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We investigated the succession process of intestinal bacteria during seed production in full-cycle aquaculture of Pacific bluefin tuna (Thunnus orientalis). During the seed production, eggs, healthy fish, rearing water, and feeds from three experimental rounds in 2012 and 2013 were collected before transferring to offshore net cages and subjected to a fragment analysis of the bacterial community structure. We identified a clear succession of intestinal bacteria in bluefin tuna during seed production. While community structures of intestinal bacteria in the early stage of larvae were relatively similar to those of rearing water and feed, the bacterial community structures seen 17 days after hatching were different. Moreover, although intestinal bacteria in the late stage of larvae were less diverse than those in the early stage of larvae, the specific bacteria were predominant, suggesting that the developed intestinal environment of the host puts selection pressure on the bacteria in the late stage. The specific bacteria in the late stage of larvae, which likely composed 'core microbiota', were also found on the egg surface. The present study highlights that proper management of the seed production process, including the preparation of rearing water, feeds, and fish eggs, is important for the aquaculture of healthy fish., Competing Interests: The authors have declared that no competing interests exist.

IgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide. Pediatric patients in Japan are diagnosed with IgAN at an early stage of the disease through annual urinary examinations. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible 14 (Fn14) have various roles, including proinflammatory effects, and modulation of several kidney diseases; however, no reports have described their roles in pediatric IgAN. In this study, we performed pathological and immunohistochemical analyses of samples from 14 pediatric IgAN patients. Additionally, gene expression arrays of glomeruli by laser-captured microdissection were performed in hemi-nephrectomized high serum IgA (HIGA) mice, a model of IgA nephropathy, to determine the role of Fn14. Glomeruli with intense Fn14 deposition were observed in 80% of mild IgAN cases; however, most severe cases showed glomeruli with little or no Fn14 deposition. Fn14 deposition was not observed in obvious mesangial proliferation or the crescent region of glomeruli, but was detected strongly in the glomerular tuft, with an intact appearance. In HIGA mice, Fn14 deposition was observed mildly beginning at 11 weeks of age, and stronger Fn14 deposition was detected at 14 weeks of age. Expression array analysis indicated that Fn14 expression was higher in HIGA mice at 6 weeks of age, increased slightly at 11 weeks, and then decreased at 26 weeks when compared with controls at equivalent ages. These findings suggest that Fn14 signaling affects early lesions but not advanced lesions in patients with IgAN. Further study of the TWEAK/Fn14 pathway will contribute to our understanding of the progression of IgAN., Competing Interests: The authors have declared that no competing interests exist.

Objective: Accumulating evidence suggests that amino acids, particularly tryptophan and glutamate, play an important role in the pathology of depression, but prospective epidemiologic data on this issue is scarce. We examined the association between circulating amino acids and the risk of depressive symptoms in a Japanese working population., Methods: Participants were 841 workers who were free from depressive symptoms and provided blood at baseline and completed 3-yr follow-up survey. 30 varieties of amino acid concentrations in serum were measured using liquid chromatography/mass spectrometry. Depressive symptoms were defined using the Center for Epidemiologic Studies Depression Scale. Logistic regression was used to calculate the odds ratios of depressive symptoms according to serum amino acids with adjustment for lifestyle factors., Results: A total of 151 (18.0%) workers were newly identified as having depressive symptoms at the follow-up. Baseline tryptophan and glutamate concentrations in serum were not appreciably associated with the risk of depressive symptoms. Risk of depressive symptoms tended to increase with increasing arginine concentrations; the multivariable-adjusted odds ratio for the highest versus lowest tertile of serum arginine was 1.65 (95% confidence interval: 0.96-2.83; P for trend = 0.07). No clear association was found for other amino acids., Conclusions: Results of the present study do not support a significant role of circulating amino acids in the development of depressive symptoms among Japanese., Competing Interests: T.K. and M.E. are health professionals at the Furukawa Electric Corporation. Author I. Kabe is a health professional at the Kubota Corporation. All authors declare no conflict of interest, patents, products in development or marketed products etc. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

In fish-farming areas, copious amounts of organic matter are released into the surrounding environment. Although it is well-known that bacterial community structures and activities are tightly coupled with organic conditions in the environment, actively growing bacteria (AGB) species that are responsible are still largely unknown. Here, we determined seasonal variations in the community structures of free-living and particle-attached AGB in surface and bottom seawater, and also in the easily resuspendable sediment boundary layer. Accordingly, we used bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) analysis. Whereas overall bacterial communities in the resuspendable sediment were quite different from those of the free-living and particle-attached bacteria, the AGB community structures were similar among them. This result suggests that sediment resuspension in aquaculture environments functions as an organic source for bacteria in the water column, and that bacterial species contributing to the environmental capacity and carbon cycle are limited. We identified 25 AGB phylotypes, belonging to Alphaproteobacteria (Roseobacter clade, nine phylotypes), Gammaproteobacteria (five phylotypes), Deltaproteobacteria (one phylotype), Bacteroidetes (seven phylotypes), and Actinobacteria (three phylotypes). Among them, some AGB phylotypes appeared throughout the year with high frequency and were also identified in other coastal environments. This result suggests that these species are responsible for the environmental capacity and carbon cycle, and are key species in this fish-farming area, as well as other coastal environments., Competing Interests: The authors have declared that no competing interests exist.

[This corrects the article DOI: 10.1371/journal.pone.0142779.].

Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133+ cell-derived EPCs. We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133+ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133+ cord blood EPCs. In contrast, hDll-1 had little effect. CD133+ cells stimulated by hJagged-1 differentiated into CD31+/KDR+ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133+ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133+ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis., Competing Interests: The authors have declared that no competing interests exist.

Aims: The control of blood glucose levels, blood pressure (BP), and low-density lipoprotein cholesterol (LDL-C) levels reduces the risk of diabetes complications; however, data are scarce on control status of these factors among workers with diabetes. The present study aimed to estimate the prevalence of participants with diabetes who meet glycated hemoglobin (HbA1c), BP, and LDL-C recommendations, and to investigate correlates of poor glycemic control in a large working population in Japan., Methods: The Japan Epidemiology Collaboration on Occupational Health (J-ECOH) Study is an ongoing cohort investigation, consisting mainly of employees in large manufacturing companies. We conducted a cross-sectional analysis of 3,070 employees with diabetes (2,854 men and 216 women) aged 20-69 years who attended periodic health examinations. BP was measured and recorded using different company protocols. Risk factor targets were defined using both American Diabetes Association (ADA) guidelines (HbA1c < 7.0%, BP < 140/90 mmHg, and LDL-C < 100 mg/dL) and Japan Diabetes Society (JDS) guidelines (HbA1c < 7.0%, BP < 130/80 mmHg, and LDL-C < 120 mg/dL). Logistic regression models were used to explore correlates of poor glycemic control (defined as HbA1c ≥ 8.0%)., Results: The percentages of participants who met ADA (and JDS) targets were 44.9% (44.9%) for HbA1c, 76.6% (36.3%) for BP, 27.1% (56.2%) for LDL-C, and 11.2% (10.8%) for simultaneous control of all three risk factors. Younger age, obesity, smoking, and uncontrolled dyslipidemia were associated with poor glycemic control. The adjusted odds ratio of poor glycemic control was 0.58 (95% confidence interval, 0.46-0.73) for participants with treated but uncontrolled hypertension, and 0.47 (0.33-0.66) for participants with treated and controlled hypertension, as compared with participants without hypertension. There was no significant difference in HbA1c levels between participants with treated but uncontrolled hypertension and those with treated and controlled hypertension., Conclusion: Data from a large working population, predominantly composed of men, suggest that achievement of HbA1c, BP, and LDL-C targets was less than optimal, especially in younger participants. Uncontrolled dyslipidemia was associated with poor glycemic control. Participants not receiving antihypertensive treatment had higher HbA1c levels.

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

Objective: Risk models and scores have been developed to predict incidence of type 2 diabetes in Western populations, but their performance may differ when applied to non-Western populations. We developed and validated a risk score for predicting 3-year incidence of type 2 diabetes in a Japanese population., Methods: Participants were 37,416 men and women, aged 30 or older, who received periodic health checkup in 2008-2009 in eight companies. Diabetes was defined as fasting plasma glucose (FPG) ≥ 126 mg/dl, random plasma glucose ≥ 200 mg/dl, glycated hemoglobin (HbA1c) ≥ 6.5%, or receiving medical treatment for diabetes. Risk scores on non-invasive and invasive models including FPG and HbA1c were developed using logistic regression in a derivation cohort and validated in the remaining cohort., Results: The area under the curve (AUC) for the non-invasive model including age, sex, body mass index, waist circumference, hypertension, and smoking status was 0.717 (95% CI, 0.703-0.731). In the invasive model in which both FPG and HbA1c were added to the non-invasive model, AUC was increased to 0.893 (95% CI, 0.883-0.902). When the risk scores were applied to the validation cohort, AUCs (95% CI) for the non-invasive and invasive model were 0.734 (0.715-0.753) and 0.882 (0.868-0.895), respectively. Participants with a non-invasive score of ≥ 15 and invasive score of ≥ 19 were projected to have >20% and >50% risk, respectively, of developing type 2 diabetes within 3 years., Conclusions: The simple risk score of the non-invasive model might be useful for predicting incident type 2 diabetes, and its predictive performance may be markedly improved by incorporating FPG and HbA1c.

Aims: To examine the association of smoking status, smoking intensity, and smoking cessation with the risk of type 2 diabetes (T2D) using a large database., Methods: The present study included 53,930 Japanese employees, aged 15 to 83 years, who received health check-up and did not have diabetes at baseline. Diabetes was defined as fasting plasma glucose ≥126 mg/dl, random plasma glucose ≥200 mg/dl, HbA1c ≥6.5% (≥48 mmol/mol), or receiving medication for diabetes. Cox proportional-hazards regression models were used to investigate the association between smoking and the risk of diabetes., Results: During 3.9 years of median follow-up, 2,441 (4.5%) individuals developed T2D. The multivariable-adjusted hazard ratios (95% CI) for diabetes were 1 (reference), 1.16 (1.04 to 1.30) and 1.34 (1.22 to 1.48) for never smokers, former smokers, and current smokers, respectively. Diabetes risk increased with increasing numbers of cigarette consumption among current smokers (P for trend <0.001). Although the relative risk of diabetes was greater among subjects with lower BMIs (< 23 kg/m2), attributable risk was greater in subjects with higher BMIs (≥ 23 kg/m2). Compared with individuals who had never smoked, former smokers who quit less than 5 years, 5 to 9 years, and 10 years or more exhibited hazards ratios for diabetes of 1.36 (1.14 to 1.62), 1.23 (1.01 to 1.51), and 1.02 (0.85 to 1.23), respectively., Conclusions: Results suggest that cigarette smoking is associated with an increased risk of T2D, which may decrease to the level of a never smoker after 10 years of smoking cessation.

Objective: Epidemiologic evidence on long working hour and diabetes has been conflicting. We examined the association between overtime work and prevalence of diabetes among Japanese workers., Methods: The subjects were 40,861 employees (35,170 men and 5,691 women), aged 16 to 83 years, of 4 companies in Japan. Hours of overtime were assessed using self-reported questionnaires. Diabetes was defined as a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l), hemoglobin A1c ≥6.5% (48 mmol/mol), or current use of anti-diabetic drug. Multiple logistic regression analysis was used to calculate odds ratio of diabetes for each category of overtime., Results: After adjustment for age, sex, company, smoking, and BMI, there was a suggestion of U-shaped relationship between overtime work and prevalence of diabetes (P for quadratic trend = 0.07). Compared with those who worked <45 hours of overtime per month, the adjusted odds ratios (95% confidence interval) of diabetes were 0.86 (0.77-0.94), 0.69 (0.53-0.89), and 1.03 (0.72-1.46) for those who worked 45-79, 80-99, and ≥100 hours of overtime per month, respectively. In one company (n = 33,807), where other potential confounders including shift work, job position, type of department, alcohol consumption, sleep duration, leisure time physical activity, and family history of diabetes was additionally adjusted for, similar result was obtained (P for quadratic trend = 0.05)., Conclusions: Long hours of overtime work may not be associated with increased prevalence of diabetes among Japanese workers.

Diabetes is associated with higher incidence of myocardial infarction (MI) and increased propensity for subsequent events post-MI. Here we conducted a temporal analysis of the influence of diabetes on cardiac dysfunction and remodeling after ischemia reperfusion (IR) injury in mice. Diabetes was induced using streptozotocin and IR performed by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for up to 42 days. We first evaluated changes in cardiac function using echocardiography after 24 hours reperfusion and observed IR injury significantly decreased the systolic function, such as ejection fraction, fractional shortening and end systolic left ventricular volume (LVESV) in both control and diabetic mice. The longitudinal systolic and diastolic strain rate were altered after IR, but there were no significant differences between diabetic mice and controls. However, a reduced ability to metabolize glucose was observed in the diabetic animals as determined by PET-CT scanning using 2-deoxy-2-((18)F)fluoro-D-glucose. Interestingly, after 24 hours reperfusion diabetic mice showed a reduced infarct size and less apoptosis indicated by TUNEL analysis in heart sections. This may be explained by increased levels of autophagy detected in diabetic mice hearts. Similar increases in IR-induced macrophage infiltration detected by CD68 staining indicated no change in inflammation between control and diabetic mice. Over time, control mice subjected to IR developed mild left ventricular dilation whereas diabetic mice exhibited a decrease in both end diastolic left ventricular volume and LVESV with a decreased intraventricular space and thicker left ventricular wall, indicating concentric hypertrophy. This was associated with marked increases in fibrosis, indicted by Masson trichrome staining, of heart sections in diabetic IR group. In summary, we demonstrate that diabetes principally influences distinct IR-induced chronic changes in cardiac function and remodeling, while a smaller infarct size and elevated levels of autophagy with similar cardiac function are observed in acute phase.

Background: Phytoplankton cultures are widely used in aquaculture for a variety of applications, especially as feed for fish larvae. Phytoplankton cultures are usually grown in outdoor tanks using natural seawater and contain probiotic or potentially pathogenic bacteria. Some Roseobacter clade isolates suppress growth of the fish pathogen Vibrio anguillarum. However, most published information concerns interactions between probiotic and pathogenic bacteria, and little information is available regarding the importance of phytoplankton in these interactions. The objectives of this study, therefore, were to identify probiotic Roseobacter clade members in phytoplankton cultures used for rearing fish larvae and to investigate their inhibitory activity towards bacterial fish pathogens in the presence of the phytoplankton Nannochloropsis oculata., Methodology/principal Findings: The fish pathogen V. anguillarum, was challenged with 6 Roseobacter clade isolates (Sulfitobacter sp. (2 strains), Thalassobius sp., Stappia sp., Rhodobacter sp., and Antarctobacter sp.) from phytoplankton cultures under 3 different nutritional conditions. In an organic nutrient-rich medium (VNSS), 6 Roseobacter clade isolates, as well as V. anguillarum, grew well (10(9) CFU/ml), even when cocultured. In contrast, in a phytoplankton culture medium (ESM) based on artificial seawater, coculture with the 6 isolates decreased the viability of V. anguillarum by approximately more than 10-fold. Excreted substances in media conditioned by growth of the phytoplankton N. oculata (NCF medium) resulted in the complete eradication of V. anguillarum when cocultured with the roseobacters. Autoclaved NCF had the same inhibitory effect. Furthermore, Sulfitobacter sp. much more efficiently incorporated (14)C- photosynthetic metabolites ((14)C-EPM) excreted by N. oculata than did V. anguillarum., Conclusion/significance: Cocultures of a phytoplankton species and Roseobacter clade members exhibited a greater antibacterial effect against an important fish pathogen (V. anguillarum) than roseobacters alone. Thus, cooperation of N. oculata, and perhaps other phytoplankton species, with certain roseobacters might provide a powerful tool for eliminating fish pathogens from fish-rearing tanks.