Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons.

The proteasome is essential for cellular responses to various physiological stressors. However, how proteasome function impacts the stress resilience of regenerative damaged motor neurons remains unclear. Here, we develop a unique mouse model using a regulatory element of the activating transcription factor (Atf3) gene to label mitochondria in a damage-induced manner while simultaneously genetically disrupting the proteasome. Using this model, we observed that in injury-induced proteasome-deficient mouse motor neurons, the increase of mitochondrial influx from soma into axons is inhibited because neurons fail to disassemble ankyrin G, an organizer of the axon initial segment (AIS), in a proteasome-dependent manner. Further, these motor neurons exhibit amyotrophic lateral sclerosis (ALS)-like degeneration despite having regenerative potential. Selectively vulnerable motor neurons in SOD1G93A ALS mice, which induce ATF3 in response to pathological damage, also fail to disrupt the AIS, limiting the number of axonal mitochondria at a pre-symptomatic stage. Thus, damage-induced proteasome-sensitive AIS disassembly could be a critical post-translational response for damaged motor neurons to temporarily transit to an immature state and meet energy demands for axon regeneration or preservation.

Pituitary adenylate cyclase-activating polypeptide deficient mice show length abnormalities of the axon initial segment.

We previously found that pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP-/-) mice exhibit dendritic spine morphology impairment and neurodevelopmental disorder (NDD)-like behaviors such as hyperactivity, increased novelty-seeking behavior, and deficient pre-pulse inhibition. Recent studies have indicated that rodent models of NDDs (e.g., attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder) show abnormalities in the axon initial segment (AIS). Here, we revealed that PACAP-/- mice exhibited a longer AIS length in layer 2/3 pyramidal neurons of the primary somatosensory barrel field compared with wild-type control mice. Further, we previously showed that a single injection of atomoxetine, an ADHD drug, improved hyperactivity in PACAP-/- mice. In this study, we found that repeated treatments of atomoxetine significantly improved AIS abnormality along with hyperactivity in PACAP-/- mice. These results suggest that AIS abnormalities are associated with NDDs-like behaviors in PACAP-/- mice. Thus, improvement in AIS abnormalities will be a novel drug therapy for NDDs.

Recombinant lectin Gg for brain imaging of glycan structure and formation in the CNS node of Ranvier.

The nodes of Ranvier are unmyelinated gaps in the axon, important for the efficient transmission of action potentials. Despite the identification of several glycoproteins involved in node formation and maintenance, glycans' structure and formation in the node remain unclear. Previously, we developed a recombinant lectin from the Clostridium botulinum neurotoxin complex, specific to the galactose and N-acetylgalactosamine terminal epitopes (Gg). Gg stained Neuro2a cells. Here, we show Gg punctuate staining in mouse brain cryosections. Thus, we hypothesized that Gg could help study glycans in the node of Ranvier. Lectin histochemistry on mouse brain cryosections confirmed that Gg binds specifically to the node of Ranvier in the central nervous system (CNS). Using a combination of lectin blotting, glycosidase treatment on tissue sections, and lectin histochemistry, Gg ligands were identified as α-galactose terminal glycoproteins in the perinodal extracellular matrix. Furthermore, we detected the spatiotemporal distribution of galactosylated glycans in the CNS node of Ranvier in mouse brain tissues at different postnatal times. Finally, we observed impaired clustering of galactosylated glycans in the nodes during demyelination and remyelination in cuprizone-induced demyelination and remyelination mouse model. In conclusion, Gg can serve as a novel brain imaging tool in glycobiology and report glycoprotein formation and alterations in the CNS node of Ranvier. Our findings might serve as a first step to establish the role of glycans in the node of Ranvier.

TMEM67 is required for the gating function of the transition zone that controls entry of membrane-associated proteins ARL13B and INPP5E into primary cilia.

Primary cilia transduce signals via transmembrane and membrane-associated proteins localized to the ciliary membrane in vertebrate cells. In humans, transmembrane protein 67 (TMEM67), a component of the multiprotein complex functioning as a gatekeeper at the transition zone (TZ) of primary cilia, is mutated in patients suffering from cilia-related pleiotropic diseases, collectively referred to as ciliopathies. The requirement of TMEM67 for the gating function of the TZ that delivers membrane proteins into the ciliary compartment has not been determined. In this study, we established hTERT-RPE1 cells with knockout (KO) of TMEM67 and examined whether cilium formation and TZ gating are affected by its ablation. TMEM67-KO cells displayed impaired ciliogenesis, elongated cilia, perturbed ciliary localization of membrane-associated proteins ARL13B and INPP5E but normal recruitment of TZ proteins CEP290, RPGRIP1L and NPHP5. The exogenous expression of ciliopathy-associated TMEM67 mutants restored ciliary localization of ARL13B and INPP5E but failed to attenuate aberrant cilium elongation in TMEM67-KO cells. Furthermore, we found that TMEM67 localization is not confined to the TZ but extends into the cilium. Our findings indicate that TMEM67 is required not only for ciliogenesis and cilium length regulation but also for the gating function of the TZ independently of RPGRIP1L/CEP290/NPHP5 recruitment to this region. They further suggest that aberrant cilium elongation underlies the pathogenesis of TMEM67-linked ciliopathies.

Strong Photoluminescence Enhancement in Molybdenum Disulfide in Aqueous Media.

The interface between conventional semiconductors and aqueous ionic solutions is an important target in chemistry and materials science. Recently, a wide variety of research has been done on transition-metal dichalcogenides (TMDCs) for use as 2D layered semiconductors, and their optoelectronic properties have been widely explored. One representative TMDC, monolayer (1L) MoS2, is known to show a photoluminescence (PL) signal of a direct band gap nature, and the PL intensity is dependent on the carrier concentration. Various methods of 1L MoS2 carrier modulation have been shown to enhance the PL intensity in dry environments. In contrast, enhancement in an aqueous environment is limited, and a strategy to design an interface with aqueous media has not yet been established. One proposed idea was an aqueous acid interface; however, the enhancement of the PL with this method was usually minimal, about 1 order of magnitude. In this study, we demonstrate a method to achieve strong PL enhancement in 1L MoS2 in an aqueous media by incorporating bis(trifluoromethane)sulfonyl anion (TFSI- ion) in an acidic environment. With the addition of the TFSI- ion in an acidic environment, the enhancement factor of the PL in 1L MoS2 is more than 100 times greater than its PL intensity in water. The molecular anion is the key factor, as the TFSI- ion facilitates the oxidation of MoS2. This anionic effect is the additional factor needed to modulate the optoelectronic properties of 2D semiconductors in aqueous media. The proposed idea could have potential applications for biochemical sensors in aqueous situations.

Twice-weekly teriparatide improves lumbar spine BMD independent of pre-treatment BMD and bone turnover marker levels.

INTRODUCTION: There have been no reports of the effects of baseline lumbar spine bone mineral density (LS-BMD) and bone turnover marker levels on the therapeutic effect of a 28.2-µg teriparatide formulation for twice-weekly use (2/W-TPTD). MATERIALS AND METHODS: An analysis was performed using data from a double-blind, randomized, non-inferiority trial (TWICE study) conducted with patients who received 2/W-TPTD or a 56.5-µg teriparatide formulation for once-weekly use (1/W-TPTD) for 48 weeks. The patients were divided into tertile groups based on baseline LS-BMD, urinary type I collagen cross-linked N-telopeptide (u-NTX), and serum type I procollagen-N-propeptide (P1NP) levels, respectively. Time profiles of these measurements were analyzed. Furthermore, whether a change in P1NP is a predictor for percentage change in BMD was assessed. RESULTS: Across all tertile groups divided based on baseline LS-BMD and levels of bone turnover markers, the LS-BMD increased significantly. The u-NTX level decreased throughout the study period in the high- and middle-u-NTX-level groups. The P1NP level increased after 4 weeks, but subsequently decreased after 12 weeks and thereafter in the high-P1NP-level group; it increased after 4 weeks and subsequently fluctuated near the baseline level in the middle-P1NP-level group. A cut-off value of 12.0 µg/L for change in P1NP after 4 weeks of 2/W-TPTD as a predictor for percentage change in LS-BMD of 3% or more after 48 weeks gave a positive predictive value of 89.6%. CONCLUSION: 2/W-TPTD, just like 1/W-TPTD, improved LS-BMD significantly, regardless of baseline LS-BMD and bone turnover marker levels.

Single-layered assembly of vanadium pentoxide nanowires on graphene for nanowire-based lithography technique.

Graphene nanoribbon (GNR)-based materials are a promising device material because of their potential high carrier mobility and atomically thin structure. Various approaches have been reported for preparing the GNR-based materials, from bottom-up chemical synthetic procedures to top-down fabrication techniques using lithography of graphene. However, it is still difficult to prepare a large-scale GNR-based material. Here, we develop a procedure to prepare a large-scale GNR network using networked single-layer inorganic nanowires. Vanadium pentoxide (V2O5) nanowires were assembled on graphene with an interfacial layer of a cationic polymer via electrostatic interaction. A large-scale nanowire network can be prepared on graphene and is stable enough for applying an oxygen plasma. Using plasma etching, a networked graphene structure can be generated. Removing the nanowires results in a networked flat structure whose both surface morphology and Raman spectrum indicate a GNR networked structure. The field-effect device indicates the semiconducting character of the GNR networked structure. This work would be useful for fabricating a large-scale GNR-based material as a platform for GNR junctions for physics and electronic circuits.

A Potential Serum N-glycan Biomarker for Hepatitis C Virus-Related Early-Stage Hepatocellular Carcinoma with Liver Cirrhosis.

Detection of early-stage hepatocellular carcinoma (HCC) is beneficial for prolonging patient survival. However, the serum markers currently used show limited ability to identify early-stage HCC. In this study, we explored human serum N-glycans as sensitive markers to diagnose HCC in patients with cirrhosis. Using a simplified fluorescence-labeled N-glycan preparation method, we examined non-sialylated and sialylated N-glycan profiles from 71 healthy controls and 111 patients with hepatitis and/or liver cirrhosis (LC) with or without HCC. We found that the level of serum N-glycan A2G1(6)FB, a biantennary N-glycan containing core fucose and bisecting GlcNAc residues, was significantly higher in hepatitis C virus (HCV)-infected cirrhotic patients with HCC than in those without HCC. In addition, A2G1(6)FB was detectable in HCV-infected patients with early-stage HCC and could be a more accurate marker than alpha-fetoprotein (AFP) or protein induced by vitamin K absence or antagonists-II (PIVKA-II). Moreover, there was no apparent correlation between the levels of A2G1(6)FB and those of AFP or PIVKA-II. Thus, simultaneous use of A2G1(6)FB and traditional biomarkers could improve the accuracy of HCC diagnosis in HCV-infected patients with LC, suggesting that A2G1(6)FB may be a reliable biomarker for early-stage HCC patients.

TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia.

The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.

Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells.

BACKGROUND: Oligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell-cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma. METHODS: Here, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens. RESULTS: We first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated. DISCUSSION: This is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma, CONCLUSION: These results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.

Sunlight caused interference in outdoor N, N-diethyl-p-phenylenediamine colorimetric measurement for residual chlorine and the solution for on-site work.

Chlorination is the most common method to control water qualities, in some case on-site outdoor measurements are required to measure easily-decaying residual chlorine concentration appropriately without delay. In this study sunlight-induced unexpected colour development (UCD) of N, N-diethyl-p-phenylenediamine (DPD) colorimetric measurement was studied under several sun exposure conditions. The colour development level was evaluated with reference to chlorine concentration (mg/L) and relationships between colour development rate (mg/L min) and intensities of solar were investigated. UCD was found to be related to both exposure intensity and time. By means of exposure experiment under specific wavelength of ultraviolet (UV), it was confirmed that both middle and short wavelength of UV radiation being responsible for such an unexpected measurement. Consequently, a simple device was designed using three commercially available anti-UV films, one of which could effectively prevent the UCD from direct sun exposure.

Branched Sialylated N-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H.

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galß1-3GlcNAcß1-2Manα1-6[Galß1-3GlcNAcß1-2Manα1-3]Manß1-4GlcNAcß1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H.

Systematic Study of Photoluminescence Enhancement in Monolayer Molybdenum Disulfide by Acid Treatment.

Monolayer molybdenum disulfide (MoS2) is an atomically thin semiconducting material with a direct band gap. This physical property is attributable to atomically thin optical devices such as sensors, light-emitting devices, and photovoltaic cells. Recently, a near-unity photoluminescence (PL) quantum yield of a monolayer MoS2 was demonstrated via a treatment with a molecular acid, bis(trifluoromethane)sulfonimide (TFSI); however, the mechanism still remains a mystery. Here, we work on PL enhancement of monolayer MoS2 by treatment of Brønsted acids (TFSI and sulfuric acid (H2SO4)) to identify the importance of the protonated environment. In TFSI as an acid, different solvents-1,2-dichloroethane (DCE), acetonitrile, and water-were studied, as they show quite different acidity in solution. All of the solvents showed PL enhancement, and the highest was observed in DCE. This behavior in DCE would be due to the higher acidity than others have. Acids from different anions can also be studied in water as a common solvent. Both TFSI and H2SO4 showed similar PL enhancement (∼4-8 enhancement) at the same proton concentration, indicating that the proton is a key factor to enhance the PL intensity. Finally, we considered another cation, Li+ from Li2SO4, instead of H2SO4, in water. Although Li and H atoms showed similar binding energy on MoS2 from theoretical calculations, Li2SO4 treatment showed little PL enhancement; only coexisting H2SO4 reproduced the enhancement. This study demonstrated the importance of a protonated environment to increase the PL intensity of monolayer MoS2. The study will lead to a solution to achieve high optical quality and to implementation for atomically thin optical devices.

Astrocyte-mediated infantile-onset leukoencephalopathy mouse model.

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile-onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte-specific Mlc1 overexpression causes infantile-onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na+ /K+ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na+ /K+ pump. In contrast, no changes in Na+ /K+ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na+ /K+ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile-onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile-onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150-168.

Sulfatase 2 Modulates Fate Change from Motor Neurons to Oligodendrocyte Precursor Cells through Coordinated Regulation of Shh Signaling with Sulfatase 1.

Sulfatases (Sulfs) are a group of endosulfatases consisting of Sulf1 and Sulf2, which specifically remove sulfate from heparan sulfate proteoglycans. Although several studies have shown that Sulf1 acts as a regulator of sonic hedgehog (Shh) signaling during embryonic ventral spinal cord development, the detailed expression pattern and function of Sulf2 in the spinal cord remains to be determined. In this study, we found that Sulf2 also modulates the cell fate change from motor neurons (MNs) to oligodendrocyte precursor cells (OPCs) by regulating Shh signaling in the mouse ventral spinal cord in coordination with Sulf1. In the mouse, Sulf mRNAs colocalize with Shh mRNA and gradually expand dorsally from embryonic day (E) 10.5 to E12.5, following strong Patched1 signals (a target gene of Shh signaling). This coordinated expression pattern led us to hypothesize that in the mouse, strong Shh signaling is induced when Shh is released by Sulf1/2, and this strong Shh signaling subsequently induces the dorsal expansion of Shh and Sulf1/2 expression. Consistent with this hypothesis, in the ventral spinal cord of Sulf1 knockout (KO) or Sulf2 KO mice, the expression patterns of Shh and Patched1 differed from that in wild-type mice. Moreover, the position of the pMN and p3 domains were shifted ventrally, MN generation was prolonged, and OPC generation was delayed at E12.5 in both Sulf1 KO and Sulf2 KO mice. These results demonstrated that in addition to Sulf1, Sulf2 also plays an important and overlapping role in the MN-to-OPC fate change by regulating Shh signaling in the ventral spinal cord. However, neither Sulf1 nor Sulf2 could compensate for the loss of the other in the developing mouse spinal cord. In vitro studies showed no evidence of an interaction between Sulf1 and Sulf2 that could increase sulfatase activity. Furthermore, Sulf1/2 double heterozygote and Sulf1/2 double KO mice exhibited phenotypes similar to the Sulf1 KO and Sulf2 KO mice. These results indicate that there is a threshold for sulfatase activity (which is likely reflected in the dose of Shh) required to induce the MN-to-OPC fate change, and Shh signaling requires the coordinated activity of Sulf1 and Sulf2 in order to reach that threshold in the mouse ventral spinal cord.

Keratan Sulfate Regulates the Switch from Motor Neuron to Oligodendrocyte Generation During Development of the Mouse Spinal Cord.

Keratan sulfate (KS) is a sulfated glycosaminoglycan and has been shown to bind to sonic hedgehog (Shh), which act as a morphogen to regulate the embryonic spinal cord development. We found highly sulfated KS was present in the floor plate (including lateral floor plate) and the notochord . This expression colocalized with Shh expression. To understand the roles of KS, we analyzed the embryonic spinal cord of GlcNAc6ST-1, KS chain synthesizing enzyme, knock-out (KO) mice. At E12.5, the pMN domain, whose formation is controlled by Shh signaling, became shifted ventrally in GlcNAc6ST-1 KO mice. In addition, the expression patterns of Patched1 and Gli1, two Shh signaling reporter genes, differed between wild type (WT) and GlcNAc6ST-1 KO mice at E12.5. Next, we focused on cell types generated from the pMN domain; namely, motor neurons and subsequently oligodendrocytes. The number of PDGFRα(+) [a marker for oligodendrocyte precursor cells (OPCs)] cells was low in the E12.5 mutant spinal cord, while motor neuron production was increased. Thus the switch from motor neuron generation to OPC generation was delayed in the pMN domain. Furthermore, we investigated the cause for this delayed switch in the pMN domain. The number of Olig2, Nkx2.2 double-positive cells was less in GlcNAc6ST-1 KO mice than in WT mice. In contrast, the number of Olig2, Neurogenin2 (Ngn2) double-positive cells related to the motor neuron specification was significantly greater in the KO mice. These results indicate that KS is important for the late phase Shh signaling and contributes to motor neuron to OPC generation switch.

The Lewis X-related α1,3-fucosyltransferase, Fut10, is required for the maintenance of stem cell populations.

Lewis X (Le(X), Galß1-4(Fucα1-3)GlcNAc) is a carbohydrate epitope that is present at the nonreducing terminus of sugar chains of glycoproteins and glycolipids, and is abundantly expressed in several stem cell populations. Le(X) antigen can be used in conjunction with fluorescence-activated cell sorting to isolate neurosphere-forming neural stem cells (NSCs) from embryonic mouse brains. However, its function in the maintenance and differentiation of stem cells remains largely unknown. In this study, we examined mice deficient for fucosyltransferase 9 (Fut9), which is thought to synthesize most, if not all, of the Le(X) moieties in the brain. We found that the number of NSCs was increased in the brain of Fut9(-/-) embryos, suggesting that Fut9-synthesized Le(X) is dispensable for the maintenance of NSCs. Another α1,3-fucosyltransferase gene, fucosyltransferase 10 (Fut10), is expressed in the ventricular zone of the embryonic brain. Overexpression of Fut10 enhanced the self-renewal of NSCs. Conversely, suppression of Fut10 expression induced the differentiation of NSCs and embryonic stem cells. In addition, knockdown of Fut10 expression in the cortical ventricular zone of the embryonic brain by in utero electroporation of Fut10-miRNAs impaired the radial migration of neural precursor cells. Our data suggest that Fut10 is involved in a unique α1,3-fucosyltransferase activity with stringent substrate specificity, and that this activity is required to maintain stem cells in an undifferentiated state.

Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.

Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex.

Oligosaccharides of glycoproteins expressed on the cell surface play important roles in cell-cell interactions, particularly sialylated N-glycans having a negative charge, which interact with sialic acid-binding immunoglobulin-like lectins (siglecs). The entire structure of sialylated N-glycans expressed in the mouse brain, particularly the linkage type of sialic acid residues attached to the backbone N-glycans, has not yet been elucidated. An improved method to analyze pyridylaminated sugar chains using high performance liquid chromatography (HPLC) was developed to determine the entire structure of sialylated N-linked sugar chains expressed in the adult and developing mouse cerebral cortices. Three classes of sialylated sugar chains were prevalent: 1) N-glycans containing α(2-3)-sialyl linkages on a type 2 antennary (Galß(1-4)GlcNAc), 2) sialylated N-glycans with α(2-6)-sialyl linkages on a type 2 antennary, and 3) a branched sialylated N-glycan with a [Galß(1-3){NeuAcα(2-6)}GlcNAc-] structure, which was absent at embryonic day 12 but then increased during development. This branched type sialylated N-glycan structure comprised approximately 2 % of the total N-glycans in the adult brain. Some N-glycans (containing type 2 antennary) were found to change their type of sialic acid linkage from α(2-6)-Gal to α(2-3)-Gal. Thus, the linkages and expression levels of sialylated N-glycans change dramatically during brain development.

Selective Isolation of Mono- to Quadlayered 2D Materials via Sonication-Assisted Micromechanical Exfoliation.

Mechanical exfoliation methods of two-dimensional materials have been an essential process for advanced devices and fundamental sciences. However, the exfoliation method usually generates various thick flakes, and a bunch of thick bulk flakes usually covers an entire substrate. Here, we developed a method to selectively isolate mono- to quadlayers of transition metal dichalcogenides (TMDCs) by sonication in organic solvents. The analysis reveals the importance of low interface energies between solvents and TMDCs, leading to the effective removal of bulk flakes under sonication. Importantly, a monolayer adjacent to bulk flakes shows cleavage at the interface, and the monolayer can be selectively isolated on the substrate. This approach can extend to preparing a monolayer device with crowded 17 electrode fingers surrounding the monolayer and for the measurement of electrostatic device performance.