in an Al–Cu–Mg alloy has been developed. The etching with the proposed etching agent was conducted on the deformed sample after being heated to 673 K for 3 h. With this etching technique, the plastic deformation zone was clearly observed even under low magnification. This was due to the change of microstructural characteristics in the plastic deformation zone after the heating process, in which there is significant precipitation of Al₂Cu and Mg₂Si, caused by the high energy arising from the severe deformation.