A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV

In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.     

Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system

We propose a novel method to prepare a DNA–protein conjugate using histidine-tag (His-tag) chemistry. Oligo-DNA was modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni²⁺. Investigations using a microplate which displayed a complementary DNA-strand revealed that a NTA-modified DNA–protein conjugate was formed and immobilized in the presence of Ni²⁺ on the microplate. We then adopted alkaline phosphatase (AP) as a model protein, and application of the DNA–AP conjugate was demonstrated in a thrombin aptamer-based detection system with a detection limit of approximately 10 nM.     

Characterization of recombinant glyoxylate reductase from thermophile Thermus thermophilus HB27

A glyoxylate reductase gene from the thermophilic bacterium Thermus thermophilus HB27 (TthGR) was cloned and expressed in Escherichia coli cells. The recombinant enzyme was highly purified to homogeneity and characterized. The purified TthGR showed thermostability up to 70 degrees C. In contrast, the maximum reaction condition was relatively mild (45 degrees C and pH 6.7). Although the kcat values against co-enzyme NADH and NADPH were similar, the Km value against co-enzyme NADH was approximately 18 times higher than that against NADPH. TthGR prefers NADPH rather than NADH as an electron donor. These results indicate that a phosphate group of a co-enzyme affects the binding affinity rather than the reaction efficiency, and TthGR demands appropriate amount of phosphate for a high activity. Furthermore, it was found that the half-lives of TthGR in the presence of 25% dimethyl sulfoxide and diethylene glycol were significantly longer than that in the absence of an organic solvent.     

Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6_ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6_ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6_ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.     

Quantification of Chloroflexi Eikelboom morphotype 1851 for prediction and control of bulking events in municipal activated sludge plants in Japan

The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200 mL g⁻¹ of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.

     

Embryology and its character evolution in Staphyleaceae

The family Staphyleaceae, which was recently reorganized into the two genera Staphylea and Dalrympelea based on molecular phylogenetic analyses, is sister to a clade of Crossosomataceae, Guamatelaceae, and Stachyuraceae in the order Crossosomatales. In contrast to Crossosomataceae and Stachyuraceae, the embryology of Staphyleaceae has been scarcely studied. We carried out a literature survey and examined three additional species of each genus in order to better understand the evolution of the embryological characters in the family. Characters were nearly identical across examined species. Staphyleaceae shared most characters of nucellus, megagametophyte, and integuments with Stachyuraceae with the exception of several embryological characters. The presence of a nucellar cap was found to be informative and supports the close relationship between Staphyleaceae and Stachyuraceae. We also investigated the evolution of variable characters among Crossosomataceae, Stachyuraceae, and Staphyleaceae. Staphyleaceae species share the presence of a single archesporial cell and absence of an aril as symplesiomorphies. The presence of less than five parietal cells in the nucellus is a possible synapomorphy for the genus Staphylea, while presence of vascular bundles in the testa is a possible synapomorphy for Staphyleaceae. Our observations support the identification of the seed coat as mesotestal. Several variant embryological characters within the family are discussed.     

Rapid G0/1 transition and cell cycle progression in CD8+ T cells compared to CD4+ T cells following in vitro stimulation

T‐cell population consists of two major subsets, CD4⁺ T cells and CD8⁺ T cells, which can be distinguished by the expression of CD4 or CD8 molecules, respectively. Although they play quite different roles in the immune system, many of their basic cellular processes such as proliferation following stimulation are presumably common. In this study, we have carefully analyzed time–course of G0/1 transition as well as cell cycle progression in the two subsets of quiescent T‐cell population following in vitro growth stimulation. We found that CD8⁺ T cells promote G0/1 transition more rapidly and drive their cell cycle progression faster compared to CD4⁺ T cells. In addition, expression of CD25 and effects of its blockade revealed that IL‐2 is implicated in the rapid progression, but not the earlier G0/1 transition, of CD8⁺ T cells.     

Spatiotemporal deep imaging of syncytium induced by the soybean cyst nematode <Emphasis Type="Italic">Heterodera glycines</Emphasis>

Parasite infections cause dramatic anatomical and ultrastructural changes in host plants. Cyst nematodes are parasites that invade host roots and induce a specific feeding structure called a syncytium. A syncytium is a large multinucleate cell formed by cell wall dissolution-mediated cell fusion. The soybean cyst nematode (SCN), Heterodera glycines, is a major soybean pathogen. To investigate SCN infection and the syncytium structure, we established an in planta deep imaging system using a clearing solution ClearSee and two-photon excitation microscopy (2PEM). Using this system, we found that several cells were incorporated into the syncytium; the nuclei increased in size and the cell wall openings began to be visible at 2 days after inoculation (DAI). Moreover, at 14 DAI, in the syncytium developed in the cortex, there were thickened concave cell wall pillars that resembled “Parthenon pillars.” In contrast, there were many thick board-like cell walls and rarely Parthenon pillars in the syncytium developed in the stele. We revealed that the syncytia were classified into two types based on the pattern of the cell wall structures, which appeared to be determined by the position of the syncytium inside roots. Our results provide new insights into the developmental process of syncytium induced by cyst nematode and a better understanding of the three-dimensional structure of the syncytium in host roots.