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High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The Glc 1 Man 9 GlcNAc 2 (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuto Nakamachi, Shinji Tanaka, Taiki Kuribara, Kiichiro Totani has patent #P7261366 issued to KH Neochem Co. Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

High-mannose-type glycans play essential biological roles, e.g., immune response and glycoprotein quality control, and preparing a series of oligomannosyl branches of high-mannose-type glycans is critical for biological studies. However, obtaining sufficient amounts of the various oligomannosyl branches is challenging. In this study, we demonstrated a partial glycosylation strategy for the single-step synthesis of various biologically relevant oligomannosyl-branched structures. First, Manα1-6(Manα1-3)Man-type oligomannosyl branch was synthesized via double glycosylation from a 3,6-di-OH mannosyl acceptor and fluorinated mannosyl donor with perfect α-selectivity. Subsequent partial glycosylation by reducing the equivalent of the mannosyl donor enabled to obtain biologically relevant Manα1-2Manα1-6(Manα1-2Manα1-3)Man, Manα1-6(Manα1-2Manα1-3)Man, Manα1-2Manα1-6(Manα1-3)Man, and Manα1-6(Manα1-3)Man in one-pot. Each oligomannosyl branch could be easily purified by liquid chromatography. The resulting structural isomers were identified by 2D-HMBC NMR. A systematic lectin affinity assay using the prepared oligomannosyl branches showed different specificities for the Galanthus nivalis lectin between structural isomers of the oligomannosyl branches with the same number of mannose residues..

In molecular biology research, a vitamin E (VE) vehicle (VE dissolved in organic solvent) is often added to water media without a stabilizer. However, the detailed behavior of VE colloids in water media is unclear. In this study, we reveal that VE nanoemulsion readily forms in water-based media through the existing protocol. The colloid size was changed from 39 nm to the submicron scale by adjusting the initial concentration of the VE solution and adding a buffer. The radical scavenging effect of the dispersed nanosized VEs is comparable to that of the water-soluble antioxidant Trolox, providing excellent antioxidant performance in colloid form. The cytoprotection effect of the VE colloids under a lipid oxidation condition largely depends on the size of the nanodispersion. Smaller dispersed particles are more efficient radical scavengers than larger particles for a constant VE amount owing to sophisticated uptake behavior of cell. This unveiled fundamental knowledge pave the way for a preparative protocol of stabilizer-free VE vehicles, which are expected to become widely used in molecular biology research.

This study aimed to evaluate the distribution of retinal pigment epithelium (RPE) melanin in patients with retinitis pigmentosa (RP) using entropy measurements by custom-made polarization-sensitive optical coherence tomography (PS-OCT) images, and compare entropy with the intensity of short-wavelength (SW) and near-infrared (NIR) autofluorescence (AF). We retrospectively reviewed the retinal images, including PS-OCT, SW-AF, and NIR-AF of patients with RP who had a hyperautofluorescent ring on AF. A total of 12 eyes of 12 patients (8 women and 4 men; mean age: 37.9 years) were included. There was a strong positive correlation between entropy value and NIR-AF intensity (r = 0.626, p < 0.001), and there was a very weak negative correlation between entropy value and SW-AF (r = - 0.197, p = 0.001). The mean values of the entropy in the foveal, temporal (2 mm from the fovea), and nasal (2 mm from the fovea) sections were 0.41 (± 0.09), 0.29 (± 0.08), and 0.26 (± 0.08), respectively. The entropy was significantly higher in the foveal section than in the temporal and nasal sections (p = 0.002 and p = 0.003, respectively). There was no significant difference between the entropies values for the temporal and nasal sections (p = 0.157). Age, logMAR best-corrected visual acuity, ellipsoid zone width, and central retinal thickness were not correlated with foveal entropy. We presented RPE melanin imaging in patients with RP using PS-OCT for the first time. PS-OCT can be a useful tool for monitoring patients with RP., (© 2022. The Author(s).)

Glycoprotein folding plays a critical role in sorting glycoprotein secretion and degradation in the endoplasmic reticulum (ER). Furthermore, relationships between glycoprotein folding and several diseases, such as type 2 diabetes and various neurodegenerative disorders, are indicated. Patients' cells with type 2 diabetes, and various neurodegenerative disorders induce ER stress, against which the cells utilize the unfolded protein response for protection. However, in some cases, chronic and/or massive ER stress causes critical damage to cells, leading to the onset of ER stress-related diseases, which are categorized into misfolding diseases. Accumulation of misfolded proteins may be a cause of ER stress, in this respect, perturbation of oligomannose-type glycan processing in the ER may occur. A great number of studies indicate the relationships between ER stress and misfolding diseases, while little evidence has been reported on the connection between oligomannose-type glycan processing and misfolding diseases. In this review, we summarize alteration of oligomannose-type glycan processing in several ER stress-related diseases, especially misfolding diseases and show the possibility of these alteration of oligomannose-type glycan processing as indicators of diseases.

To generate three-dimensional tissue in vitro, promoting vasculogenesis in cell aggregates is an important factor. Here, we found that ultrasound promoted vasculogenesis of human umbilical vein endothelial cells (HUVECs). Promotion of HUVEC network formation and lumen formation were observed using our method. In addition to morphological evaluations, protein expression was quantified by western blot assays. As a result, expression of proteins related to vasculogenesis and the response to mechanical stress on cells was enhanced by exposure to ultrasound. Although several previous studies have shown that ultrasound may promote vasculogenesis, the effect of ultrasound was unclear because of unregulated ultrasound, the complex culture environment, or two-dimensional-cultured HUVECs that cannot form a lumen structure. In this study, regulated ultrasound was propagated on three-dimensional-monocultured HUVECs, which clarified the effect of ultrasound on vasculogenesis. We believe this finding may be an innovation in the tissue engineering field., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

About half of all newly synthesized proteins have N-linked glycans. These glycans play pivotal roles in controlling the folding, sorting, and degradation of glycoproteins via several glycan-related proteins. The glycan-mediated protein quality control system is important for cellular homeostasis. In this review, we summarize recent advances in our understanding of the system and discuss structural insights from chemical and biological perspectives. In particular, we focus on the mechanisms by which these mediators respond to several folding states of glycoproteins., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A tri-antennary Man9GlcNAc2 glycan on the surface of endoplasmic reticulum (ER) glycoproteins functions as a glycoprotein secretion or degradation signal after regioselective cleavage of the terminal α-1,2-mannose residue of each branch. Four α-1,2-mannosidases-ER mannosidase I, ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1), EDEM2, and EDEM3-are involved in the production of these signal glycans. Although selective production of signal glycans is important in determining the fate of glycoproteins, the branch-discrimination abilities of the α-1,2-mannosidases are not well understood. A structural feature of the Man9GlcNAc2 glycan is that all terminal glycosidic linkages of the three branches are of the α-1,2 type, while the adjacent inner glycosidic linkages are different. In this study, we examined whether the α-1,2-mannosidases showed branch specificity by discriminating between different inner glycosides. Four trisaccharides with different glycosidic linkages [Manα1-2Manα1-2Man (natural A-branch), Manα1-2Manα1-3Man (natural B-branch), Manα1-2Manα1-6Man (natural C-branch), and Manα1-2Manα1-4Man (unnatural D-branch)] were synthesized and used to evaluate the hypothesis. When synthesizing these oligosaccharides, highly stereoselective glycosylation was achieved with a high yield in each case by adding a weak base or tuning the polarity of the mixed solvent. Enzymatic hydrolysis of the synthetic trisaccharides by a mouse liver ER fraction containing the target enzymes showed that the ER α-1,2-mannosidases had clear specificity for the trisaccharides in the order of A-branch > B-branch > C-branch ≈ D-branch. Various competitive experiments have revealed for the first time that α-1,2-mannosidase with inner glycoside specificity is present in the ER. Our findings suggest that exo-acting ER α-1,2-mannosidases can discriminate between endo-glycosidic linkages.

N-glycans are attached to newly synthesised polypeptides and are involved in the folding, secretion, and degradation of N-linked glycoproteins. In particular, the calnexin/calreticulin cycle, which is the central mechanism of the entry and release of N-linked glycoproteins depending on the folding sates, has been well studied. In addition to biological studies on the calnexin/calreticulin cycle, several studies have revealed complementary roles of in vitro chemistry-based research in the structure-based understanding of the cycle. In this mini-review, we summarise chemistry-based results and highlight their importance for further understanding of the cycle., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are enzymes that degrade polysaccharides with an oxidative mechanism and contributed to the efficiency in biomass degradation by glycoside hydrolases (GHs). In this study, the substrate and reaction specificity of SgLPMO10A that was an auxiliary activity family 10 (AA10) enzyme with a carbohydrate binding module family 2 (CBM2) domain from Streptomyces griseus, was analyzed. This enzyme produced oxidized cello-oligosaccharides from cellulose and boosted cellulose degradation by cellulases. Detailed study of the AA10 and CBM2 domains revealed that the binding ability of SgLPMO10A depended on CBM2 and that only the AA10 domain functions more effectively in the presence of a certain amount of substrates., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are redox-active enzymes that cleave insoluble polysaccharides by an oxidative reaction. In the present study, we have characterized four recombinant putative chitin-active LPMOs from Streptomyces griseus ( Sg LPMO10B, -C, -D, and -F) and evaluated their potential in enhancing hydrolysis of α- and β-chitin by three families of 18 chitinases of Serratia marcescens , Sm ChiA, -B, and -C. All four recombinant Sg LPMO10s showed oxidative activity toward both α- and β-chitin but exhibited different abilities to promote the release of chitobiose from chitin by chitinases depending on both the chitinase and the chitin type. These effects were observed under conditions where the amount of LPMO in the reaction was not rate-limiting, showing that the observed functional differences relate to different abilities of the LPMOs to interact with and act on the substrate. These results show that four seemingly similar LPMOs carrying out the same reaction, cleavage of chitin by C1 oxidation, may have different roles in natural chitin conversion, which provides a rationale for the multiplicity of these enzymes within the same organism. The ability of the LPMOs to act on more natural substrates was demonstrated by showing that Sg LPMO10B improved chitin solubilization in dried powdered shrimp shells.

Imaging of melanin in the eye is important as the melanin is structurally associated with some ocular diseases, such as age-related macular degeneration. Although optical coherence tomography (OCT) cannot distinguish tissues containing the melanin from other tissues intrinsically, polarization-sensitive OCT (PS-OCT) can detect the melanin through spatial depolarization of the backscattered light from the melanin granules. Entropy is one of the depolarization metrics that can be used to detect malanin granules in PS-OCT and valuable quantitative information on ocular tissue abnormalities can be retrived by correlating entropy with the melanin concentration. In this study, we investigate a relationship between the melanin concentration and some depolarization metrics including the entropy, and show that the entropy is linearly proportional to the melanin concentration in double logarithmic scale when noise bias is corrected for the entropy. In addition, we also confirm that the entropy does not depend on the incident state of polarization using the experimental data, which is one of important attributes that depolarization metrics should have. The dependence on the incident state of polarization is also analyzed for other depolarization metrics.

Immune attacks are key issues for cell transplantation. To assess the safety and the immune reactions after iPS cells-derived retinal pigment epithelium (iPS-RPE) transplantation, we transplanted HLA homozygote iPS-RPE cells established at an iPS bank in HLA-matched patients with exudative age-related macular degeneration. In addition, local steroids without immunosuppressive medications were administered. We monitored immune rejections by routine ocular examinations as well as by lymphocytes-graft cells immune reaction (LGIR) tests using graft RPE and the patient's blood cells. In all five of the cases that underwent iPS-RPE transplantation, the presence of graft cells was indicated by clumps or an area of increased pigmentation at 6 months, which became stable with no further abnormal growth in the graft during the 1-year observation period. Adverse events observed included corneal erosion, epiretinal membrane, retinal edema due to epiretinal membrane, elevated intraocular pressure, endophthalmitis, and mild immune rejection in the eye. In the one case exhibiting positive LGIR tests along with a slight fluid recurrence, we administrated local steroid therapy that subsequently resolved the suspected immune attacks. Although the cell delivery strategy must be further optimized, the present results suggest that it is possible to achieve stable survival and safety of iPS-RPE cell transplantation for a year.

Deglucosylation and reglucosylation of glycoproteins by glucosidase II and uridine diphosphate-glucose: glycoprotein glucosyltransferase 1 (UGGT1), respectively, are important steps in glycoprotein quality control. Misfolded glycoprotein accumulation is associated with endoplasmic reticulum stress and can lead to protein misfolding diseases such as metabolic syndrome. Here, we analyzed the expression and activities of glucosidase II and UGGT1 in rat models of obesity and obese type 2 diabetes, and phenotypes associated with moderate and severe metabolic syndrome, respectively. In obesity, the mRNA and protein levels of glucosidase II and UGGT1 are decreased and their activities are reduced. In obese type 2 diabetes, the mRNA and protein levels of these enzymes are increased, and glucosidase II activity is slightly recovered, although UGGT1 activity is reduced. Our findings suggest that metabolic syndrome affects deglucosylation/reglucosylation enzymes according to disease severity., (© 2020 Federation of European Biochemical Societies.)

Transplantation of autologous human induced pluripotent stem cell-derived retinal pigment epithelial (hiPSC-RPE) sheets is a promising therapy for age-related macular degeneration (AMD). As melanin content is a representative feature of healthy RPE, we used polarization-sensitive optical coherence tomography (PS-OCT) to estimate the relative melanin content of RPE in diseased and non-diseased area, and in human iPSC-RPE sheets in vitro and in vivo by evaluating the randomness of polarization (entropy). Two aged Japanese women, one with neovascular AMD that underwent transplantation of an autologous hiPSC-RPE cell sheet and another with binocular dry AMD, were selected for this study. Entropy value was minimal in cells containing no melanin, whereas that of human RPE and hiPSC-RPE sheets was high. En face entropy of the cultured hiPSC-RPE sheet was compared with its grey-scale photo and its values were found to be inversely correlated with the extent of absence of pigmentation in vitro. En face entropy maps were compared to colour fundus photographs, fundus autofluorescence images, and fluorescein angiography images from patients. Entropy values of intact and defective RPEs and of iPSC-RPE transplant areas were determined in vivo using PS-OCT B-scan images. PS-OCT was found to be applicable in the estimation of relative melanin content of cultured and transplanted RPEs in regenerative medicine.

We demonstrate the preferential orders of molecular chaperones glucose-regulated protein 94 (GRP94), binding immunoglobulin protein (BiP), and calreticulin (CRT) in an endoplasmic reticulum (ER) fraction from rat liver using columns conjugated with denatured myoglobin, RNase A, or β-lactoglobulin as client proteins in the presence or absence of ATP. The results showed that BiP, CRT, and GRP94 preferentially contributed myoglobin, RNase A, and β-lactoglobulin, respectively, in the presence of ATP. In the absence of ATP, GRP94 and CRT preferentially recognized misfolded myoglobin (α-helix-rich protein), whereas BiP preferentially recognized misfolded RNase A (α-helix/β-sheet mixed protein) and β-lactoglobulin (β-sheet-rich protein). The preferential order of ER chaperones may be dynamically regulated by ER conditions and the higher-order structure of client proteins., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0-7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages., (2020 by The Japanese Society of Applied Glycoscience.)

Cancer research is increasingly focused on discovering strategies to induce cancer cell apoptosis without affecting surrounding normal cells. One potential biocompatible method is mechanical vibration, which has been developed as part of the emerging field of mechanomedicine. Previous studies of mechanical vibration have employed high-frequency vibration, which damages healthy cells. In this study, we examined the effects of brief (1 h) low-frequency (20 Hz) mechanical vibration on glucose consumption and survival (apoptosis, necrosis, HMGB1 release) of the human epidermoid carcinoma cell line A431. We found that apoptosis, but not necrosis, was significantly increased at 48 h after mechanical vibration compared with cells maintained in static culture. In keeping with this, extracellular release of HMGB1, a necrosis marker, was lower in cultures of A431 cells subjected to mechanical vibration compared with control cells. Glucose consumption was increased in the first 24 h after mechanical vibration but returned to control levels before the onset of apoptosis. Although the precise intracellular mechanisms by which low-frequency mechanical vibration triggers apoptosis of A431 cells is unknown, these results suggest a possible role for metabolic pathways. Mechanical vibration may thus represent a novel application of mechanomedicine to cancer therapy., Competing Interests: The authors have declared no conflict of interest., (© 2020 The Authors. Engineering in Life Sciences published by WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Golgi endo-α-mannosidase (G-EM) catalyzes an alternative deglucosylation process for N-glycans and plays important roles in the post-endoplasmic reticulum (ER) quality control pathway. To understand the post-ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G-EM based on a fluorescence quenching assay. The probe was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and a 2,4-dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G-EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G-EM activity to be evaluated easily and quantitatively., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We characterized an α-glucosidase belonging to the glycoside hydrolase family 31 from Aspergillus sojae. The α-glucosidase gene was cloned using the whole genome sequence of A. sojae , and the recombinant enzyme was expressed in Aspergillus nidulans . The enzyme was purified using affinity chromatography. The enzyme showed an optimum pH of 5.5 and was stable between pH 6.0 and 10.0. The optimum temperature was approximately 55 °C. The enzyme was stable up to 50 °C, but lost its activity at 70 °C. The enzyme acted on a broad range of maltooligosaccharides and isomaltooligosaccharides, soluble starch, and dextran, and released glucose from these substrates. When maltose was used as substrate, the enzyme catalyzed transglucosylation to produce oligosaccharides consisting of α-1,6-glucosidic linkages as the major products. The transglucosylation pattern with maltopentaose was also analyzed, indicating that the enzyme mainly produced oligosaccharides with molecular weights higher than that of maltopentaose and containing continuous α-1,6-glucosidic linkages. These results demonstrate that the enzyme is a novel α-glucosidase that acts on both maltooligosaccharides and isomaltooligosaccharides, and efficiently produces oligosaccharides containing continuous α-1,6-glucosidic linkages., Competing Interests: The authors declare no conflict of interests., (2019 by The Japanese Society of Applied Glycoscience.)

Cellular aggregates that mimic cell-cell interactions in vitro are essential for biological research. This study introduces a method to form large scaffold-free 3-D aggregates in a clinically ubiquitous cell culture dish using kilohertz-order ultrasound standing wave trapping (USWT). We fabricated an aggregate formation system in which a 60-mm dish was set above a Langevin transducer via water. The transducer was excited at 110.8 kHz, and then C2C12 myoblasts were injected into the dish and trapped at the node position of the standing wave. The diameter and thickness of the formed aggregate were 8 and 2.7 mm, respectively, which are larger than those of aggregates formed previously by USWT. Moreover, we confirmed that >94% of cells constituting the aggregates survived 9 h, and the protein expression of cells was not altered significantly. This method can be applied to form aggregates with high functionality, which contributes to the development of biological research methodology., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell patterning methods have been previously reported for cell culture. However, these methods use inclusions or devices that are not used in general cell culture and that might affect cell functionality. Here, we report a cell patterning method that can be conducted on a general cell culture dish without any inclusions by employing a resonance vibration of a disk-shaped ultrasonic transducer located under the dish. A resonance vibration with a single nodal circle patterned C2C12 myoblasts into a circular shape on the dish with 10-min exposure of the vibration with maximum peak-peak amplitude of 10 μm[Formula: see text]. Furthermore, the relationship between the amplitude distribution of the transducer and the cell density in the patterned sample could be expressed as a linear function, and there was a clear threshold of amplitude for cell adhesion. To evaluate the cell function of the patterned cells, we conducted proliferation and protein assays at 120-h culture after patterning. Our results showed that the cell proliferation rate did not decrease and the expression of cellular proteins was unchanged. Thus, we conclude, this method can successfully pattern cells in the clinically ubiquitous culture dish, while maintaining cell functionality.

The understanding of the basic physicochemical properties of trehalose lipid is indispensable to extending their availability. In this study, the hydrate crystal (Cr), the liquid crystalline (LC) phase and the glassy state formations of 6-O-acyl trehalose fatty acid monoester (TREn) were examined under in a desiccated system. TREn (n = 10, 12, 14, 16) formed monohydrate Cr and showed the hydrate Cr-glassy fluid lamellar LC (Lα) phase transition via dehydration in the heating process. Here, Lα phase for TRE10 and TRE12 was kinetically formed by the dehydration below the glass transition temperature (T g ). On the dehydration temperature (T deh ), T g s, and heat capacity changes (ΔC p s) at these T g s, no distinct effects by the difference of the acyl chain length were recognized, possibly because the core structure of containing sugar hydrate Cr or sugar moieties should be similar regardless of the acyl chain length. Besides, TRE10 having a relatively high hydrophilic/hydrophobic balance (HLB) afforded to form the cubic LC (Q) phase and the corresponding glassy phase, while TRE14 and TRE16 having low HLB afforded the Lα phase as well as the corresponding glassy phases above T g . TRE12 having middle HLB afforded both LCs and the corresponding glassy phase by controlling the kinetics of LC-LC phase transition between Lα and Q phases. Furthermore, the anomalous reversible phase transition during both the heating and cooling processes was also ascertained in the glassy phase for TRE16, which was considered the phase transition between glassy Lα and glassy lamellar gel (Lβ) phase. It greatly empathizes the two-dimensional trehalose glass layer and fluid hydrocarbon chains in the TREn glassy phase. Thus, in this study, it was demonstrated that TREn as the simplest trehalose lipid exhibited the glassy formation performance as well as the hydrate Cr formation, which showed less chain length dependence, together with the LC formation and the phase transition between glassy Lα and glassy Lβ phase, which depended on chain length greatly., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An unambiguous structural characterization of the water-soluble Aureobasidium pullulans β-(1→3, 1→6)-glucan is yet to be achieved, although this β-(1→3, 1→6)-glucan is expected to exhibit excellent biofunctional properties. Thus, we herein report the elucidation of the primary structure of the A. pullulans β-(1→3, 1→6)-glucan using nuclear magnetic resonance spectroscopy, followed by comparison of the obtained structure with that of schizophyllan (SPG). Structural characterization of the A. pullulans β-(1→3, 1→6)-glucan revealed that the structural units are a β-(1→3)-d-glucan backbone with four β-(1→6)-d-glucosyl side branching units every six residues. In addition, circular dichroism spectroscopic analysis revealed that the β-(1→3, 1→6)-glucan interacted with polyadenylic acid (poly(A)) chains in DMSO solution to form a complex similar to that obtained in the complexation of SPG/poly(A). This finding indicates that β-(1→3, 1→6)-glucan forms a triple-helical conformation in aqueous solution but exhibits a random coil structure in DMSO solution, which is similar to the behavior of SPG., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This article contains two-dimensional (2D) NMR experimental data, obtained by the Bruker BioSpin 500 MHz NMR spectrometer (Germany) which can used for the determination of primary structures of schizophyllan from Schizophyllum commune (SPG) and a water-soluble β-(1→3, 1→6)-glucan from Aureobasidium pullulans . Data include analyzed the 2D NMR spectra of these β-glucans, which are related to the subject of an article in Carbohydrate Polymers , entitled "NMR spectroscopic structural characterization of a water-soluble β-(1→3, 1→6)-glucan from A. pullulans " (Kono et al., 2017) [1]. Data can help to assign the 1 H and 13 C chemical shifts of the structurally complex polysaccharides.

Cell detachment is an essential process in adherent cell culture. However, trypsinization, which is the most popular detachment technique used in culture, damages cellular membranes. Reducing cellular membrane damage during detachment should improve the quality of cell culture. In this article, we propose an enzyme-free cell detachment method based on resonance vibration with temperature modulation. We developed a culture device that can excite a resonance vibration and control temperature. We then evaluated the cell detachment ratio and the growth response, observed the morphology, and analyzed the cellular protein of the collected cells-mouse myoblast cell line (C2C12). With the temperature of 10°C and the maximum vibration amplitude of 2 μm, 77.9% of cells in number were successfully detached compared with traditional trypsinization. The 72-h proliferation ratio of the reseeded cells was similar to that with trypsinization, whereas the proliferation ratio of proposed method was 12.6% greater than that of trypsinization after freezing and thawing. Moreover, the cells can be collected relatively intact and both intracellular and cell surface proteins in the proposed method were less damaged than in trypsinization. These results show that this method has definite advantages over trypsinization, which indicates that it could be applied to subcultures of cells that are more susceptible to trypsin damage for mass culture of sustainable clinical use. Biotechnol. Bioeng. 2017;114: 2279-2288. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

We have investigated whether acidity can be used to control the physicochemical properties of chitin nanofibers (ChNFs). In this study, we define acidity as the molar ratio of dissociated protons from the acid to the amino groups in the raw chitin powder. The effect of acidity on the physicochemical properties of α- and β-ChNFs was compared. The transmittance and viscosity of the β-ChNFs drastically and continuously increased with increasing acidity, while those of the α-ChNFs were not affected by acidity. These differences are because of the higher ability for cationization based on the more flexible crystal structure of β-chitin than α-chitin. In addition, the effect of the acid species on the transmittance of β-ChNFs was investigated. The transmittance of β-ChNFs can be expressed by the acidity regardless of the acid species, such as hydrochloric acid, phosphoric acid, and acetic acid. These results indicate that the acidity defined in this work is an effective parameter to define and control the physicochemical properties of ChNFs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo-α-mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo-α-mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α-glycosyl-fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1- 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high-mannose-type undecasaccharide (Glc 1 Man 9 GlcNAc 2 )., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)