Polypropylene-Rendered Antiviral by Three-Dimensionally Surface-Grafted Poly(N-benzyl-4-vinylpyridinium bromide).

To inhibit viral infection, it is necessary for the surface of polypropylene (PP), a polymer of significant industrial relevance, to possess biocidal properties. However, due to its low surface energy, PP weakly interacts with other organic molecules. The biocidal effects of quaternary ammonium compounds (QACs) have inspired the development of nonwoven PP fibers with surface-bound quaternary ammonium (QA). Despite this advancement, there is limited knowledge regarding the durability of these coatings against scratching and abrasion. It is hypothesized that the durability could be improved if the thickness of the coating layer were controlled and increased. We herein functionalized PP with three-dimensionally surface-grafted poly(N-benzyl-4-vinylpyridinium bromide) (PBVP) by a simple and rapid method involving graft polymerization and benzylation and examined the influence of different factors on the antiviral effect of the resulting plastic by using a plaque assay. The thickness of the PBVP coating, surface roughness, and amount of QACs, which jointly determine biocidal activity, could be controlled by adjusting the duration and intensity of the ultraviolet irradiation used for grafting. The best-performing sample reduced the viral infection titer of an enveloped model virus (bacteriophage ϕ6) by approximately 5 orders of magnitude after 60 min of contact and retained its antiviral activity after surface polishing-simulated scratching and abrasion, which indicated the localization of QACs across the coating interior. Our method may expand the scope of application to resin plates as well as fibers of PP. Given that the developed approach is not limited to PP and may be applied to other low-surface-energy olefinic polymers such as polyethylene and polybutene, our work paves the way for the fabrication of a wide range of biocidal surfaces for use in diverse environments, helping to prevent viral infection.

The origins of binding specificity of a lanthanide ion binding peptide.

Lanthanide ions (Ln3+) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln3+, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln3+. Our results clearly show that LBT3-Ln3+ binding can be divided into three, and the large affinity difference is based on the ability of Ln3+ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln3+ with a larger ionic radius (La3+ to Sm3+), a water molecule can interact with Ln3+ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln3+, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln3+ (Sm3+ to Yb3+), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln3+, namely Lu3+, although it completely surrounds Lu3+, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb3+. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

Ordered silica mineralization by regulating local reaction conditions.

Using cationic peptides with tetramethyl orthosilicate, a silica nano-film >100 µm in size with <100 nm thickness was constructed under physiological conditions. Control of silica nucleation speed and location was found to be the dominant factor affecting the ordered architecture. Our approach adds new insight into bottom-up nanomaterial construction and contributes to evaluating the silica mineralization system in living organisms.

Expression of the human UDP-galactose transporter gene hUGT1 in tobacco plants' enhanced plant hardness.

We reported previously that tobacco plants transformed with the human UDP-galactose transporter 1 gene (hUGT1) had enhanced growth, displayed characteristic traits, and had an increased proportion of galactose (hyper-galactosylation) in the cell wall matrix polysaccharides. Here, we report that hUGT1-transgenic plants have an enhanced hardness. As determined by breaking and bending tests, the leaves and stems of hUGT1-transgenic plants were harder than those of control plants. Transmission electron microscopy revealed that the cell walls of palisade cells in leaves, and those of cortex cells and xylem fibers in stems of hUGT1-transgenic plants, were thicker than those of control plants. The increased amounts of total cell wall materials extracted from the leaves and stems of hUGT1-transgenic plants supported the increased cell wall thickness. In addition, the cell walls of the hUGT1-transgenic plants showed an increased lignin contents, which was supported by the up-regulation of lignin biosynthetic genes. Thus, the heterologous expression of hUGT1 enhanced the accumulation of cell wall materials, which was accompanied by the increased lignin content, resulting in the increased hardness of the leaves and stems of hUGT1-trangenic plants. The enhanced accumulation of cell wall materials might be related to the hyper-galactosylation of cell wall matrix polysaccharides, most notably arabinogalactan, because of the enhanced UDP-galactose transport from the cytosol to the Golgi apparatus by hUGT1, as suggested in our previous report.

UDP-galactose transporter gene hUGT1 expression in tobacco plants leads to hyper-galactosylated cell wall components.

We reported previously that tobacco plants transformed with the human UDP-galactose transporter 1 gene (hUGT1-transgenic plants) displayed morphological, architectural, and physiological alterations, such as enhanced growth, increased accumulation of chlorophyll and lignin, and a gibberellin-responsive phenotype. In the present study, we demonstrated that hUGT1 expression altered the monosaccharide composition of cell wall matrix polysaccharides, such as pectic and hemicellulosic polysaccharides, which are biosynthesized in the Golgi lumen. An analysis of the monosaccharide composition of the cell wall matrix polysaccharides revealed that the ratio of galactose to total monosaccharides was significantly elevated in the hemicellulose II and pectin fractions of hUGT1-transgenic plants compared with that of control plants. A hyper-galactosylated xyloglucan structure was detected in hemicellulose II using oligosaccharide mass profiling. These results indicated that, because of the enhanced UDP-galactose transport from the cytosol to the Golgi apparatus by hUGT1, galactose incorporation in the cell wall matrix polysaccharides increased. This increased galactose incorporation may have contributed to increased galactose tolerance in hUGT1-transgenic plants.

Investigation of utilization of the algal biomass residue after oil extraction to lower the total production cost of biodiesel.

In this study, component analysis of a novel biodiesel-producing alga, Pseudochoricystis ellipsoidea, was performed. The component analysis results indicated that proteins and amino acids are abundant in P. ellipsoidea while the sugar content is relatively low. Rather than its use as a carbon source, the use of the algal biomass residue after oil extraction as a nutrient source provided a new way for lowering the total production cost of biodiesel. In both lactic acid and ethanol fermentations, the use of the residue instead of high-cost nutrient yeast extract allowed a significant saving, showing the promise of the algal biomass residue for use as a fermentation nutrient source.

Ionic liquid/water interfacial localization of a green fluorescent protein fused to a tryptophan-rich peptide.

We report that several tryptophan-rich peptides exhibit an affinity for a hydrophobic ionic liquid (IL) (1-ethyl-3-methylimidazolium bis-trifluoromethanesulfonyl imide), and that green fluorescent protein (GFP) fused to a peptides, "SSSWWSWWWW" (SW1) or "SWWWWSWWWW" (SW2), containing serine (S) and tryptophan (W) at the C terminus localized at the IL/water interface. While GFPs without W-rich peptide distributed only in water phase, SW1- and SW2-GFPs were accumulated at the interface. The localization of SW1-GFP showed biphasic behavior, and most distinctive localization was observed at 7.1 µM. The localization of SW2-GFP presumably occurred at largely lower concentration (≤0.5 µM) than that of SW1-GFP, which difference was due to the higher hydrophobicity of SW2 peptide.

The impact of the overexpression of human UDP-galactose transporter gene hUGT1 in tobacco plants.

When the human UDP-galactose transporter 1 gene (hUGT1) was introduced into tobacco plants, the plants displayed enhanced growth during cultivation, and axillary shoots had an altered determinate growth habit, elongating beyond the primary shoots and having a sympodial growth pattern similar to that observed in tomatoes at a late cultivation stage. The architecture and properties of tissues in hUGT1-transgenic plants were also altered. The leaves had an increase in thickness, due to an increased amount of spongy tissue, and a higher content of chlorophyll a and b; the stems had an increased number of xylem vessels and accumulated lignin and arabinogalactan proteins (AGPs). Some of these characteristics resembled a gibberellin (GA)-responsive phenotype, suggesting involvement of GA. RT-PCR-based analysis of genes involved in GA biosynthesis suggested that the GA biosynthetic pathway was not activated. However, an increase in the proportion of galactose in polysaccharide side chains of AGPs was detected. These results suggested that because of higher UDP-galactose transport from the cytosol to the Golgi apparatus, galactose incorporation into polysaccharide side chains of AGP is involved in the gibberellin response, resulting in morphological and architectural changes.

Analysis of CMP-sialic acid transporter-like proteins in plants.

It is commonly accepted that sialic acids do not exist in plants. However, putative gene homologs of animal sialyltransferases and CMP-sialic acid transporters have been detected in the genomes of some plants. To elucidate the physiological functions of these genes, we cloned 2 cDNAs from Oryza sativa (Japanese rice), each of which encodes a CMP-sialic acid transporter-like protein designated as OsCSTLP1 and OsCSTLP2. To examine the CMP-sialic acid transporter activity of OsCSTLP1 and OsCSTLP2, we introduced their expression vectors into CMP-sialic acid transporter activity-deficient Lec2 cells. Transfection with OsCSTLP1 resulted in recovery of the deficit phenotype of Lec2 cells, but transfection with OsCSTLP2 did not. We also performed an in vitro nucleotide sugar transport assay using a yeast expression system. Among the nucleotide sugars examined, the OsCSTLP1-containing yeast microsomal membrane vesicles specifically incorporated CMP-sialic acid, indicating that OsCSTLP1 has CMP-sialic acid transporter activity. On the other hand, OsCSTLP2 did not exhibit any nucleotide sugar transporter activity. T-DNA insertion lines of Arabidopsis thaliana targeting the homologs of the OsCSTLP1 and OsCSTLP2 genes exhibited a lethal phenotype, suggesting that these proteins play important roles in plant development and may transport important nucleotide sugars such as CMP-Kdo in physiological conditions.

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human.

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

Notch deficiency implicated in the pathogenesis of congenital disorder of glycosylation IIc.

Congenital disorder of glycosylation IIc (CDG IIc), also termed leukocyte adhesion deficiency II, is a recessive syndrome characterized by slowed growth, mental retardation, and severe immunodeficiency. Recently, the gene responsible for CDG IIc was found to encode a GDP-fucose transporter. Here, we investigated the possible cause of the developmental defects in CDG IIc patients by using a Drosophila model. Biochemically, we demonstrated that a Drosophila homolog of the GDP-fucose transporter, the Golgi GDP-fucose transporter (Gfr), specifically transports GDP-fucose in vitro. To understand the function of the Gfr gene, we generated null mutants of Gfr in Drosophila. The phenotypes of the Drosophila Gfr mutants were rescued by the human GDP-fucose transporter transgene. Our phenotype analyses revealed that Notch (N) signaling was deficient in these Gfr mutants. GDP-fucose is known to be essential for the fucosylation of N-linked glycans and for O-fucosylation, and both fucose modifications are present on N. Our results suggest that Gfr is involved in the fucosylation of N-linked glycans on N and its O-fucosylation, as well as those of bulk proteins. However, despite the essential role of N O-fucosylation during development, the Gfr homozygote was viable. Thus, our results also indicate that the Drosophila genome encodes at least another GDP-fucose transporter that is involved in the O-fucosylation of N. Finally, we found that mammalian Gfr is required for N signaling in mammalian cultured cells. Therefore, our results implicate reduced N signaling in the pathology of CDG IIc.

Hypoxia induces adhesion molecules on cancer cells: A missing link between Warburg effect and induction of selectin-ligand carbohydrates.

Cancer cells undergo distinct metabolic changes to cope with their hypoxic environment. These changes are achieved at least partly by the action of transcriptional factors called hypoxia-inducible factors (HIFs). We investigated gene expression in cultured human colon cancer cells induced by hypoxic conditions with special reference to cell-adhesion molecules and carbohydrate determinants having cell-adhesive activity by using DNA-microarray and RT-PCR techniques. Hypoxic culture of colon cancer cells induced a marked increase in expression of selectin ligands, the sialyl Lewis x and sialyl Lewis a determinants at the cell surface, which led to a definite increase in cancer cell adhesion to endothelial E-selectin. The transcription of genes for fucosyltransferase VII (FUT7), sialyltransferase ST3Gal-I (ST3O), and UDP-galactose transporter-1 (UGT1), which are all known to be involved in the synthesis of the carbohydrate ligands for E-selectin, was significantly induced in cancer cells by hypoxic culture. In addition, a remarkable induction was detected in the genes for syndecan-4 (SDC4) and alpha5-integrin (ITGA5), the cell-adhesion molecules involved in the enhanced adhesion of cancer cells to fibronectin. The transcriptional induction by hypoxia was reproduced in the luciferase-reporter assays for these genes, which were significantly suppressed by the co-transfection of a dominant-negative form of HIF. These results indicate that the metabolic shifts of cancer cells partly mediated by HIFs significantly enhance their adhesion to vascular endothelial cells, through both selectin- and integrin-mediated pathways, and suggest that this enhancement further facilitates hematogenous metastasis of cancers and tumor angiogenesis.

Molecular physiology and pathology of the nucleotide sugar transporter family (SLC35).

The solute carrier family SLC35 consists of at least 17 molecular species in humans. The family members so far characterized encode nucleotide sugar transporters localizing at the Golgi apparatus and/or the endoplasmic reticulum (ER). These transporters transport nucleotide sugars pooled in the cytosol into the lumen of these organelles, where most glycoconjugate synthesis occurs. Pathological analyses and developmental studies of small, multicellular organisms deficient in nucleotide sugar transporters have shown these transporters to be involved in tumour metastasis, cellular immunity, organogenesis and morphogenesis. Leukocyte adhesion deficiency type II (LAD II) or the congenital disorder of glycosylation type IIc (CDG IIc) are the sole human congenital disorders known to date that are caused by a defect of GDP-fucose transport. Along with LAD II, the possible involvement of nucleotide sugar transporters in disorders of connective tissues and muscles is also discussed.