Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

Conductive Iron Oxides Promote Methanogenic Acetate Degradation by Microbial Communities in a High-Temperature Petroleum Reservoir.

Supplementation with conductive magnetite particles promoted methanogenic acetate degradation by microbial communities enriched from the production water of a high-temperature petroleum reservoir. A microbial community analysis revealed that Petrothermobacter spp. (phylum Deferribacteres), known as thermophilic Fe(III) reducers, predominated in the magnetite-supplemented enrichment, whereas other types of Fe(III) reducers, such as Thermincola spp. and Thermotoga spp., were dominant under ferrihydrite-reducing conditions. These results suggest that magnetite induced interspecies electron transfer via electric currents through conductive particles between Petrothermobacter spp. and methanogens. This is the first evidence for possible electric syntrophy in high-temperature subsurface environments.

Diversity of lactic acid bacteria from Miang, a traditional fermented tea leaf in northern Thailand and their tannin-tolerant ability in tea extract.

The microbiota of lactic acid bacteria (LAB) in thirty-five samples of Miang, a traditional fermented tea leaf product, collected from twenty-two different regions of eight provinces in upper northern Thailand was revealed through the culture-dependent technique. A total of 311 presumptive LAB strains were isolated and subjected to clustering analysis based on repetitive genomic element-PCR (rep-PCR) fingerprinting profiles. The majority of the strains belonged to the Lactobacillus genera with an overwhelming predominance of the Lb. plantarum group. Further studies of species-specific PCR showed that 201 of 252 isolates in the Lb. plantarum group were Lb. plantarum which were thus considered as the predominant LAB in Miang, while the other 51 isolates belonged to Lb. pentosus. In contrast to Lb. plantarum, there is a lack of information on the tannase gene and the tea tannin-tolerant ability of Lb. pentosus. Of the 51 Lb. pentosus isolates, 33 were found to harbor the genes encoding tannase and shared 93-99% amino acid identity with tannase obtained from Lb. pentosus ATCC 8041T. Among 33 tannase gene-positive isolates, 23 isolates exhibited high tannin- tolerant capabilities when cultivated on de Man Rogosa and Sharpe agar-containing bromocresol purple (0.02 g/L, MRS-BCP) supplemented with 20% (v/v) crude tea extract, which corresponded to 2.5% (w/v) tannins. These Lb. pentosus isolates with high tannin-tolerant capacity are expected to be the high potential strains for functional tannase production involved in Miang fermentation as they will bring about certain benefits and could be used to improve the fermentation of tea products.

Restoration of the growth of Escherichia coli under K+-deficient conditions by Cs+ incorporation via the K+ transporter Kup.

Biological incorporation of cesium ions (Cs+) has recently attracted significant attention in terms of the possible applications for bioremediation of radiocesium and their significant roles in biogeochemical cycling. Although high concentrations of Cs+ exhibit cytotoxicity on microorganisms, there are a few reports on the promotive effects of Cs+ on microbial growth under K+-deficient conditions. However, whether this growth-promoting effect is a common phenomenon remains uncertain, and direct correlation between growth promotion and Cs+ uptake abilities has not been confirmed yet. Here, we validated the growth promotive effects of Cs+ uptake under K+-deficient conditions using an Escherichia coli strain with an inducible expression of the Kup K+ transporter that has nonspecific Cs+ transport activities (strain kup-IE). The strain kup-IE exhibited superior growth under the Cs+-supplemented and K+-deficient conditions compared to the wild type and the kup null strains. The intracellular Cs+ levels were significantly higher in strain kup-IE than in the other strains, and were well correlated with their growth yields. Furthermore, induction levels of the kup gene, intracellular Cs+ concentrations, and the growth stimulation by Cs+ also correlated positively. These results clearly demonstrated that Cs+ incorporation via Kup transporter restores growth defects of E. coli under K+-deficient conditions.

Enrichment and isolation of Flavobacterium strains with tolerance to high concentrations of cesium ion.

Interest in the interaction of microorganisms with cesium ions (Cs(+)) has arisen, especially in terms of their potent ability for radiocesium bioaccumulation and their important roles in biogeochemical cycling. Although high concentrations of Cs(+) display toxic effects on microorganisms, there have been only limited reports for Cs(+)-tolerant microorganisms. Here we report enrichment and isolation of Cs(+)-tolerant microorganisms from soil microbiota. Microbial community analysis revealed that bacteria within the phylum Bacteroidetes, especially Flavobacterium spp., dominated in enrichment cultures in the medium supplemented with 50 or 200 mM Cs(+), while Gammaproteobacteria was dominant in the control enrichment cultures (in the presence of 50 and 200 mM K(+) instead of Cs(+)). The dominant Flavobacterium sp. was successfully isolated from the enrichment culture and was closely related to Flavobacterium chungbukense with 99.5% identity. Growth experiments clearly demonstrated that the isolate has significantly higher tolerance to Cs(+) compared to its close relatives, suggesting the Cs(+)-tolerance is a specific trait of this strain, but not a universal trait in the genus Flavobacterium. Measurement of intracellular K(+) and Cs(+) concentrations of the Cs(+)-tolerant isolate and its close relatives suggested that the ability to maintain low intracellular Cs(+) concentration confers the tolerance against high concentrations of external Cs(+).

Acute effect of soybean beta-conglycinin hydrolysate ingestion on appetite sensations in healthy humans.

A hydrolysate prepared from soybean beta-conglycinin reduced food intake through cholecystokinin release in rats; however, effects of the hydrolysate on human appetites are unknown. In this study, healthy volunteers ingested 3g of the beta-conglycinin hydrolysate (BconB) and/or a soy protein hydrolysate (HN) contained in a beverage or in a jelly. Appetite profiles (hunger, fullness and prospective consumption) after the ingestion and palatability of test jellies were recorded. Fullness was rated higher, and hunger was rated lower after BconB ingestion as compared to HN ingestion. These results demonstrate that 3g of BconB is effective to enhance fullness and reduce hunger sensations in healthy humans.

Soybean ß-conglycinin bromelain hydrolysate stimulates cholecystokinin secretion by enteroendocrine STC-1 cells to suppress the appetite of rats under meal-feeding conditions.

A peptic digest of soybean ß-conglycinin (BconP) suppresses the appetite in rats through cholecystokinin (CCK) secretion by enteroendocrine cells. We investigate in this study more appetite-suppressing hydrolysates. ß-Conglycinin hydrolyzed with food-processing proteases thermolysin (BconT), bromelain (BconB), chymotrypsin, protease S, and protease M was examined for CCK-secreting activity in a CCK-producing cell line for comparison with BconP. The potent CCK-releasing hydrolysates were then tested for their suppression of the food intake by rats. BconB, BconT, and BconP stimulated high CCK secretion, with the highest by BconB. Orogastric preloading by BconB, but not by BconT, suppressed the 60-min food intake. A meal-feeding trial twice a day in the morning (a.m.) and evening (p.m.) for 10 d showed that BconB preloading before every meal attenuated the p.m. meal size, but not that a.m., resulting in an overall reduction of the daily meal size. These results demonstrate that the bromelain hydrolysate of ß-conglycinin having potent CCK-releasing activity suppressed the appetite of rats under meal-feeding conditions.

Production of equol from daidzein by gram-positive rod-shaped bacterium isolated from rat intestine.

Isoflavones (mainly daidzein and genistin) belong to the flavonoid group of compounds and are classified as phytoestrogens. In the intestine, daidzin is converted to daidzein by beta-glucosidase, and then daidzein is converted to O-desmethylangolensin (O-DMA) or equol via dihydrodaidzein by enzymes of intestinal bacteria. We isolated, for the first time, an anaerobic gram-positive rod-shaped strain capable of producing equol from daidzein. Its 16S rDNA gene sequence (1428 bp) showed 99% similarity with that of the human intestinal bacterium SNU-Julong 732 (AY310748) and 93% similarity with that of Eggerthella lenta ATCC 25559(T) (AF292375). This strain converted daidzein to equol via dihydrodaidzein in an equol-assay medium anaerobically. The addition of butyric acid and arginine increased the conversion ratio of daidzein to equol 4.7- and 4.5-fold, respectively.

Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve.

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.

Effects of difructose anhydride III (DFA III) administration on rat intestinal microbiota.

The effects of difructose anhydride III (di-D-fructofuranose-1,2':2,3'-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA III-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340T (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.

Effects of difructose anhydride III (DFA III) administration on bile acids and growth of DFA III-assimilating bacterium Ruminococcus productus on rat intestine.

The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 10(8)-10(9) cells/g dry feces within a week from 10(6) cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.