A Metabologenomic Approach Reveals Changes in the Intestinal Environment of Mice Fed on American Diet.

Intestinal microbiota and their metabolites are strongly associated with host physiology. Developments in DNA sequencing and mass spectrometry technologies have allowed us to obtain additional data that enhance our understanding of the interactions among microbiota, metabolites, and the host. However, the strategies used to analyze these datasets are not yet well developed. Here, we describe an original analytical strategy, metabologenomics, consisting of an integrated analysis of mass spectrometry-based metabolome data and high-throughput-sequencing-based microbiome data. Using this approach, we compared data obtained from C57BL/6J mice fed an American diet (AD), which contained higher amounts of fat and fiber, to those from mice fed control rodent diet. The feces of the AD mice contained higher amounts of butyrate and propionate, and higher relative abundances of Oscillospira and Ruminococcus. The amount of butyrate positively correlated with the abundance of these bacterial genera. Furthermore, integrated analysis of the metabolome data and the predicted metagenomic data from Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) indicated that the abundance of genes associated with butyrate metabolism positively correlated with butyrate amounts. Thus, our metabologenomic approach is expected to provide new insights and understanding of intestinal metabolic dynamics in complex microbial ecosystems.

[Factors related to frequent emergency hospital refusal of ambulance transport calls].

Purpose　Among the factors that adversely affect the time needed for ambulance transport, one factor for which intervention is possible is the time it takes to choose a medical receiving facility. This research clarified the characteristics of the time, location, and severity of medical emergencies in which the choice of hospital was difficult.Methods　The research covered a 1-year period from January 1 through December 31, 2014, investigating all incidents of ambulance transport for patients in Wakayama Prefecture, except for those involving pediatric patients, amounting to a total of 41,574 incidents. Of those, the number of referrals was missing in 129 cases and the remaining 41,445 incidents were analyzed. Cases with 4 or more referrals were adopted as an indicator of difficulty in choosing a hospital. Using binary logistic regression analysis, odds ratios (ORs) with 95% confidence intervals (CIs) were computed for the times and locations of emergency calls, and the degree of injury or illness. A comprehensive analysis was performed, and stratified analysis by Major Diagnostic Category (MDC) was performed, examining external injuries, burns, and poisoning; nervous system disorders; digestive system, liver, biliary tract, and pancreatic disorders; respiratory disorders; and circulatory system disorders.Results　The distribution of numbers of referrals ranged from 1 to 12, and 79.6% of cases involved 1 referral, while cases with 4 or more referrals made up 3.5% of the total. In the overall analysis, for cases with 4 or more referrals, call times were examined using weekday working hours as a reference; all other times had high ORs (at 95%CI). The highest OR (95%CI) of 4.0 (3.2-5.0) was for late nights during weekends and holidays. Regarding the degree of injury or illness, using moderate and mild injuries as a reference, the number of referrals (3 times or fewer) was significantly lower for severe injuries and incidents of death (0.8;0.7-0.9). However, in the results of the stratified analysis by MDC, external injuries, burns, and poisoning; severe injury; and death each had an OR (95%CI) of 1.4 (1.0-1.8).Conclusion　The relationship between the time, location, and severity of medical emergencies was examined using the number of referrals as an indicator of difficulty with hospital choice in ambulance transport. This research clarified that cases late at night during weekends and holidays, and moderate and mild injuries caused the most difficulty, and that the problems depended on the secondary medical district.

Cardiac overexpression of perilipin 2 induces dynamic steatosis: prevention by hormone-sensitive lipase.

Cardiac intracellular lipid accumulation (steatosis) is a pathophysiological phenomenon observed in starvation and diabetes mellitus. Perilipin 2 (PLIN2) is a lipid droplet (LD)-associated protein expressed in nonadipose tissues, including the heart. To explore the pathophysiological function of myocardial PLIN2, we generated transgenic (Tg) mice by cardiac-specific overexpression of PLIN2. Tg hearts showed accumulation of numerous small LDs associated with mitochondrial chains and high cardiac triacylglycerol (TAG) content [8-fold greater than wild-type (WT) mice]. Despite massive steatosis, cardiac uptake of glucose, fatty acids and VLDL, systolic function, and expression of metabolic genes were comparable in the two genotypes, and no morphological changes were observed by electron microscopy in the Tg hearts. Twenty-four hours of fasting markedly reduced steatosis in Tg hearts, whereas WT mice showed accumulation of LDs. Although activity of adipose triglyceride lipase in heart homogenate was comparable between WT and Tg mice, activity of hormone-sensitive lipase (HSL) was 40-50% less in Tg than WT mice under both feeding and fasting conditions, suggesting interference of PLIN2 with HSL. Mice generated through crossing of PLIN2-Tg mice and HSL-Tg mice showed cardiac-specific HSL overexpression and complete lack of steatosis. The results suggest that cardiac PLIN2 plays an important pathophysiological role in the development of dynamic steatosis and that the latter was prevented by upregulation of intracellular lipases, including HSL.

Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress.

Fat-specific protein 27 (FSP27), a member of the cell death-inducing DNA fragmentation factor α-like effector (Cide) family, is highly expressed in adipose tissues and is a lipid droplet (LD)-associated protein that induces the accumulation of LDs. Using a yeast two-hybrid system to examine potential interactions of FSP27 with other proteins, a direct interaction with the N-terminal region of nuclear factor of activated T cells 5 (NFAT5) was identified. NFAT5 is a transcription factor that induces osmoprotective and inflammatory genes after its translocation to the nucleus. The interaction between FSP27 and NFAT5 was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation. Using immunocytochemistry, NFAT5 is detected in the cytoplasm and in the nucleus under isotonic conditions; however, overexpression of FSP27 inhibited the hypertonic-induced nuclear translocation of NFAT5. Consistent with the suppression of NFAT5 nuclear translocation, in cells transfected with a reporter construct containing the NFAT5 response element from the monocyte chemoattractant protein 1 (MCP1) promoter, FSP27 overexpression repressed hypertonic-induced luciferase activity and the expression of NFAT5 target genes. Knockdown of FSP27 in differentiated 3T3-L1 adipocytes increased the NFAT5-mediated rise in MCP1. These results suggest that FSP27 not only modulates LD homeostasis but also modulates the response to osmotic stress via a physical interaction with NFAT5 at the LD surface.

Commercial-scale utilization of greenhouse residues.

Development of techniques utilizing waste without any additional energy or rare catalysts is a starting point for becoming sustainable. In the present work, the complex utilization of greenhouse residues was studied on a commercial scale. Only the energy produced by the process (8%) was used to run the technology, thanks to multilevel heat recuperation and high methane yields (over 340 m(3) volatile solid t(-1) ). Manifestations of labile carbon in relation to available nitrogen, methane yields, and the formation of inhibitors were investigated in detail. The results sweep away many false beliefs about the ratios of carbon to nitrogen and highlight the role of the availability of carbon in phytomass utilization.

Establishment of a human microbiome- and immune system-reconstituted dual-humanized mouse model.

Humanized mice are widely used to study the human immune system in vivo and investigate therapeutic targets for various human diseases. Immunodeficient NOD/Shi-scid-IL2rγnull (NOG) mice transferred with human hematopoietic stem cells are a useful model for studying human immune systems and analyzing engrafted human immune cells. The gut microbiota plays a significant role in the development and function of immune cells and the maintenance of immune homeostasis; however, there is currently no available animal model that has been reconstituted with human gut microbiota and immune systems in vivo. In this study, we established a new model of CD34+ cell-transferred humanized germ-free NOG mice using an aseptic method. Flow cytometric analysis revealed that the germ-free humanized mice exhibited a lower level of human CD3+ T cells than the SPF humanized mice. Additionally, we found that the human CD3+ T cells slightly increased after transplanting human gut microbiota into the germ-free humanized mice, suggesting that the human microbiota supports T cell proliferation or maintenance in humanized mice colonized by the gut microbiota. Consequently, the dual-humanized mice may be useful for investigating the physiological role of the gut microbiota in human immunity in vivo and for application as a new humanized mouse model in cancer immunology.

The use of underwater high-voltage discharges to improve the efficiency of Jatropha curcas L. biodiesel production.

Underwater high-voltage discharges (3.5 kV) resulting in 4.9 kJ shock waves (50-60 MPa) were studied at the laboratory scale as a Jatropha curcas L. seed disintegration method. Grinding and macerating in an excess of methanol (3.5:1) was advantageous because methanol acts both as a liquid carrier for the pressure shock waves and as a solvent that increases the efficiency of oil extraction while remaining usable for esterification. The influence of the number of shock waves and the intensity of methanol maceration on the heat values of the pressed cake are stated in detail. Soxhlet extraction demonstrated that a greater than 94% oil extraction was achieved. The increased disintegration of vacuoles rich in oil was documented by surface area analysis, mineralization kinetics analysis, and electron microscopy. The working volumes were small, and the proportion of energy inadequate compared to the yields released; however, much can be improved by upgrading the process.

Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes.

BACKGROUND: While an increase in bone marrow adiposity is associated with age-related bone disease, the function of bone marrow adipocytes has not been studied. The aim of this study was to characterize and compare the age-related gene expression profiles in bone marrow adipocytes and epididymal adipocytes. RESULTS: A total of 3918 (13.7%) genes were differentially expressed in bone marrow adipocytes compared to epididymal adipocytes. Bone marrow adipocytes revealed a distinct gene profile with low expression of adipocyte-specific genes peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid binding protein 4 (FABP4), perilipin (Plin1), adipsin (CFD) and high expression of genes associated with early adipocyte differentiation (CCAAT/enhancer binding protein beta (C/EBPß), regulator of G-protein signaling 2 (RGS2). In addition, a number of genes including secreted frizzled related protein 4 (SFRP4), tumor necrosis factor α (TNFα), transforming growth factor beta 1(TGFß1), G-protein coupled receptor 109A (GPR109A) and interleukin 6 (IL-6), that could affect adipose-derived signaling to bone are markedly increased in bone marrow adipocytes. Age had a substantial effect on genes associated with mitochondria function and inflammation in bone marrow adipocytes. Twenty seven genes were significantly changed with age in both adipocyte depots. Among these genes, IL6 and GPR109A were significantly reduced with age in both adipocyte depots. CONCLUSIONS: Overall, gene profiling reveals a unique phenotype for primary bone marrow adipocytes characterized by low adipose-specific gene expression and high expression of inflammatory response genes. Bone marrow and epididymal adipocytes share a common pathway in response to aging in mice, but age has a greater impact on global gene expression in epididymal than in bone marrow adipocytes. Genes that are differentially expressed at greater levels in the bone marrow are highly regulated with age.

Creation of an experimental rearing environment for microbiome animal research using an individually ventilated cage system and bioBUBBLE enclosure.

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.

Identification and characterization of a novel Enterococcus bacteriophage with potential to ameliorate murine colitis.

Increase of the enteric bacteriophages (phage), components of the enteric virome, has been associated with the development of inflammatory bowel diseases. However, little is known about how a given phage contributes to the regulation of intestinal inflammation. In this study, we isolated a new phage associated with Enterococcus gallinarum, named phiEG37k, the level of which was increased in C57BL/6 mice with colitis development. We found that, irrespective of the state of inflammation, over 95% of the E. gallinarum population in the mice contained phiEG37k prophage within their genome and the phiEG37k titers were proportional to that of E. gallinarum in the gut. To explore whether phiEG37k impacts intestinal homeostasis and/or inflammation, we generated mice colonized either with E. gallinarum with or without the prophage phiEG37k. We found that the mice colonized with the bacteria with phiEG37k produced more Mucin 2 (MUC2) that serves to protect the intestinal epithelium, as compared to those colonized with the phage-free bacteria. Consistently, the former mice were less sensitive to experimental colitis than the latter mice. These results suggest that the newly isolated phage has the potential to protect the host by strengthening mucosal integrity. Our study may have clinical implication in further understanding of how bacteriophages contribute to the gut homeostasis and pathogenesis.

Two Japanese pepper (Zanthoxylum piperitum) fruit-derived compounds attenuate IgE-mediated allergic response in vitro and in vivo via inhibition of mast cell degranulation.

Zanthoxylum piperitum (ZP, 'Japanese pepper') is a traditional medicine and pepper used in Asian countries such as Japan. Hydroxy-α-sanshool, a pungent-tasting substance contained within ZP, has been reported to slightly suppress immunoglobulin E (IgE)-mediated mast cell degranulation. The current study aims to newly identify anti-allergic compounds derived from ZP. We examine the inhibitory mechanisms behind IgE-mediated mast cell degranulation. By inhibitory effect-guided isolation, we identified degranulation inhibitory compounds derived from ZP fruit: 1-acetoxy-7-hydroxy-3, 7-dimethylocta-2E, 5E-diene (ZP1) and 8-hydroxygeranyl acetate (ZP2). ZP1 and ZP2 inhibited IgE-mediated degranulation and A23187-mediated degranulation in RBL-2H3 mast cells. Our findings suggest the inhibition of degranulation by ZP1 and ZP2 was by inhibition of Lyn phosphorylation, followed by inhibition of intracellular Ca2+ mobilization, protein kinase C alpha phosphorylation, membrane ruffling, and granule-to-plasma membrane fusion. Oral administration of ZP1 or ZP2 attenuated an IgE-mediated passive cutaneous anaphylactic reaction in mice. Histological observation suggests that this effect occurred via inhibition of mast cell degranulation. These findings indicate that ZP1 and ZP2 attenuate allergic reaction via inhibition of IgE-mediated mast cell degranulation.

Association of household smoking status in childhood with young adults' educational attainment and smoking status: Results from a series of population-based cross-sectional surveys in Japan.

Smoking in young adulthood is a risk factor for future health-related disabilities and a cause of expanding health inequalities. Education and smoking are inversely associated. Using population-based representative data, this study aimed to clarify how the presence of household smokers during childhood related to both current smoking status and educational attainment among young adults. Surveys were distributed to young adults (19-20 years) invited to coming-of-age ceremonies in 2014-2017 in a rural area in Japan. Data were collected on low educational attainment (defined as ≤ 12 years of education), current smoking status, and childhood household smoking status. We used logistic regression models to calculate odds ratios (ORs) of low educational attainment for household smoking status. A total of 17.6% of men (n = 1077) and 3.8% of women (n = 1021) were current smokers. Current smoking was more common among participants from households with smokers (P < 0.001 for both men and women). The odds of low educational attainment were significantly higher for participants from smoking households (OR: 1.59, 95% confidence interval [CI]: 1.17-2.17 for men; OR: 2.29, 95% CI: 1.61-3.24, for women). All associations were characterized by a dose-response relationship with the number of household smokers. The number of household smokers in childhood was positively associated with current smoking and negatively associated with level of educational attainment among young adults. Controlling for year and geographical area, exposure to family smokers in childhood appears to be a risk factor for the intergenerational transmission of health inequalities.

Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding.

Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by ∼6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.

Glucose deprivation accelerates VLDL receptor-mediated TG-rich lipoprotein uptake by AMPK activation in skeletal muscle cells.

Glucose and fatty acids are major energy sources in skeletal muscle. Very low-density lipoprotein receptor (VLDL-R), which is highly expressed in heart, skeletal muscle and adipose tissue, plays a crucial role in metabolism of triglyceride (TG)-rich lipoproteins. To explore energy switching between glucose and fatty acids, we studied expression of VLDL-R and lipoprotein uptake in rat L6 myoblasts. l-Glucose or d-glucose deprivation in the medium noticeably induced the AMPK (AMP-activated protein kinase) activation and VLDL-R expression. Dose-dependent induction of VLDL-R expression was observed when d-glucose was less than 4.2mM. The same phenomenon was also observed in rat primary skeletal myoblasts and cultured vascular smooth muscle cells. The uptake of beta-VLDL but not LDL was accompanied by induction of VLDL-R expression. Our study suggests that the VLDL-R-mediated uptake of TG-rich lipoproteins might compensate for glucose shortfall through AMPK activation in skeletal muscle.

Experimental infection studies of Pasteurella pneumotropica and V-factor dependent Pasteurellaceae for F344-rnu rats.

To investigate the pathogenicities of P. pneumotropica (Pp) and V-factor dependent Pasteurellaceae (VFDP) in immunodeficient rats, experimental infections of F344-rnu rats were performed using 3 strains (ATCC 35149, CNP 160 and RPZ) of Pp and 4 strains (V6, V7, V8 and V9) of VFDP. Four animals per experimental group were inoculated twice on day 0 and post-inoculation day (PID) 14 with bacterial suspension intranasally. Two animals from each group were sacrificed on PID 60 and 120, and examined. In the animals inoculated with strains of Pp, sneezing was observed in some animals inoculated with strains ATCC 35149 and CNP 160 until PID 31. No clinical signs were observed in other animals. The strains were mainly isolated from the nasal cavity and trachea on PID 60, and the nasal cavity, trachea and lung on PID 120. Inflammation and necrosis of nasal cavity mucosa were observed in all animals inoculated with strains ATCC 35149 and CNP 160 in a histopathologic examination. No histopathological changes were observed in any other animal. In the animals inoculated with strains of VFDP, neither clinical disorder nor histopathological change was observed. The strains were mainly isolated from the trachea on PID 60, and from the trachea and lungs on PID 120. From these results, the pathogenicity of Pp in immunodeficient rats appears to differ by strain, and VFDP appears to be non-pathogenic in immunodeficient rats.

A Metabolomic-Based Evaluation of the Role of Commensal Microbiota throughout the Gastrointestinal Tract in Mice.

Commensal microbiota colonize the surface of our bodies. The inside of the gastrointestinal tract is one such surface that provides a habitat for them. The gastrointestinal tract is a long organ system comprising of various parts, and each part possesses various functions. It has been reported that the composition of intestinal luminal metabolites between the small and large intestine are different; however, comprehensive metabolomic and commensal microbiota profiles specific to each part of the gastrointestinal lumen remain obscure. In this study, by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome and 16S rRNA gene-based microbiome analyses of specific pathogen-free (SPF) and germ-free (GF) murine gastrointestinal luminal profiles, we observed the different roles of commensal microbiota in each part of the gastrointestinal tract involved in carbohydrate metabolism and nutrient production. We found that the concentrations of most amino acids in the SPF small intestine were higher than those in the GF small intestine. Furthermore, sugar alcohols such as mannitol and sorbitol accumulated only in the GF large intestine, but not in the SPF large intestine. On the other hand, pentoses, such as arabinose and xylose, gradually accumulated from the cecum to the colon only in SPF mice, but were undetected in GF mice. Correlation network analysis between the gastrointestinal microbes and metabolites showed that niacin metabolism might be correlated to Methylobacteriaceae. Collectively, commensal microbiota partially affects the gastrointestinal luminal metabolite composition based on their metabolic dynamics, in cooperation with host digestion and absorption.

Biological and epidemiological evidence of anti-allergic effects of traditional Japanese food ume (Prunus mume).

Japanese apricot (Prunus mume; ume) is a traditional food in Japan that has been shown to have various beneficial health effects. There is some evidence to suggest that ume is also effective against allergic disease. Here, we conducted a cross-sectional epidemiological pilot study to examine the association between ume intake frequency and allergic symptoms including rhinitis in 563 adults (288 men and 275 women) who resided in Wakayama, Japan. After adjusting for age, present illness and medication, women with high ume intake had significantly lower odds ratio (OR) for the presence of symptoms of allergy [OR: 0.49 with 95% confidence interval (CI): 0.25-0.97]. Therefore, we investigated the anti-allergic effect of ume on passive cutaneous anaphylaxis (PCA) reaction in immunoglobulin E (IgE)-sensitized mice. The animal study demonstrated that oral administration of ume extract attenuated the PCA reaction and mast cell degranulation. Furthermore, RBL-2H3 mast cells were used to identify anti-allergic ume compounds. The following ume compounds inhibited IgE-mediated mast cell degranulation: vanillin, syringic acid, protocatechuic aldehyde, lyoniresinol and p-coumaric acid. These results suggested that ume has the potential to inhibit mast cell degranulation and may be associated with reduced risk of allergic symptoms in women.

Composition of fecal microbiota of laboratory mice derived from Japanese commercial breeders using 16S rRNA gene clone libraries.

The fecal microbiota of six mice derived from three Japanese commercial breeders was analyzed by using 16S rRNA gene clone libraries to construct a database for analyzing the gut microbiota of laboratory mice. The 566 clones were obtained from the clone libraries generated from the fecal DNA samples derived from BALB/c, C57BL/6N, DBA/2 and ICR mice. Among these 566 clones, there were 446 unique 16S rRNA gene sequences. When grouped at the 98% similarity level, the 446 unique sequences consisted of 103 Clostridiales, 43 Bacteroidales, 5 Lactobacillus and 3 Erysipelotricaceae, as well as sequences from 11 other phyla.

Constitutive equation of friction based on the subloading-surface concept.

The subloading-friction model is capable of describing static friction, the smooth transition from static to kinetic friction and the recovery to static friction after sliding stops or sliding velocity decreases. This causes a negative rate sensitivity (i.e. a decrease in friction resistance with increasing sliding velocity). A generalized subloading-friction model is formulated in this article by incorporating the concept of overstress for viscoplastic sliding velocity into the subloading-friction model to describe not only negative rate sensitivity but also positive rate sensitivity (i.e. an increase in friction resistance with increasing sliding velocity) at a general sliding velocity ranging from quasi-static to impact sliding. The validity of the model is verified by numerical experiments and comparisons with test data obtained from friction tests using a lubricated steel specimen.