Extracellular Vesicles Produced by Bifidobacterium longum Export Mucin-Binding Proteins.

Extracellular proteins are important factors in host-microbe interactions; however, the specific factors that enable bifidobacterial adhesion and survival in the gastrointestinal (GI) tract are not fully characterized. Here, we discovered that Bifidobacterium longum NCC2705 cultured in bacterium-free supernatants of human fecal fermentation broth released a myriad of particles into the extracellular environment. The aim of this study was to characterize the physiological properties of these extracellular particles. The particles, approximately 50 to 80 nm in diameter, had high protein and double-stranded DNA contents, suggesting that they were extracellular vesicles (EVs). A proteomic analysis showed that the EVs primarily consisted of cytoplasmic proteins with crucial functions in essential cellular processes. We identified several mucin-binding proteins by performing a biomolecular interaction analysis of phosphoketolase, GroEL, elongation factor Tu (EF-Tu), phosphoglycerate kinase, transaldolase (Tal), and heat shock protein 20 (Hsp20). The recombinant GroEL and Tal proteins showed high binding affinities to mucin. Furthermore, the immobilization of these proteins on microbeads affected the permanence of the microbeads in the murine GI tract. These results suggest that bifidobacterial exposure conditions that mimic the intestine stimulate B. longum EV production. The resulting EVs exported several cytoplasmic proteins that may have promoted B. longum adhesion. This study improved our understanding of the Bifidobacterium colonization strategy in the intestinal microbiome.IMPORTANCEBifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. Morphological observations revealed that extracellular appendages of bifidobacteria in complex microbial communities are important for understanding its adaptations to the GI tract environment. We identified dynamic extracellular vesicle (EV) production by Bifidobacterium longum in bacterium-free fecal fermentation broth that was strongly suggestive of differing bifidobacterial extracellular appendages in the GI tract. In addition, export of the adhesive moonlighting proteins mediated by EVs may promote bifidobacterial colonization. This study provides new insight into the roles of EVs in bifidobacterial colonization processes as these bacteria adapt to the GI environment.

Next-generation sequencing-based miRNA expression analysis in Parp1-deficient embryonic stem cell-derived exosomes.

Poly (ADP-ribose) polymerase family, member 1 (Parp1) has pleiotropic and disparate functions in multiple cellular signaling pathways through post-translational protein modification. It contributes to the regulation of various cellular processes, including DNA damage repair, cell death, and cell differentiation, genetically or epigenetically. Meanwhile, the functions of Parp1 in intercellular signaling remain to be established. To examine the functions of Parp1 in intercellular signaling, we examined microRNA (miRNA) regulation in exosomes derived from Parp1-deficient (Parp1-/-) embryonic stem (ES) cells. The percentages of miRNAs among total RNAs, including small RNAs such as miRNAs, snRNAs, snoRNAs, tRNAs, exonic RNAs, and intronic RNAs, in Parp1+/+ and Parp1-/- ES cell-derived exosomes were 8.2% and 3.5%, respectively. Overall, 329 distinct miRNAs exhibited ≥2-fold changes (118 upregulated; 211 downregulated). The upregulated miRNAs targeted 810 candidate genes, and the downregulated miRNAs targeted 716 candidate genes. Pathway analyses revealed that the upregulated miRNAs were significantly associated with five pathways including MAPK signaling cascades (p < 0.05), indicating that the target genes in these pathways were suppressed in Parp1-/- ES cells. In quantitative analyses of miRNA expression, miR365-3p, let-7a-5p, miR196b-5p, miR203-3p, miR98-5p, and miR146a-5p were increased by ≥ 2-fold in Parp1-/- ES cell-derived exosomes. Gene ontology enrichment analyses revealed that the upregulated miRNAs were significantly annotated for growth and stress-related cell signaling and cell communication (p < 0.05). Parp1 deficiency in ES cells led to inhibition of cell-cell communication, possibly by intercellular signal transduction, suggesting that Parp1 functions extracellularly by regulating exosomal miRNAs.

Catechins in tea suppress the activity of cytochrome P450 1A1 through the aryl hydrocarbon receptor activation pathway in rat livers.

Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) develop various adverse effects through activation of an aryl hydrocarbon receptor (AhR). The suppressive effects of brewed green tea and black tea on 3-methylcholanthrene (MC)-induced AhR activation and its downstream events were examined in the liver of rats. Ad-libitum drinking of green tea and black tea suppressed MC-induced AhR activation and elevation of ethoxyresorufin O-deethylase activity in the liver, whereas the teas themselves did not induce them. Tea showed a suppressive fashion on the expression of cytochrome P450 1A1 (CYP1A1). Tea suppressed the AhR activation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ex vivo. A part of catechins and theaflavins was present in plasma and liver as conjugated and intact forms. The results of this study suggested that active component(s) of tea are incorporated in the liver and suppress the activity of CYP1As through the AhR activation pathway.

Evaluating functionalities of food components by a model simulating human intestinal microbiota constructed at Kobe University.

In this era of pandemics, reducing the risk of lifestyle-related diseases (LRD) by functional foods is of paramount importance. The conventional process of functional food development almost invariably involves in vitro, animal, and human intervention trials, but differences in intestinal environments between humans and experimental animals make it difficult to develop functional foods that are truly effective in humans. Thus, it is necessary to construct a model that simulates the human intestinal environment to evaluate the functionality of any food component before subjecting it to a human intervention trial. In this review, we provide an overview of a model simulating human intestinal microbiota constructed at Kobe University and its use as a tool to identify food components that contribute to the prevention and treatment of LRD.

New Implications of Metabolites and Free Fatty Acids in Quality Control of Crossbred Wagyu Beef during Wet Aging Cold Storage.

Efficient cold-chain delivery is essential for maintaining a sustainable global food supply. This study used metabolomic analysis to examine meat quality changes during the "wet aging" of crossbred Wagyu beef during cold storage. The longissimus thoracic (Loin) and adductor muscles (Round) of hybrid Wagyu beef, a cross between the Japanese Black and Holstein-Friesian breeds, were packaged in vacuum film and refrigerated for up to 40 days. Sensory evaluation indicated an increase in the umami and kokumi taste owing to wet aging. Comprehensive analysis using gas chromatography-mass spectrometry identified metabolite changes during wet aging. In the Loin, 94 metabolites increased, and 24 decreased; in the Round, 91 increased and 18 decreased. Metabolites contributing to the umami taste of the meat showed different profiles during wet aging. Glutamic acid increased in a cold storage-dependent manner, whereas creatinine and inosinic acid degraded rapidly even during cold storage. In terms of lipids, wet aging led to an increase in free fatty acids. In particular, linoleic acid, a polyunsaturated fatty acid, increased significantly among the free fatty acids. These results provide new insight into the effects of wet aging on Wagyu-type beef, emphasizing the role of free amino acids, organic acids, and free fatty acids generated during cold storage.

Establishment of porcine fecal-derived ex vivo microbial communities to evaluate the impact of livestock feed on gut microbiome.

Sustainable livestock production requires reducing competition for food and feed resources and increasing the utilization of food by-products in livestock feed. This study describes the establishment of an anaerobic batch culture model to simulate pig microbiota and evaluate the effects of a food by-product, wakame seaweed stalks, on ex vivo microbial communities. We selected one of the nine media to support the growth of a bacterial community most similar in composition and diversity to that observed in pig donor feces. Supplementation with wakame altered the microbial profile and short-chain fatty acid composition in the ex vivo model, and a similar trajectory was observed in the in vivo pig experimental validation. Notably, the presence of wakame increased the abundance of Lactobacillus species, which may have been due to cross-feeding with Bacteroides. These results suggest the potential of wakame as a livestock feed capable of modulating the pig microbiome. Collectively, this study highlights the ability to estimate the microbiome changes that occur when pigs are fed a specific feed using an ex vivo culture model.

Isolation and identification of hyaluronan-degrading bacteria from Japanese fecal microbiota.

Hyaluronan (HA) is a high-molecular-weight glycosaminoglycan and widely distributed in all connective tissues and organs with diverse biological functions. HA has been increasingly used as dietary supplements targeted to joint and skin health for humans. We here first report isolation of bacteria from human feces that are capable of degrading HA to lower molecular weight HA oligosaccharides (oligo-HAs). The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted feces of healthy Japanese donors were individually incubated in an enrichment medium containing HA, followed by the isolation of candidate strains from streaked HA-containing agar plates and selection of HA-degrading strains by measuring HA using an ELISA. Subsequent genomic and biochemical assays identified the strains as Bacteroides finegoldii, B. caccae, B. thetaiotaomicron, and Fusobacterium mortiferum. Furthermore, our HPLC analysis revealed that the strains degraded HA to oligo-HAs of various lengths. Subsequent quantitative PCR assay targeting the HA degrading bacteria showed that their distribution in the Japanese donors varied. The evidence suggests that dietary HA is degraded by the human gut microbiota with individual variation to oligo-HAs components, which are more absorbable than HA, thereby exerting its beneficial effects.

Pre- and Post-Harvest Conditions Affect Polyphenol Content in Strawberry (Fragaria × ananassa).

The strawberry fruit contains abundant polyphenols, such as anthocyanins, flavan-3-ol, and ellagitannin. Polyphenol enrichment improves the quality of strawberries and leads to a better understanding of the polyphenol induction process. We measured the total polyphenol content of strawberry fruits under different growth conditions, developmental stages, and treatment conditions during pre-harvest and post-harvest periods. High fruit polyphenol content was observed in cold treatment, which was selected for further analysis and optimization. A transcriptome analysis of cold-treated fruits suggested that the candidate components of polyphenols may exist in the phenylpropanoid pathway. Coverage with a porous film bag excluded the effects of drought stress and produced polyphenol-rich strawberry fruits without affecting quality or quantity. The degree of stress was assessed using known stress indicators. A rapid accumulation of abscisic acid was followed by an increase in superoxide dismutase and DPPH (2,2-Diphenyl-1-picrylhydrazyl) activity, suggesting that the strawberry fruits responded to cold stress immediately, reaching the climax at around 6 days, a trend consistent with that of polyphenol content. These findings enhance our understanding of the mechanism of post-harvest polyphenol accumulation and the value of strawberries as a functional food.

Cardamonin stimulates glucose uptake through translocation of glucose transporter-4 in L6 myotubes.

Glucose transporter-4 (GLUT4) is a transmembrane protein that plays a major role in insulin-mediated glucose transport in muscle and adipocytes. For glucose transport to occur, the GLUT4 protein needs to be translocated from the intracellular pool to the plasma membrane, and certain compounds may enhance this process. The present study investigated the promotion of glucose uptake in differentiated L6 myotubes by cardamonin, isolated from Alpinia katsumadai. Cardamonin increased translocation of GLUT4 to the plasma membrane in L6 cells, but did not activate protein kinase C ζ/λ, Akt, or AMP-activated protein-kinase, all of which are known to regulate GLUT4 translocation. The glucose-uptake-promoting activity of cardamonin was not lowered by treatment with a phosphatidylinositol 3'-kinase inhibitor. These results suggest that cardamonin is a promising active compound for maintaining glucose homeostasis, and that it acts via an unknown mechanism that does not involve activation of the downstream insulin signal and AMP-activated protein kinase.

Milk oligosaccharide-mediated cross-feeding between Enterococcus gallinarum and lactobacilli in the gut microbiota of infant rats.

We investigated bacteria that have a nutritional symbiotic relationship with respect to milk oligosaccharides in gut microbiota of suckling rats, with specific reference to sialyllactose (SL) degrading Enterococcus gallinarum. Our next generation sequencing analysis of the colonic contents of 12-day-old suckling rats revealed that almost half of the bacteria in the microbiota belonged to the Lactobacillaceae family. Major Lactobacillus species in the contents were identified as L. johnsonii, L. murinus, and L. reuteri. We then monitored changes in numbers of the above Lactobacillus species, E. gallinarum, and the bacteria belonging to the family Enterobacteriaceae (i.e., enterobacteria) in the colonic contents of infant rats at 7, 12, 21, 28, and 35 days of age by using real-time PCR assays targeting these bacterial groups. The 7-day-old infant rats had a gut microbiota in which enterobacteria were predominant. Such dominance was replaced by L. johnsonii and the concomitant E. gallinarum markedly increased in those of 12 and 21 days of ages. During this period, the number of enterobacteria declined dramatically, but that of L. reuteri surged dramatically. Our separate in vitro experiment showed that supplementation of culture media with SL promoted the growth of L. johnsonii and E. gallinarum, with marked production of lactic acid. These findings revealed possible milk oligosaccharide-mediated cross-feeding between E. gallinarum and L. johnsonii, with the former degrading SL to release lactose to be utilized by the latter.

Isolation and identification of milk oligosaccharide-degrading bacteria from the intestinal contents of suckling rats.

We report the isolation of bacteria capable of degrading milk oligosaccharides from suckling infant rats. The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted intestinal contents of infant rats were individually incubated in an enrichment medium containing 3'-sialyllactose (3'-SL), followed by the isolation of candidate strains from streaked agar plates and selection of 3'-SL-degrading strains using thin-layer chromatography. Subsequent genomic and phenotypic analyses identified all strains as Enterococcus gallinarum. The strains were capable of degrading both 3'-SL and 6'-SL, which was not observed with the type strain of E. gallinarum used as a reference. Furthermore, a time-course study combining high-performance anion-exchange chromatography with pulsed amperometric detection revealed that the representative strain AH4 degraded 3'-SL completely to yield an equimolar amount of lactose and an approximately one-fourth equimolar amount of sialic acid after 24 hr of anaerobic incubation. These findings point to a possibility that the enterococci degrade rat milk oligosaccharides to "cross-feed" their degradants to other members of concomitant bacteria in the gut of the infant rat.

Suppression mechanisms of flavonoids on aryl hydrocarbon receptor-mediated signal transduction.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological and toxicological effects by binding to its agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Previously we demonstrated that flavonoids suppressed the TCDD-induced DNA-binding activity of the AhR in a structure-dependent manner. In this study, we investigated the mechanisms by which flavonoids suppressed the AhR-mediated signal transduction in mouse hepatoma Hepa-1c1c7 cells. Flavones and flavonols suppressed the TCDD-induced nuclear translocation of the AhR and dissociation of its partner proteins, heat shock protein 90 and X-associated protein 2, whereas flavanones and catechins did not. Flavonoids of all these four subclasses suppressed the phosphorylation of both AhR and Arnt and the formation of a heterodimer consisting of these proteins. Since certain flavonoids are known to inhibit mitogen-activated protein kinases (MAPKs), we confirmed the contribution of MAPK/ERK kinase (MEK) to the AhR-mediated signal transduction by using U0126, an inhibitor of MEK1/2. U0126 suppressed TCDD-induced phosphorylation of the AhR and Arnt followed by the DNA-binding activity of the AhR. Flavanones and catechins suppressed the TCDD-induced phosphorylation of ERK1/2. The inhibition of MEK/ERK phosphorylation is one of the mechanisms by which flavanones and catechins suppress the AhR-mediated signal transduction in Hepa-1c1c7 cells.

Suppression of cytochrome P450 1A1 expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse hepatoma hepa-1c1c7 cells treated with serum of (-)-epigallocatechin-3-gallate- and green tea extract-administered rats.

The suppression of cytochrome P450 1A1 (CYP1A1) expression was examined in mouse hepatoma Hepa-1c1c7 cells treated with serum prepared from (-)-epigallocatechin-3-gallate- and green tea extract-administered rats. Catechins were found in the rat plasma after the administration. In Hepa-1c1c7 cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 expression was suppressed by treatment with the rat serum. It is concluded that catechins can possibly modulate CYP1A1 expression.

Butyryl-CoA:acetate CoA-transferase gene associated with the genus Roseburia is decreased in the gut microbiota of Japanese patients with ulcerative colitis.

Microbial production of butyrate is impaired in patients with ulcerative colitis (UC); however, this inhibition is not well understood in Japanese UC patients. Therefore, we quantitatively analyzed genes encoding butyryl-CoA:acetate CoA-transferase (but) and butyrate kinase (buk) in the gut microbiota of Japanese patients with UC and healthy volunteers (HVs). But showed higher levels than buk. Moreover, patients with UC showed significantly decreased levels of but associated with Roseburia sp./Eubacterium rectale compared with HVs. But, which is associated with Faecalibacterium sp., was maintained in patients with UC, with an unchanged relative abundance of Faecalibacterium sp. microorganisms in patients with UC compared with HVs.

Construction of a Model Culture System of Human Colonic Microbiota to Detect Decreased Lachnospiraceae Abundance and Butyrogenesis in the Feces of Ulcerative Colitis Patients.

Compositional alteration of the gut microbiota is associated with ulcerative colitis (UC). Here, a model culture system is established for the in vitro human colonic microbiota of UC, which will be helpful for determining medical interventions. 16S ribosomal RNA sequencing confirms that UC models are successfully developed from fecal inoculum and retain the bacterial species biodiversity of UC feces. The UC models closely reproduce the microbial components and successfully preserve distinct clusters from the healthy subjects (HS), as observed in the feces. The relative abundance of bacteria belonging to the family Lachnospiraceae significantly decreases in the UC models compared to that in HS, as observed in the feces. The system detects significantly lower butyrogenesis in the UC models than that in HS, correlating with the decreased abundance of Lachnospiraceae. Interestingly, the relative abundance of Lachnospiraceae does not correlate with disease activity (defined as partial Mayo score), suggesting that Lachnospiraceae persists in UC patients at a decreased level, irrespective of the alteration in disease activity. Moreover, the system shows that administration of Clostridium butyricum MIYAIRI restores butyrogenesis in the UC model. Hence, the model detects deregulation in the intestinal environment in UC patients and may be useful for simulating the effect of probiotics.

Epigallocatechin gallate promotes GLUT4 translocation in skeletal muscle.

In this study, we investigated whether epigallocatechin gallate (EGCg) affects glucose uptake activity and the translocation of insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. A single oral administration of EGCg at 75 mg/kg body weight promoted GLUT4 translocation in skeletal muscle of rats. EGCg significantly increased glucose uptake accompanying GLUT4 translocation in L6 myotubes at 1 nM. The translocation of GLUT4 was also observed both in skeletal muscle of mice and rats ex vivo and in insulin-resistant L6 myotubes. Wortmannin, an inhibitor of phosphatidylinositol 3'-kinase, inhibited both EGCg- and insulin-increased glucose uptakes, while genistein, an inhibitor of tyrosine kinase, failed to inhibit the EGCg-increased uptake. Therefore, EGCg may improve hyperglycemia by promoting GLUT4 translocation in skeletal muscle with partially different mechanism from insulin.

Antagonistic and agonistic effects of indigoids on the transformation of an aryl hydrocarbon receptor.

Halogenated and polycyclic aromatic hydrocarbons, exogenous ligands of the aryl hydrocarbon receptor (AhR), cause various toxicological effects through the transformation of the AhR. In this study, we investigated the antagonistic effects of indigoids on the transformation in addition to their agonistic ones. In a cell-free system, indigoids induced the transformation dose-dependently, but suppressed the transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the binding of 3-methylcholanthrene to the AhR. In mouse hepatoma Hepa-1c1c7 cells, indigoids, especially indirubin, suppressed the transformation and expression of CYP1A1 by inhibiting the translocation of AhR into the nucleus. When orally administered to mice at 10mg/kg BW/day for three successive days, indigoids did not induce AhR transformation and expression of the CYP1A subfamily in the liver, while indirubin and indigo upregulated quinone reductase activity. These results indicate that indigoids are able to bind to the AhR as ligands and exhibit antagonistic effects at lower concentrations in mammalian cells.

High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function.

An update on the dietary ligands of the AhR.

BACKGROUND: Halogenated aromatic hydrocarbons including dioxins and non-halogenated polycyclic aromatic hydrocarbons are ligands of an aryl hydrocarbon receptor (AhR) and stimulate its transformation. Exposure to these environmental contaminants occurs mainly through diet. Recent articles demonstrated that certain food factors regulate the AhR transformation and expression of downstream drug-metabolizing enzymes. OBJECTIVE: To explain the actions of these food factors on the AhR transformation, as the mechanisms underlying are not fully understood. METHODS: This review introduces recent articles that have demonstrated the molecular mechanisms by which food factors regulate the AhR transformation and downstream drug-metabolizing enzymes. RESULTS/CONCLUSION: The role of classical ligands including dioxins as agonists of the receptor is well documented. As to the food factors, they act as antagonists because they basically suppress the AhR transformation by different mechanisms. Moreover, the fate and metabolism of food factors are important to understand their mechanisms.

Low amounts of dietary fibre increase in vitro production of short-chain fatty acids without changing human colonic microbiota structure.

This study investigated the effect of various prebiotics (indigestible dextrin, α-cyclodextrin, and dextran) on human colonic microbiota at a dosage corresponding to a daily intake of 6 g of prebiotics per person (0.2% of dietary intake). We used an in vitro human colonic microbiota model based on batch fermentation starting from a faecal inoculum. Bacterial 16S rRNA gene sequence analysis showed that addition of 0.2% prebiotics did not change the diversity and composition of colonic microbiota. This finding coincided with results from a clinical study showing that the microbiota composition of human faecal samples remained unchanged following administration of 6 g of prebiotics over seven days. However, compared to absence of prebiotics, their addition reduced the pH and increased the generation of acetate and propionate in the in vitro system. Thus, even at such relatively low amounts, prebiotics appear capable of activating the metabolism of colonic microbiota.