Examination of the microbiota of normal cow milk using MinIONTM nanopore sequencing

The aim of this study was to evaluate the microbiota of normal milk in dairy cows and their relationship with host factors, such as the age of the cow (Age), somatic cell counts in milk (SCCs), and days in milk (DIM). We investigated 48 milk samples from 22 cows with no systemic or local clinical signs using MinION^TM nanopore sequencing for a 16S rRNA gene amplicon. Bacterial richness was positively correlated with the DIM (P=0.043), and both the Shannon-Wiener Index and Simpson’s Index, which are metrics of alpha-diversity, were also significantly positively correlated with the SCC (P<0.001). The composition ratios of both Actinobacteria at the phylum level and Kocuria spp. at the genus level in the milk microbiota were significantly correlated with the SCC (P<0.001 and P<0.001, respectively). In the beta-diversity test, the one-way analysis of similarities test showed a significant difference (P=0.0051) between the low- and high-SCC groups. This study clarified that the composition of the normal milk microbiota in this herd was related to the SCC. It also raised the possibility of variations in bacterial genera in the normal milk microbiota between the low- and high-SCC groups. However, to clarify the actual condition of the milk microbiota and to elucidate the relationship with the SCC, it is necessary to perform further analyses taking into account not only the relative abundance, but also the absolute abundance of microbes.     

Enhancement of saccharification by overexpression of poplar cellulase in sengon

Lignocellulosic material from trees has great potential to form the basis of the second generation for bioethanol production because trees produce most of the biomass on the earth. We modified the wall structure of sengon (Paraserianthes falcataria) through overexpression of poplar cellulase in the cell walls. The overexpression did not decrease cellulose content but caused a decrease in xyloglucan bound to the walls. The level of saccharification and successive ethanol production was increased in the wood of the transgenic sengon overexpressing poplar cellulase compared with that of the wild type plant, and even after delignification of the wood. We propose that a xyloglucan intercalated into cellulose microfibrils could be one of the recalcitrant components in the saccharification of lignocelluloses.     

Proteomic analysis of the G-layer in poplar tension wood

Angiosperm trees bend their stems by forming tension wood at the upper side of leaning stems. Most tension wood has a cellulose-rich G-layer in the innermost surface of the fiber cell wall. Strong tensile stress is considered to occur in the G-layer. This study undertook to identify the proteins involved in G-layer formation and function through a proteomic analysis of G-layer-localized protein. G-layers of poplar were loosened by sonication and isolated as doughnut-shaped pieces of thinly sliced transverse sections. The proteins, once extracted with urea/detergent solution, were separated by two-dimensional polyacrylamide gel electrophoresis, and 110 spots were subjected to liquid chromatography tandem mass spectrometry (LC/MS/MS). A database search for these spots’ mass spectrum patterns identified 72 proteins. In addition, all peptide digestion mixtures of G-layer proteins were separated by strong cation exchange chromatography and 39 proteins were identified using LC/MS/MS analysis. Proteins involved in wall formation, such as lignin biosynthesis-related protein, xyloglucan endotransglucosylase, and fasciclin-like arabinogalactan protein, were notably detected in the G-layer.     

Formaldehyde adsorption by karamatsu (Larix leptolepis) bark

Formaldehyde adsorption and release tests of karamatsu (Larix leptolepis) bark and bark ingredients were performed. Karamatsu bark had good formaldehyde adsorption ability that was better than microcrystalline cellulose. An acetone-soluble fraction from karamatsu bark, which might contain bark tannin, had excellent formaldehyde adsorption ability, and was found to release a trace amount of the adsorbed formaldehyde, suggesting that its formaldehyde adsorption was predominantly due to chemical adsorption. It was confi rmed that the acetone-soluble fraction played an important part in formaldehyde adsorption by karamatsu bark. An acetone-insoluble fraction from karamatsu bark had good formaldehyde adsorption ability as well as the bark, in spite of the residue after the removal of the acetone-soluble fraction.     

Mechanism of formaldehyde adsorption of (+)-catechin

The reaction of (+)-catechin with formaldehyde vapor was investigated as a model reaction of formaldehyde adsorption of (+)-catechin. It was found by H nuclear magnetic resonance spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and gel permeation chromatography of the product that the vapor reaction of (+)-catechin with formaldehyde without a solvent or a catalyst proceeds via methylolation, condensation, and polymerization. This is the same as the reaction in a solvent in the presence of an acid or a base. The formation of polyoxymethylenes such as paraformaldehyde was not confirmed.     

Babesia microti-like parasites detected in Eurasian red squirrels (Sciurus vulgaris orientis) in Hokkaido, Japan

Six Eurasian red squirrels (Sciurus vulgaris orientis), victims of road traffic found during 2002 and 2004 near the Noppro Forest Park in Ebetsu, Hokkaido, Japan, were examined for the presence of Babesia parasites. Three of the six squirrels exhibited positive signals by nested PCRs targeting both the 18S rRNA and beta-tubulin genes. Three squirrels proved to be infected with a B. microti-like parasite as evidenced by sequencing the amplified DNAs and by the morphology of the intraerythrocytic parasites. Genotypically, however, the parasite appeared to be of a new type, as it was clearly distinguishable from any of the known types that have previously been reported in various wild animals. This is the first report showing molecular evidence for the presence of B. microti-like parasites in Sciuridae.     

Comparative study of the plasma globulin level, CD21(-) B-cell counts and FOXP3 mRNA expression level in CD4(+) T-cells for different clinical stages of feline immunodeficiency virus infected cats

Feline immunodeficiency virus (FIV) infection leads to hypergammaglobulinemia through mechanisms that remain poorly understood. We investigated changes in plasma globulin level, B cells, and T cells with progression of the clinical stage of FIV-infected cats. We classified FIV-infected cats into the stage of Asymptomatic carrier (AC) and AIDS-related complex (ARC) based on the clinical symptoms, and measured the plasma globulin level, the CD4(+) T-cell counts, and analyzed surface markers of B cells. We investigated the relationship between the plasma globulin level and regulatory T cells (Tregs) using the Forkhead box P3 (FOXP3) mRNA expression level. In FIV-infected cats, the plasma globulin level and the surface immunoglobulin (sIg)(+) CD21(-) B-cell counts were increased, whereas the CD4(+) T-cell counts were decreased compared with specific-pathogen free (SPF) cats. The mRNA expression of Blimp-1 (master gene of plasma cells) was increased in peripheral blood, and the FOXP3 mRNA expression level was decreased in CD4(+) T-cells. These immunological changes were marked in the ARC stage. These data indicate that the decrease of Tregs and the increase of plasma cells lead to hypergammaglobulinemia.     

Growth and root sucker ability of field-grown transgenic poplars overexpressing xyloglucanase

Xyloglucan is thought to be a key hemicellulose cross-linking adjacent cellulose microfibrils in plant cell walls. The growth traits of transgenic poplars (Populus alba) with decreased xyloglucan from overexpression of Aspergillus aculeatus xyloglucanase were characterized during a 4-year field trial. The field-trial site consisted of two blocks, a fertile soil block and a non-fertile soil block, determined by soil analysis. In the fertile block, the growth of aboveground biomass of the transgenic poplars was reduced to 24?C44?% compared to that of wild-type poplars, in contrast to the growth seen in chamber and greenhouse conditions. In the non-fertile block, the aboveground biomass of transgenic poplars was also smaller than that of the wild-type poplars. Because poplars reproduce asexually by root suckers, we also compared the formation of root suckers from transgenic and wild-type poplars. Root suckers formed less frequently from transgenic poplars than from wild-type poplars. The growth rates of root suckers from transgenic poplars were also slower than those from wild-type poplars. The results showed that constitutive degradation of xyloglucan impairs poplar growth and vegetative reproduction ability.