Lit-LAMP-DNA-vaccine for shrimp allergy prevents anaphylactic symptoms in a murine model.

Allergen-specific immunotherapy (AIT) is a promising therapeutic approach to food allergy but requires optimization in terms of both efficacy and safety due to the risk of undesired anaphylactic reactions. Here, we investigated the potential of a single DNA plasmid vaccine (Lit-LAMP-DNA-vaccine) encoding multivalent shrimp antigens (Lit v (Litopenaeus vannamei; Whiteleg shrimp) 1, Lit v4, and Lit v3) and a lysosomal-associated membrane protein (LAMP) as the next generation of AIT for patients with allergy. We first confirmed the expression of the LAMP-1-Lit v1-Lit v4-Lit v3 fusion protein in human cells transfected with the Lit-LAMP-DNA-vaccine, and the induction of anti-Lit v1, Lit v3, and Lit v4 IgG2a antibody production as well as Th1 response in Lit-LAMP-DNA-vaccine-treated mice. Next, we established an anaphylaxis model in mice epicutaneously sensitized with a crude shrimp protein extract (SPE) and investigated both the efficacy of Lit-LAMP-DNA-vaccine, and the difference in the mechanism of action (MOA) from oral immunotherapy (OIT). In the mouse shrimp allergy model, Lit-LAMP-DNA-vaccine potently suppressed anaphylactic reactions and mast cell activation with robust antigen-specific IgG2a production. The IgG1:IgG2a ratio was significantly lower than that of OIT. This suppressive effect was also confirmed by plasma transfer from mice previously vaccinated with the Lit-LAMP-DNA-vaccine. These results suggest that this Lit-LAMP-DNA-vaccine may represent a promising therapeutic strategy for human shrimp allergy which acts via the efficient induction of antigen-specific IgG with antagonism.

Projections of surface air temperature required to sustain permafrost and importance of adaptation to climate change in the Daisetsu Mountains, Japan.

Permafrost is known to occur in high mountainous areas such as the Daisetsu Mountains in Japan, which are located at the southernmost limit of the permafrost distribution in the world. In this study, areas with climatic conditions suitable for sustaining permafrost in the Daisetsu Mountains are projected using bias-corrected and downscaled climate model outputs and statistical relationships between surface air temperatures and permafrost areas. Using freezing and thawing indices, the size of the area in the Daisetsu Mountains where climatic conditions were suitable for permafrost were estimated to be approximately 150 km2 in 2010. Under the RCP8.5 scenario, this area is projected to decrease to about 30 km2 by 2050 and it is projected to disappear by around 2070. Under the RCP2.6 scenario, the area is projected to decrease to approximately 20 km2 by 2100. The degradation of mountain permafrost could potentially affect the stability of trekking trails due to slope displacement, and it may also have deleterious effects on current alpine ecosystems. It is therefore important to accurately monitor changes in the mountain ecosystem environment and to implement measures to adapt to an environment that is projected to change significantly in the future.

Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus.

Human norovirus is the leading cause of acute nonbacterial gastroenteritis in people of all ages worldwide. Currently, no licensed norovirus vaccine, pharmaceutical drug, or therapy is available for the control of norovirus infection. Here, we used a rice transgenic system, MucoRice, to produce a variable domain of a llama heavy-chain antibody fragment (VHH) specific for human norovirus (MucoRice-VHH). VHH is a small heat- and acid-stable protein that resembles a monoclonal antibody. Consequently, VHHs have become attractive and useful antibodies (Abs) for oral immunotherapy against intestinal infectious diseases. MucoRice-VHH constructs were generated at high yields in rice seeds by using an overexpression system with RNA interference to suppress the production of the major rice endogenous storage proteins. The average production levels of monomeric VHH (7C6) to GII.4 norovirus and heterodimeric VHH (7C6-1E4) to GII.4 and GII.17 noroviruses in rice seed were 0.54 and 0.28% (w/w), respectively, as phosphate buffered saline (PBS)-soluble VHHs. By using a human norovirus propagation system in human induced pluripotent stem-cell-derived intestinal epithelial cells (IECs), we demonstrated the high neutralizing activity of MucoRice expressing monomeric VHH (7C6) against GII.4 norovirus and of heterodimeric VHH (7C6-1E4) against both GII.4 and GII.17 noroviruses. In addition, MucoRice-VHH (7C6-1E4) retained neutralizing activity even after heat treatment at 90°C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and for prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly.

Discovery of a novel 9-position modified second-generation anti-HCV candidate via bioconversion and semi-synthesis of FR901459.

Evidence that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in the virus replication cycle has increased attention on cyclophilin inhibitors as attractive therapeutic targets in the treatment of HCV. Previous reports have described a number of non-immunosuppressive cyclophilin inhibitors, most of which require many synthetic steps for their preparation. Sasamura et al. have previously reported the isolation of bioconversion derivative 4. This analog is a convenient starting point for optimization due to the presence of the readily modifiable primary hydroxyl group and because it shows moderate anti-HCV activity and decreased immunosuppressive activity. We have also established an efficient C-alkylation reaction at the 3-position. Through a detailed structure-activity relationship study, we discovered a new type of clinical candidate 14 which requires a short synthetic process and has potent anti-HCV activity and reduced immunosuppressive activity, as well as improved aqueous solubility and pharmacokinetics.

Reduced bioavailability of polycyclic aromatic hydrocarbons (PAHs) in sediments impacted by carbon manufacturing plant effluent: Evaluation by ex situ passive sampling method.

Potential risks of polycyclic aromatic hydrocarbons (PAHs) in sediments of a Japanese bay contaminated by carbon manufacturing plant effluent were evaluated by calculating toxicity units (TUs). TUs calculated from the measured whole-sediment concentrations (Cwhole) were often higher than or close to 1, signaling a possible toxicity concern to benthic organisms. In contrast, TUs based on the freely dissolved pore water concentrations (Cfree) measured by an ex-situ passive sampling method with polyethylene strips were 0.0007-0.005, much lower than 1, indicating no effect. We also found that the fractions of black carbon in sediments of the contaminated bay were significantly higher than those of reference sites. Overall, we conclude that carbon manufacturing plant effluent substantially increases Cwhole of PAHs in sediments but also increases the fraction of carbonaceous particles that strongly retain PAHs. As a combined result, bioavailable concentrations (as expressed by pore water Cfree) of PAHs do not increase as much as Cwhole. The results of this study indicate that ecotoxicological risks of PAH contamination by carbon manufacturing plants should be evaluated by directly measuring pore water Cfree instead of Cwhole.

Signs of biological activities of 28,000-year-old mammoth nuclei in mouse oocytes visualized by live-cell imaging.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Carbon storage reservoirs in watersheds support stream food webs via periphyton production.

We measured the natural abundances of radiocarbon (delta14C) in macroinvertebrates, fishes, and their potential food sources, collected from the upper and lower reaches of six temperate streams in Lake Biwa basin (central Japan), three of which flow on limestone bedrock. Several carbon storage reservoirs in the watersheds show distinctive delta14C signatures (e.g., ancient carbonate rocks, -1000 per thousand; modern atmospheric CO2, +50 per thousand). Our analyses showed that the delta14C values for periphytic algae range from -361 per thousand to +21 per thousand, reflecting 14C-depleted signals from watershed storage reservoirs (carbonate rocks and/or soils). In contrast, the delta14C values for coarse particulate organic matter (CPOM) range from -6 per thousand to +62 per thousand, reflecting 14C-enriched signals from modern atmospheric CO2. The periphyton from streams on limestone bedrock was more 14C-depleted than that from streams in non-limestone areas, although the delta14C values for periphyton from the latter were less than modern atmospheric 14CO2 concentration. The delta14C values for most of the consumers were between those for periphyton and CPOM. Based on a delta14C two-source mixing models, the results suggested that the grazers rely on periphyton, while the carbon source for collectors and predators shifts from CPOM in the upper reaches of streams to periphyton in the lower reaches. The delta14C signature can trace carbon from watershed storage reservoirs to benthic production, which suggests that stream food webs are composed of mixtures of carbon originating from various sources of different ages.

Increasing summer rainfall in arid eastern-Central Asia over the past 8500 years.

A detailed and well-dated proxy record of summer rainfall variation in arid Central Asia is lacking. Here, we report a long-term, high resolution record of summer rainfall extracted from a peat bog in arid eastern-Central Asia (AECA). The record indicates a slowly but steadily increasing trend of summer rainfall in the AECA over the past 8500 years. On this long-term trend are superimposed several abrupt increases in rainfall on millennial timescales that correspond to rapid cooling events in the North Atlantic. During the last millennium, the hydrological climate pattern of the AECA underwent a major change. The rainfall in the past century has reached its highest level over the 8500-year history, highlighting the significant impact of the human-induced greenhouse effect on the hydrological climate in the AECA. Our results demonstrate that even in very dry eastern-Central Asia, the climate can become wetter under global warming.

Contributions of detrital subsidies to aboveground spiders during secondary succession, revealed by radiocarbon and stable isotope signatures.

Prey subsidies originating from detritus add nutrients and energy to arboreal communities. Measurement of this subsidy is required in the understanding of how food web dynamics respond to changes in surrounding environments. Shrub spiders are one of the key predators involved in food web coupling. We evaluate the effects of potential changes in prey availabilities during secondary succession on the contribution of subsidy from detrital food webs to shrub spiders and how different spider feeding guilds used the subsidy of prey from detrital food webs. We measured the relative importance of the subsidy for the spider feeding guilds, using the ratios of stable isotopes of C (δ(13)C), and N (δ(15)N) and C isotope discrimination (Δ(14)C). Diet age was calculated from Δ(14)C values, because old diet ages of spiders indicate that the spiders consume prey from detrital food sources. Dominant aerial prey (Diptera) had a distinctively old diet age compared with arboreal prey, which indicates that aerial prey were subsidized from detrital food webs. Sit-and-wait spiders tended to have an older diet age than active hunting spiders, which indicates that sit-and-wait spiders depended more on subsidies. Diet age varied only slightly for spiders in stands of different ages, indicating that rates at which spiders use grazing and detrital prey are probably determined more by foraging strategies and not by stand age. A dominance of sit-and-wait predators will lead to higher detrital subsidy inputs in shrub habitats. This study highlights the effect of shrub spider community structure (feeding guild composition) on the volume of the subsidy received from the detrital food web.

Radiocarbon (14C) diurnal variations in fine particles at sites downwind from Tokyo, Japan in summer.

The radiocarbon ((14)C) of total carbon (TC) in atmospheric fine particles was measured at 6 h or 12 h intervals at two sites, 50 and 100 km downwind from Tokyo, Japan (Kisai and Maebashi) in summer 2007. The percent modern carbon (pMC) showed clear diurnal variations with minimums in the daytime. The mean pMC values at Maebashi were 28 ± 7 in the daytime and 45 ± 16 at night (37 ± 15 for the overall period). Those at Kisai were 26 ± 9 in the daytime and 44 ± 8 at night (37 ± 12 for the overall period). This data indicates that fossil sources were major contributors to the daytime TC, while fossil and modern sources had comparable contributions to nighttime TC in the suburban areas. At both sites, the concentration of fossil carbon as well as O(3) and the estimated secondary organic carbon increased in the daytime. These results suggest that fossil sources around Tokyo contributed significantly to the high daytime concentration of secondary organic aerosols (SOA) at the two suburban sites. A comparison of pMC and the ratio of elemental carbon/TC from our particulate samples with those from three end-member sources corroborates the dominant role of fossil SOA in the daytime.

Carbon isotope composition of dissolved humic and fulvic acids in the Tokachi River system.

This study reports carbon isotopic ratios (Δ(14)C and δ(13)C) of dissolved humic and fulvic acids in the Tokachi River system, northern Japan. These acids have a refractory feature and they represent the largest fraction of dissolved organic matter in aquatic environments. The acids were isolated using the XAD extraction method from river water samples collected at three sites (on the upper and lower Tokachi River, and from one of its tributaries) in June 2004 and 2005. δ(13)C values were -27.8 to -26.9 ‰ for humic and fulvic acids. On the other hand, the Δ(14)C values ranged from -247 to +26 ‰ and the average values were -170 ± 79 ‰ for humic acid and -44 ± 73 ‰ for fulvic acid. The difference was attributed to the residence time of fulvic acid in the watershed being shorter than that of humic acid. The large variation suggested that humic substances have a different pathway in each watershed environment.

Contrasting diurnal variations in fossil and nonfossil secondary organic aerosol in urban outflow, Japan.

Diurnal variations of fossil secondary organic carbon (SOC) and nonfossil SOC were determined for the first time using a combination of several carbonaceous aerosol measurement techniques, including radiocarbon (¹4C) determinations by accelerator mass spectrometry, and a receptor model (chemical mass balance, CMB) at a site downwind of Tokyo during the summer of 2007. Fossil SOC showed distinct diurnal variation with a maximum during daytime, whereas diurnal variation of nonfossil SOC was relatively small. This behavior was reproduced by a chemical transport model (CTM). However, the CTM underestimated the concentration of anthropogenic secondary organic aerosol (ASOA) by a factor of 4-7, suggesting that ASOA enhancement during daytime is not explained by production from volatile organic compounds that are traditionally considered major ASOA precursors. This result suggests that unidentified semivolatile organic compounds or multiphase chemistry may contribute largely to ASOA production. As our knowledge of production pathways of secondary organic aerosol (SOA) is still limited, diurnal variations of fossil and nonfossil SOC in our estimate give an important experimental constraint for future development of SOA models.

Compound class specific 14C analysis of polycyclic aromatic hydrocarbons associated with PM10 and PM1.1 aerosols from residential areas of suburban Tokyo.

Compound class specific radiocarbon analysis (CCSRA) was performed for polycyclic aromatic hydrocarbons (PAHs) associated with airborne particulate matter (APM) with diameter <10 microm (PM10) and <1.1 microm (PM1.1) collected from a residential area of suburban Tokyo, Japan, and seasonal and particle-size radiocarbon variations were investigated. Source diagnostic isomer pair ratios indicated mixed contributions from petroleum combustion and from biomass and coal combustion to the PAHs in APM. The delta14C- PAHs in APM, ranging from -787 to -514 per thousand, indicated dominance of fossil fuel combustion. The delta14C of 5-6 rings (HMW) PAHs were higher than the 3-4 rings (LMW) species in both PM10 and PM1.1 samples. The delta14C of HMW-PAHs indicated greater biomass-burning contributions in summer than in winter and no apparent particle-size variation. Conversely, the delta14C of LMW species showed a greater contribution from fossil sources in summer and in larger particles (PM10). This finding could be tentatively attributed to the recondensation of fossil-PAHs vaporized from petroleum sources. A 14C isotopic mass balance approach estimated that biomass burning contributes 17-45% of the PAH burden in suburban Tokyo, and that the increase in the biomass-PAH accounts for approximately 27% and 22% of winter-time elevation of LMW- and HMW-PAHs, respectively. These are far exceeding what is expected from the emission statistics for CO2 and combusted materials in Japan and emphasizing the importance of biomass-burning as a source of PAHs; which, in turn, demonstrates the utility and the significance of field-based source assessment by using CCSRA for an effective regulation of atmospheric pollution by PAHs.

Measurement of HCV RdRp activity with C-terminal 21 aa truncated NS5b protein: optimization of assay conditions.

The non-structural protein 5b (NS5b) of hepatitis C virus (HCV), bearing an RNA-dependent RNA polymerase (RdRp) activity, is considered as a new target of antiviral therapy. We expressed and purified the C-terminal 21 amino acid truncated NS5b protein fused with glutathione S-transferase (GST-5bC21) using Escherichia coli. With the highly purified GST-5bC21 protein, we established an in vitro assay system for RdRp activity by using poly(C) as the template and a 12 mer oligo(rG) as the primer. The optimal conditions for testing various concentrations of template, primer and proteins were determined to 22 degrees C and a pH of 7.5. The addition of 2.5 mM Mn(2+) increased the activity profoundly, to a level fivefold higher than that in the presence of 10 mM Mg(2+). At higher concentrations of Mn(2+), GST-5bC21 is stable as compared with previously reported full-length NS5b expressed using insect cells or NS5b protein with the C-terminal 18 amino acids deleted. This sensitive and easy to use quantitative assay system will provide a stable system for the screening of inhibitors for HCV RdRp.

Innate form of HCV core protein plays an important role in the localization and the function of HCV core protein.

Hepatitis C Virus (HCV) has been identified as the major causative agent of non-A, non-B hepatitis. Core protein is not only a capsid protein of HCV but also a regulator of cellular functions, and plays an important role in the pathogenesis of HCV. Core protein is produced as an innate form (amino acids [a.a.] 1-191), and following processing produces a mature form (a.a. 1-173). This study demonstrates that the innate form regulates subcellular localization of the mature form, and that the innate form in the cytoplasm enhances p21 expression; on the other hand, the mature form in the nucleus suppresses p21 expression. These observations suggest that the innate form is not only a precursor of the mature form but also a regulator of the localization and functions of core protein.

Hepatitis C virus core protein binds to a C-terminal region of NS5B RNA polymerase.

Hepatitis C virus (HCV) NS5B has been shown to exhibit RNA-dependent RNA polymerase activity for its viral RNA replication. In this study, we demonstrated the formation of a complex between NS5B and the core protein (NS5B-core protein complex) in mammalian cells, as determined by indirect immunofluorescence and immunoprecipitation analyses. The localization of the core protein was observed to change to the same locus in ER as NS5B locates by its coexpression with NS5B, indicating that the localization of the core protein is determined by NS5B. The truncated NS5B molecule lacking the C-terminal region did not form a complex with the core protein, suggesting that the C-terminal region of NS5B is essential for its interaction with the core protein. Moreover, the change in NS5B localization because of C-terminal deletion indicates that this region includes a certain signal for NS5B retention in ER.