Brewer's yeast cell wall affects microbiota composition and decreases Bacteroides fragilis populations in an anaerobic gut intestinal model

Brewer's yeast cell wall (BYC) has been reported to have prebiotic activity that improves the microbiotal composition of the human gut. To understand the precise effect of BYC on gut microbiota and its metabolism, we used a three-stage continuous-flow reactor system that mimicked the environment of the large intestine. The reactor system was able to maintain the bacterial community stably for a week. The Bacteroides fragilis population decreased drastically after the addition of BYC into this system while the number of Lactobacillus was stably maintained. In addition, propionate and acetate levels increased drastically. This metabolic change correlated with an increase in a number of specific operational taxonomic units annotated to the genus Veillonella and Megasphaella. These results suggest that BYC affects the composition of microbiota in an in vitro model system.     

Aryl hydrocarbon receptor plays a significant role in mediating airborne particulate-induced carcinogenesis in mice

Urban particulate air pollution is associated with an increased incidence of cancers, and especially lung cancer. Organic extracts of airborne particulate matter (APM) cause cancer in mice, and PAHs adsorbed to APM are associated with particle-induced carcinogenesis. PAHs are agonists for AhR and are predominantly responsible for lung cancer through induction of highly carcinogenic metabolites. PAH metabolization requires CYP1A1 induction through activation of AhR, and therefore we hypothesized that carcinogenesis due to PAHs in APM would be reduced in AhR-/- mice. To examine this hypothesis, we performed a long-term continuous-application study of carcinogenesis in AhR-/- mice using airborne particulate extract (APE) of APM collected in Sapporo. Tumor development (squamous cell carcinoma) occurred in 8 of 17 AhR+/+ mice (47%), but no tumors were found in AhR-/-mice, and CYP1A1 was induced in AhR+/+ mice but not in AhR-/- mice. These results demonstrate that AhR plays a significant role in APE-induced carcinogenesis in AhR+/+ mice and CYP1A1 activation of carcinogenic PAHs is also of importance. Therefore, measurement of CYP1A1 induction in vitro may be useful for assessment of APM-induced carcinogenesis in humans. We also show that PAH-like compounds are major contributors to AhR-mediated carcinogenesis, whereas TCDD and related compounds make a smaller contribution.     

Hydrolysis of a mixture of saccharides by cellulase from Aspergillus niger and its application for visible-light-induced hydrogen gas production system using Mg chlorophyll-a and platinum nanoparticles

Cellulase obtained from Aspergillus niger was used to hydrolyze a mixture of saccharides containing sucrose, maltose, and cellobiose; the reduced form of nicotinamide-adenine dinucleotide (NAD⁺), which is NADH, was produced during hydrolysis of the mixture of saccharides in the presence of NAD⁺ and glucose dehydrogenase (GDH). We have developed a visible-light-induced enzymatic biohydrogen production system involving the combination of cellulase-mediated hydrolysis of the mixture of saccharides and hydrogen production by platinum nanoparticles using photosensitization of Mg chlorophyll-a (Mg Chl-a). Continuous production of hydrogen gas was observed when the reaction mixture containing saccharides, cellulase, GDH, NAD⁺, Mg Chl-a, methylviologen (MV²⁺, an electron donor), and platinum nanoparticles was irradiated by visible light. After 120 min of irradiation, the amount of hydrogen produced from the mixture of saccharides was approximately 2.8 μmol.     

Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.)

Cucurbitacins are a group of bitter‐tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter‐tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non‐bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure

An acidulocomposting system for the treatment of cattle manure with little emission of ammonia gas was developed, and the structure of its microbial community was investigated by denaturing gradient gel electrophoresis (DGGE) and clone library construction. An acidulocomposting apparatus (BC20, 20 L) was operated for 79 days to treat 2 kg (wet wt) of garbage per 1 or 2 days. On day 80 of operation, the substrate was changed from garbage to cattle manure (1 kg of beef cattle manure was added to the apparatus every 2 or 3 days), and the system continued operating from days 80 to 158. The compost in the vessel was under acidic conditions at pH 5.2–5.8, and ammonia emission was below the detectable level (< 5 ppm) throughout the period of cattle manure feeding. Total nitrogen and total carbon in the compost were 26–29 and 439 – 466 mg/g of dry weight, respectively, which are higher than those in general cattle manure compost. The main acids accumulated during operation were lactic and acetic. Sequencing analysis targeting the 16S rRNA gene revealed the stable dominance of the bacterial phylum Firmicutes, with a high proportion of the isolates belonging to the genus Bacillus. Using a culturing method with MRS agar, we isolated lactic acid bacteria (LAB) related to Pediococcus acidilactici, Weissella paramesenteroides, and Lactobacillus salivarius, indicating the existence of LAB in the system. These results indicate that acidulocomposting treatment of cattle manure is not accompanied by ammonia emission and that Bacillus and LAB may be the key components in the system.     

Heat Transfer Characteristics of Gaseous Flows in Micro-Channel with Negative Heat Flux

Two-dimensional compressible momentum and energy equations are solved to obtain the heat transfer characteristics of gaseous flows in micro-channels with constant heat flux for which the value is negative for no-slip flow. The numerical methodology is based on the Arbitrary-Lagrangian-Eulerian method. The computations are performed for channels with constant heat flux ranging from ?10⁴ to ?10² W/m². The channel height ranges from 10 to 100 μ m and the aspect ratio of the channel height and length is 200. The stagnation pressure is chosen such that the exit Mach number ranges from 0.1 to 0.7. The outlet pressure is fixed at the atmosphere. The wall and bulk temperatures in micro-channels with negative heat flux are compared with those of positive heat flux cases obtained in our previous work and also those of the incompressible flow in a conventional sized channel. In the case of fast flow, temperatures normalized by heat flux have different trends whether heat flux value is positive or negative. A correlation for the prediction of the wall temperature of the gaseous flow in the micro-channel is proposed. The rarefaction effect is investigated for the cases of channel height of 10 μ m with slip boundary conditions. The magnitudes of viscous dissipation term and compressibility term are also investigated. The effect of each term on heat transfer characteristics is discussed.     

Screening and characterization of transposon-insertion mutants in a pseudohyphal strain of Saccharomyces cerevisiae

Strains of Saccharomyces cerevisiae with sigma1278b background are widely used for elucidation of pseudohyphal differentiation and signal transduction. However, information and resources on the strains are limited compared to the S288C strains. To facilitate functional analysis of strains with sigma1278b background, mutant strains were generated by using a mini-transposon3-3 x HA/LacZ (mTn3)-mutagenized library. Mutants with mTn3 insertions were screened for expression of beta-galactosidase activity under nitrogen starvation and the insertion sites were identified. One hundred and five heterozygous diploid strains were selected and subjected to tetrad analysis. Insertion of mTn3 in 11 genes was lethal in the strain, including three genes, HAC1, TPS1 and UME6, which are non-essential genes according to the Saccharomyces Genome Database. Viable haploid strains with mTn3 insertions were examined for invasive growth, which is a differentiation process in haploid strains including agar penetration on rich medium, and cell morphology during invasive growth. We also examined homozygous diploid strains with mTn3-insertions for filamentous growth and sporulation.     

Saccharomyces cerevisiae QNS1 codes for NAD(+) synthetase that is functionally conserved in mammals

NAD(+), an essential molecule involved in a variety of cellular processes, is synthesized through de novo and salvage pathways. NAD(+) synthetase catalyses the final step in both pathways. Here we show that this enzyme is encoded by the QNS1 gene in Saccharomyces cerevisiae. Expression of Escherichia coli or Bacillus subtilis NAD(+) synthetases was able to suppress the lethality of a qns1 deletion, while a B. subtilis NAD(+) synthetase mutant with lowered catalytic activity was not. Overexpression of QNS1 tagged with HA led to elevated levels of NAD(+) synthetase activity in yeast extracts, and this activity can be recovered by immunoprecipitation using anti-HA antibody. An allele of QNS1 was constructed that carries a point mutation predicted to reduce the catalytic activity. Overexpression of this allele, qns1(G521E), failed to elevate NAD(+) synthetase levels and qns1(G521E) could not rescue the lethality caused by the depletion of Qns1p. These results demonstrate that NAD(+) synthetase activity is essential for cell viability. A GFP-tagged version of Qns1p displayed a diffuse localization in both the nucleus and the cytosol. Finally, the rat homologue of QNS1 was cloned and shown to functionally replace yeast QNS1, indicating that NAD(+) synthetase is functionally conserved from bacteria to yeast and mammals.     

Rapid selection of nonhotspot mutants among hisD+ revertants of Salmonella typhimurium TA98 in Ames test by peptide nucleic acid (PNA)-mediated PCR clamping

Ames test is the most popular method of assessing mutagenicity using Salmonella typhimurium as an indicator. Recently, sequence analyses have been introduced for the investigation of mutation mechanisms. Most revertants (>70%) carry 2-bp deletion within an 8-bp CG repeat in hisD (hotspot mutation) in the Ames test using S. typhimurium TA98. We developed a new specific amplification method for nonhotspot mutants by peptide nucleic acid (PNA)-mediated PCR clamping. It markedly reduces the labor and cost of this kind of studies.     

Methane reforming with CO2 in molten salt using FeO catalyst

Methane dry reforming with CO₂ using FeO powder in molten salt has been investigated at various flow rates of CH₄/CO₂ mixed gases (CH₄/CO₂=1) between 50 and 400 ml/min at 1223 K in an infrared furnace. This work is carried out to determine the usefulness of this method for the chemical storage of solar energy. The CH₄/CO₂ mixed gases passing through the molten salt (Na₂CO₃/K₂CO₃=1) containing the FeO powder were catalytically decomposed into CO, H₂ and H₂O. The product gas mole ratios, CO/H₂/H₂O, were shown to be 3:1:1 for a high flow rate of 200 ml/min and to be CO/H₂=2:1 for a low flow rate of 50 ml/min. The results were explained in terms of the kinetics of the CH₄-reforming reaction and the thermodynamics of the redox process of FeO powder mixed in the molten salt;

CH₄+2FeO2Fe+H₂+CO+H₂O

Fe+CO₂FeO+CO

for a high flow rate, and

FeO+CH₄Fe+2H₂+CO

Fe+CO₂FeO+CO

for a low flow rate.