In vivo role of nectin-1 in entry of herpes simplex virus type 1 (HSV-1) and HSV-2 through the vaginal mucosa

Herpes simplex virus type 2 (HSV-2) is transmitted through the genital mucosa during sexual encounters. In recent years, HSV-1 has also become commonly associated with primary genital herpes. The mechanism of viral entry of HSV-1 and HSV-2 in the female genital tract is unknown. In order to understand the molecular interactions required for HSV entry into the vaginal epithelium, we examined the expression of herpesvirus entry mediator nectin-1 in the vagina of human and mouse at different stages of their hormonal cycle. Nectin-1 was highly expressed in the epithelium of human vagina throughout the menstrual cycle, whereas the mouse vaginal epithelium expressed nectin-1 only during the stages of the estrous cycle in which mice are susceptible to vaginal HSV infection. Furthermore, the ability of nectin-1 to mediate viral entry following intravaginal inoculation was examined in a mouse model of genital herpes. Vaginal infection with either HSV-1 or HSV-2 was blocked by preincubation of the virus with soluble recombinant nectin-1. Viral entry through the vaginal mucosa was also inhibited by preincubation of HSV-2 with antibody against gD. Together, these results suggest the importance of nectin-1 in mediating viral entry for both HSV-1 and HSV-2 in the genital mucosa in female hosts.     

A review on fisheries and conservation status of Asian horseshoe crabs

Horseshoe crabs are the only extant xiphosurans and are believed to be morphologically unchanged for more than 200 million years. Of the four extant species namely, Limulus polyphemus, Tachypleus tridentatus, Tapinauchenius gigas and Carcinoscorpius rotundicauda, the latter three are found in Asian waters. Recent evidences showed that Asian horseshoe crabs are facing serious threats such as degradation of their spawning grounds and habitat, environmental pollution, overexploitation as a culinary delicacy and biomedical bleeding practices. Baseline data on the distribution and existing population of the wild horseshoe crabs remain poorly known in several Asian regions. Several studies have clearly revealed that pressure due to over-fishing of wild stock has increased tremendously in the last decade. Due to an increase in demand for Tachypleus Amebocyte Lysate (TAL) analogous to Limulus Amebocyte Lysate (LAL) in the United States, there is an urgent need to comprehensively address their fishing and conservation measures in the Asian region. This review addresses the overall studies on three species of Asian horseshoe crabs in relation to their fishing practices, local exploitation of their wild stock either for human consumption (or) by biomedical industries. The authors have structured the discussion on an international scale to address the existing problems in fishing and conservation of horseshoe crabs. Since no specific regulatory force or legislative protection act or a policy to preserve their natural stock are available to this date, this paper strongly recommends representative countries to include horseshoe crabs under their wildlife protection act to avoid further unsustainable exploitation of their wild populations.     

Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.     

Environmental DNA enables detection of terrestrial mammals from forest pond water

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500‐ml water samples from ponds in two cool‐temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.     

Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1

Neutrophils contribute to pathogen clearance by producing neutrophil extracellular traps (NETs), which are genomic DNA-based net-like structures that capture bacteria and fungi. Although NETs also express antiviral factors, such as myeloperoxidase and α-defensin, the involvement of NETs in antiviral responses remains unclear. We show that NETs capture human immunodeficiency virus (HIV)-1 and promote HIV-1 elimination through myeloperoxidase and α-defensin. Neutrophils detect HIV-1 by Toll-like receptors (TLRs) TLR7 and TLR8, which recognize viral nucleic acids. Engagement of TLR7 and TLR8 induces the generation of reactive oxygen species that trigger NET formation, leading to NET-dependent HIV-1 elimination. However, HIV-1 counteracts this response by inducing C-type lectin CD209-dependent production of interleukin (IL)-10 by dendritic cells to inhibit NET formation. IL-10 suppresses the reactive oxygen species-dependent generation of NETs induced upon TLR7 and TLR8 engagement, resulting in disrupted NET-dependent HIV-1 elimination. Therefore, NET formation is an antiviral response that is counteracted by HIV-1.