Caged Xanthones and Biphenyls Isolated from the Tropical Plant Garcinia lateriflora.

Four cytotoxic heptacyclic caged-xanthones [gambogefic acids B-E (1-4)], a cytotoxic hexacyclic caged-xanthone [garcilatelic acid (5)], and four biphenyl derivatives [garcilatelibiphenyls A-D (6-9)] were newly isolated in a phytochemical study of a 50% MeOH/CH2Cl2 extract of Garcinia lateriflora (Clusiaceae). The isolated compounds were evaluated for antiproliferative activity against five human tumor cell lines including a vincristine-resistant line. The new caged-xanthones displayed potent activity with IC50 values from 0.5 to 6.7 µM against all tested tumor cell lines.

Sign-constrained linear regression for prediction of microbe concentration based on water quality datasets.

This study presents a novel methodology for estimating the concentration of environmental pollutants in water, such as pathogens, based on environmental parameters. The scientific uniqueness of this study is the prevention of excess conformity in the model fitting by applying domain knowledge, which is the accumulated scientific knowledge regarding the correlations between response and explanatory variables. Sign constraints were used to express domain knowledge, and the effect of the sign constraints on the prediction performance using censored datasets was investigated. As a result, we confirmed that sign constraints made prediction more accurate compared to conventional sign-free approaches. The most remarkable technical contribution of this study is the finding that the sign constraints can be incorporated in the estimation of the correlation coefficient in Tobit analysis. We developed effective and numerically stable algorithms for fitting a model to datasets under the sign constraints. This novel algorithm is applicable to a wide variety of the prediction of pollutant contamination level, including the pathogen concentrations in water.

Cell-free analysis of polyQ-dependent protein aggregation and its inhibition by chaperone proteins.

Protein misfolding and aggregation is one of the major causes of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and Huntington's disease. So far protein aggregation related to these diseases has been studied using animals, cultured cells or purified proteins. In this study, we show that a newly synthesized polyglutamine protein implicated in Huntington's disease forms large aggregates in HeLa cells, and successfully recapitulate the process of this aggregation using a translation-based system derived from HeLa cell extracts. When the cell-free translation system was pre-incubated with recombinant human cytosolic chaperonin CCT, or the Hsc70 chaperone system (Hsc70s: Hsc70, Hsp40, and Hsp110), aggregate formation was inhibited in a dose-dependent manner. In contrast, when these chaperone proteins were added in a post-translational manner, aggregation was not prevented. These data led us to suggest that chaperonin CCT and Hsc70s interact with nascent polyglutamine proteins co-translationally or immediately after their synthesis in a fashion that prevents intra- and intermolecular interactions of aggregation-prone polyglutamine proteins. We conclude that the in vitro approach described here can be usefully employed to analyze the mechanisms that provoke polyglutamine-driven protein aggregation and to screen for molecules to prevent it.

Estimation of concentration ratio of indicator to pathogen-related gene in environmental water based on left-censored data.

A stochastic model for estimating the ratio between a fecal indicator and a pathogen based on left-censored data, which includes a substantially high number of non-detects, was constructed. River water samples were taken for 16 months at six points in a river watershed, and conventional fecal indicators (total coliforms and general Escherichia coli), genetic markers (Bacteroides spp.), and virulence genes (eaeA of enteropathogenic E. coli and ciaB of Campylobacter jejuni) were quantified. The quantification of general E. coli failed to predict the presence of the virulence gene from enteropathogenic E. coli, different from what happened with genetic markers (Total Bac and Human Bac). A Bayesian model that was adapted to left-censored data with a varying analytical quantification limit was applied to the quantitative data, and the posterior predictive distributions of the concentration ratio were predicted. When the sample size was 144, simulations conducted in this study suggested that 39 detects were enough to accurately estimate the distribution of the concentration ratio, when combined with a dataset with a positive rate higher than 99%. To evaluate the level of accuracy in the estimation, it is desirable to perform a simulation using an artificially generated left-censored dataset that has the identical number of non-detects as the actual data.

Peyer's patch innate lymphoid cells regulate commensal bacteria expansion.

Anatomical containment of commensal bacteria in the intestinal mucosa is promoted by innate lymphoid cells (ILCs). However, the mechanism by which ILCs regulate bacterial localization to specific regions remains unknown. Here we show that Peyer's patch (PP) ILCs robustly produce IL-22 and IFN-γ in the absence of exogenous stimuli. Antibiotic treatment of mice decreased both IL-22+ and IFN-γ+ cells in PPs. Blockade of both IL-2 and IL-23 signaling in vitro lowered IL-22 and IFN-γ production. PP ILCs induced mRNA expression of the antibacterial proteins RegIIIß and RegIIIγ in intestinal epithelial cells. Furthermore, in vivo depletion of ILCs rather than T cells altered bacterial composition and allowed bacterial proliferation in PPs. Collectively, our results show that ILCs regulate the expansion of commensal bacteria in PPs.

IL-33 activates eosinophils of visceral adipose tissue both directly and via innate lymphoid cells.

Eosinophils are multifunctional leukocytes involved in allergic reactions as well as adipose tissue regulation. IL-5 is required for eosinophil survival; however, the in vivo mechanisms of eosinophil regulation are not fully understood. A tg mouse model with il5 promoter-driven EGFP expression was established for detecting the IL-5-producing cells in vivo. Il5-egfp tg mice expressed high levels of EGFP in gonadal adipose tissue (GAT) cells. EGFP(+) cells in GAT were mainly group 2 innate lymphoid cells (ILCs). IL-33 preferentially expanded EGFP(+) cells and eosinophils in GAT in vivo. EGFP(+) ILCs were found to upregulate prg2 mRNA expression in GAT eosinophils. These results demonstrate that ILCs activate eosinophils in GAT. The blockage of IL-33Rα, on the other hand, did not impair EGFP(+) ILC numbers but did impair eosinophil numbers in vivo. GAT eosinophils expressed IL-33Rα and IL-33 expanded eosinophil numbers in CD90(+) cell-depleted mice. IL-33 was further observed to induce the expression of retnla and epx mRNA in eosinophils. These findings demonstrate that IL-33 directly activates eosinophils in GAT, and together with our other findings described above, our findings show that IL-33 has dual pathways via which it activates eosinophils in vivo: a direct activation pathway and a group 2 ILC-mediated pathway.

Microfluidic quantitative PCR for simultaneous quantification of multiple viruses in environmental water samples.

To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/µl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.

Water quality monitoring and risk assessment by simultaneous multipathogen quantification.

Water quality monitoring and microbial risk assessment are important to ensure safe water for drinking, recreational, and agricultural purposes. In this study, we applied a microfluidic quantitative PCR (MFQPCR) approach to simultaneously quantify multiple waterborne pathogens in a natural freshwater lake in Hokkaido, Japan, from April to November, 2012. Tens of thousands of geese stopped over at this lake during their migration in spring and fall. Because lake water is used for irrigation of the surrounding agricultural area, we assessed infection risks through irrigation water usage based on pathogen concentrations directly measured by MFQPCR. We detected various pathogens in the lake water, particularly during the bird migration seasons, suggesting that migratory birds were the main source of the pathogens. However, neither counts of geese nor fecal indicator bacteria were good predictors of pathogen concentrations. On the basis of quantitative microbial risk assessment, concentrations of Campylobacter jejuni and Shigella spp. in water samples were above the concentrations that can potentially cause 10(-4) infections per person per year when water is used to grow fresh vegetables. These results suggest that direct and simultaneous multipathogen quantification can provide more reliable and comprehensive information for risk assessment than the current fecal indicator-based approach.

NFκB attenuates IL-5 production and upregulates T-box transcription factors in Th2-like T cells.

IL-5 plays important roles in eosinophil differentiation, expansion, and recruitment. The regulation of IL-5 seems critical for the treatment of eosinophil-mediated allergic reactions. However, the precise mechanisms for IL-5 regulation remain unknown. In this study, we investigated how IL-5 production is regulated. The transduction of GATA-3 into a murine T cell hybridoma resulted in acquiring the ability to produce IL-5 in response to an antigenic stimulus like Th2 cells. This production was dependent on the cAMP-PKA pathway, but not on p38 activation. Transduction of NIK largely impaired IL-5 production. RelA and RelB similarly impaired IL-5 production. RelA decreased not only IL-5 protein amount but mRNA. RelA also inhibited Il5-luciferase reporter activity. The transduction of GATA-3 decreased the expression of Tbx21 and Eomes, but the additional transduction of RelA abrogated the decreased expression of GATA-3-induced Tbx21 and Eomes. Furthermore, the transduction of T-bet or Eomes into the GATA-3-transduced T cell hybridoma impaired IL-5 production. These results suggested that strong enhancement of the NFκB pathway downregulates IL-5 production and upregulates T-box protein expression to shift an immune response from Th2 to inflammatory Th1.

Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers.

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.

CD2-mediated regulation of peripheral CD4(+) CD25(+) regulatory T-cell apoptosis accompanied by down-regulation of Bim.

Extensive studies on CD4(+)  CD25(+) regulatory T (Treg) cells suggest that they are important in regulating immune responses. However, mechanisms of peripheral Treg cell homeostasis are unknown. We found that stromal cells isolated from secondary lymphoid organs such as spleen and lymph nodes could support the survival of Treg cells. This was dependent on CD2 engagement and a direct interaction between Treg cells and stromal cells. In the presence of stromal cells, Bim, a pro-apoptotic factor, was partially decreased in Treg cells. This effect could be inhibited by anti-CD2 blocking antibodies, indicating that stimulation through CD2 on Treg cells regulates Bim expression, which may be relevant to Treg cell apoptosis. Therefore, Treg cell interactions with stromal cells through CD2 may be essential for Treg cell survival. Surprisingly, the expression of CD2 ligands on stromal cells was not detected. Hence, it is not clear how CD2 on Treg cells contributes to a direct interaction with the stromal cells and participates in survival support for Treg cells. Taken together, CD2 stimuli were mandatory for Treg cell survival with reduced Bim expression, but CD2 may not function as a direct receptor for molecules on stromal cells.

Effects of temperature and predator on the persistence of host-specific Bacteroides-Prevotella genetic markers in water.

Genetic markers derived from Bacteroidales spp. have been proposed as promising indicators for fecal contamination in the water environment. However, little is known about the persistency of Bacteroidales spp. 16S rRNA genetic markers in the natural environment, which hampers the precise identification of fecal contamination sources. In this study, the persistency of human-specific Bacteroidales spp. genetic markers in river water was investigated during a 3-week agitation. The copy number of Bacteroidales spp. genetic marker was decreased with agitation time, and was very sensitive to water temperature. After the 3-week agitation, three clones of 18S rRNA gene related to Glaucoma scintillans, Spumella-like flagellate, and Colpidium campylum were acquired. The presence of predators that can prey on target bacteria could also be a critical factor affecting the quantified value of genetic markers. It is very important to take these factors, water temperature and the presence of predator, into account for predicting the fate of genetic markers to accurately identify fecal pollution sources.

Chicken- and duck-associated Bacteroides-Prevotella genetic markers for detecting fecal contamination in environmental water.

Bacteroides-Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides-Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61 %, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70 % of chicken feces, while detected from 39 % of cow feces, 8.3 % of pig feces, and 12 % of swan feces. Duck-Bac marker was detected from 85 % of wild duck feces and cross-reacted with 31 % of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides-Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.

Differentiation stage-specific requirement in hypoxia-inducible factor-1alpha-regulated glycolytic pathway during murine B cell development in bone marrow.

Hypoxia-inducible factor (HIF)-1alpha plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that an HIF-1alpha gene deficiency caused abnormal B cell development and autoimmunity. In this study we show that HIF-1alpha-enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1alpha-deficient B220(+) bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3, was greatly reduced in HIF-1alpha-deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory chain-related genes and TCA cycle-related genes in HIF-1alpha-deficient cells than in wild-type cells. In agreement with these findings, HIF-1alpha-deficient cells used pyruvate more efficiently than wild-type cells. The key role of HIF-1alpha-enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1alpha plays an important role in B cell development.