Culture-Independent Evaluation of Nonenveloped-Virus Infectivity Reduced by Free-Chlorine Disinfection

The inability of molecular detection methods to distinguish disinfected virions from infectious ones has hampered the assessment of infectivity for enteric viruses caused by disinfection practices. In the present study, the reduction of infectivity of murine norovirus S7-PP3 and mengovirus vMC0, surrogates of human noroviruses and enteroviruses, respectively, caused by free-chlorine treatment was characterized culture independently by detecting carbonyl groups on viral capsid protein. The amount of carbonyls on viral capsid protein was evaluated by the proportion of biotinylated virions trapped by avidin-immobilized gel (percent adsorbed). This culture-independent approach demonstrated that the percent adsorbed was significantly correlated with the logarithm of the infectious titer of tested viruses. Taken together with the results of previous reports, the result obtained in this study indicates that the amount of carbonyls on viral capsid protein of four important families of waterborne pathogenic viruses, Astroviridae, Reoviridae, Caliciviridae, and Picornaviridae, is increased in proportion to the received oxidative stress of free chlorine. There was also a significant correlation between the percent adsorbed and the logarithm of the ratio of genome copy number to PFU, which enables estimation of the infectious titer of a subject virus by measuring values of the total genome copy number and the percent adsorbed. The proposed method is applicable when the validation of a 4-log reduction of viruses, a requirement in U.S. EPA guidelines for virus removal from water, is needed along with clear evidence of the oxidation of virus particles with chlorine-based disinfectants.     

Early‐shared Mycobacterium bovis bacillus Calmette–Guérin sub‐strains induce Th1 cytokine production in vivo

Interleukin‐12 is one of the cytokines that induce acquired immunity by progressing the differentiation of T cells. When antigens are presented by APCs, including macrophages and DCs, T cells are activated and produce the Th1 cytokines IL‐2 and IFN‐γ. We have previously reported greater IL‐12 production from macrophages infected with early‐shared BCG sub‐strains (ex. BCG‐Japan, ‐Sweden) than from those infected with late‐shared BCG (ex. BCG‐Pasteur and ‐Connaught) 1 . In this study, we investigated the Th1 cytokine‐inducing activity of splenocytes co‐cultured with BCG‐infected DCs. Early‐shared BCG‐infected DCs produced IL‐12 and TNF‐α? Furthermore, when they were co‐cultured with purified protein derivative‐stimulated DCs, the splenocytes of mice immunized with BCG‐Tokyo/Japan produced more Th1 cytokine than did those of mice immunized with BCG‐Connaught. In conclusion, early‐shared BCG sub‐strains more strongly induce Th1 cytokine production in vivo. This study provides basic information to inform the selection of candidates for primary vaccination.

     

Analysis of crystal growth of methane hydrate using molecular dynamics simulation

Methane hydrate is a crystalline compound with methane molecules enclosed in cages formed by hydrogen-bonded water molecules. Understanding the mechanism of nucleation and crystal growth from methane vapour and liquid water is important for all hydrate applications. However, processes near the water/methane interface are still unclear. In this work, we focused on the crystal growth of methane hydrate seeds located near the water/methane interface. We performed molecular dynamics (MD) simulation and analysed the crystal growth of the hydrate seed at the interface. New cages formed in the liquid water phase were stabilised when they shared faces with the hydrate seed. We also investigated the crystal growth rate as the time development of the number of methane molecules trapped in hydrate cages, based on the trajectory of the MD simulation. The calculated growth rate in the direction that covers the interface was 1.38 times that in the direction towards the inside of the water phase.     

High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence

Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo‐dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and β‐1,3‐glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus.     

Correlative Light and Scanning Electron Microscopy for Observing the Three-Dimensional Ultrastructure of Membranous Cell Organelles in Relation to Their Molecular Components

Although the osmium maceration method has been used to observe three-dimensional (3D) structures of membranous cell organelles with scanning electron microscopy (SEM), the use of osmium tetroxide for membrane fixation and the removal of cytosolic soluble proteins largely impairs the antigenicity of molecules in the specimens. In the present study, we developed a novel method to combine cryosectioning with the maceration method for correlative immunocytochemical analysis. We first immunocytochemically stained a semi-thin cryosection cut from a pituitary tissue block with a cryo-ultramicrotome, according to the Tokuyasu method, before preparing an osmium-macerated specimen from the remaining tissue block. Correlative microscopy was performed by observing the same area between the immunostained section and the adjacent face of the tissue block. Using this correlative method, we could accurately identify the gonadotropes of pituitary glands in various experimental conditions with SEM. At 4 weeks after castration, dilated cisternae of rough endoplasmic reticulum (RER) were distributed throughout the cytoplasm. On the other hand, an extremely dilated cisterna of the RER occupied the large region of the cytoplasm at 12 weeks after castration. This novel method has the potential to analyze the relationship between the distribution of functional molecules and the 3D ultrastructure in different composite tissues.     

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.