Broad-Spectrum Detection of H5 Subtype Influenza A Viruses with a New Fluorescent Immunochromatography System

Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 10³ pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10–100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans.     

Vacuole-localized berberine bridge enzyme-like proteins are required for a late step of nicotine biosynthesis in tobacco

Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots and accumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signaling activates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. The biosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with the N-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, we report that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family (BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobacco hairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of green fluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involved in a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.     

Aligning cells in arbitrary directions on a membrane sheet using locally formed microwrinkles

Sheets of cells can be used for tissue regenerative medicine. Cell alignment within the sheet is now a key factor in the next generation of this technology. Anisotropic cell sheets without random cell orientations have been conventionally produced with photolithographically, microfabricated substrates using special facilities and equipment. Here we demonstrate a more accessible approach to the fabrication of anisotropic substrates. We locally deformed part of an elastic membrane and simultaneously oxidized the surface to create microwrinkles as well as to enable adhesion to the extracellular matrix. The approach with the local loading made it possible to orient cells in controlled directions within a single membrane sheet depending on the strains determined by the controllable deformation. This technique potentially enables a versatile design of microwrinkles for target-compatible cell alignments.     

Morphological Features of Adult Rats of IS/Kyo and IS-Tlk/Kyo Strains with Lumbar and Caudal Vertebral Anomalies

IS-Tlk/Kyo, a mutant derived from IS/Kyo strain, exhibits a kinked and/or short tail, in addition to the congenital lumbar vertebral anomaly. Homozygotes of Tlk dominant gene are known to die during embryonic development. We previously reported the morphological features of the skeleton in IS/Kyo and IS-Tlk/Kyo fetuses and of the heart in IS/Kyo fetuses [19]. This study was conducted to clarify the morphological features of the skeleton in both adult rats and of the heart in adult IS/Kyo rats. Ventricular septal defect (VSD) was observed in 3 out of 10 IS/Kyo rats. Neither splitting of lumbar vertebra and supernumerary rib (in both strains) nor fused or absent caudal cartilage (in IS-Tlk/Kyo strain) was detected in adult rats. Fusion of lumbar vertebrae was observed in almost all specimens together with lumbarization of sacral vertebrae in a few specimens in both adult rats as well as fusion of sacral and caudal vertebrae only in adult IS-Tlk/Kyo rats. In addition, a severe reduction in the ossified sacral and caudal vertebrae was noted in adult IS-Tlk/Kyo rats (mean number: 20.6) and IS/Kyo rats (31.8), and the difference was similar to that in the length of sacral and caudal vertebrae. These results suggest that the Tlk gene may be involved in both the congenital and acquired abnormal formation of the lower vertebral centra as well as the persistent occurrence of VSD by the background gene in IS/Kyo strain.     

Microcontact Peeling as a New Method for Cell Micropatterning

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.     

Molecular cloning of N-methylputrescine oxidase from tobacco

Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-methylputrescine, catalyzed by N-methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.     

Response of a Wild Edible Plant to Human Disturbance: Harvesting Can Enhance the Subsequent Yield of Bamboo Shoots

Wild edible plants, ecological foodstuffs obtained from forest ecosystems, grow in natural fields, and their productivity depends on their response to harvesting by humans. Addressing exactly how wild edible plants respond to harvesting is critical because this knowledge will provide insights into how to obtain effective and sustainable ecosystem services from these plants. We focused on bamboo shoots of Sasa kurilensis, a popular wild edible plant in Japan. We examined the effects of harvesting on bamboo shoot productivity by conducting an experimental manipulation of bamboo shoot harvesting. Twenty experimental plots were prepared in the Teshio Experimental Forest of Hokkaido University and were assigned into two groups: a harvest treatment, in which newly emerged edible bamboo shoots were harvested (n = 10); and a control treatment, in which bamboo shoots were maintained without harvesting (n = 10). In the first year of harvesting (2013), bamboo shoot productivities were examined twice; i.e., the productivity one day after harvesting and the subsequent post-harvest productivity (2–46 days after harvesting), and we observed no difference in productivity between treatments. This means that there was no difference in original bamboo shoot productivity between treatments, and that harvesting did not influence productivity in the initial year. In contrast, in the following year (2014), the number of bamboo shoots in the harvested plots was 2.4-fold greater than in the control plots. These results indicate that over-compensatory growth occurred in the harvested plots in the year following harvesting. Whereas previous research has emphasized the negative impact of harvesting, this study provides the first experimental evidence that harvesting can enhance the productivity of a wild edible plant. This suggests that exploiting compensatory growth, which really amounts to less of a decline in productivity, may be s a key for the effective use of wild edible plants.