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.

Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells.

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

Genome sequence of Enterococcus gallinarum AH4, a milk oligosaccharide-degrading strain isolated from suckling rats.

We had previously isolated Enterococcus gallinarum AH4, a strain capable of degrading rat milk oligosaccharides. In this study, we determined the whole-genome sequence of AH4. This whole-genome information will expand our understanding of milk oligosaccharide-mediated symbioses between bacteria and host mammals.

H2-induced transient upregulation of phospholipids with suppression of energy metabolism.

Molecular hydrogen (H2) is an antioxidant and anti-inflammatory agent; however, the molecular mechanisms underlying its biological effects are largely unknown. Similar to other gaseous molecules such as inhalation anesthetics, H2 is more soluble in lipids than in water. A recent study demonstrated that H2 reduces radical polymerization-induced cellular damage by suppressing fatty acid peroxidation and membrane permeability. Thus, we sought to examine the effects of short exposure to H2 on lipid composition and associated physiological changes in SH-SY5Y neuroblastoma cells. We analyzed cells by liquid chromatography-high-resolution mass spectrometry to define changes in lipid components. Lipid class analysis of cells exposed to H2 for 1 hour revealed transient increases in glycerophospholipids including phosphatidylethanolamine, phosphatidylinositol, and cardiolipin. Metabolomic analysis also showed that H2 exposure for 1 hour transiently suppressed overall energy metabolism accompanied by a decrease in glutathione. We further observed alterations to endosomal morphology by staining with specific antibodies. Endosomal transport of cholera toxin B to recycling endosomes localized around the Golgi body was delayed in H2-exposed cells. We speculate that H2-induced modification of lipid composition depresses energy production and endosomal transport concomitant with enhancement of oxidative stress, which transiently stimulates stress response pathways to protect cells.

Drinking hydrogen water improves photoreceptor structure and function in retinal degeneration 6 mice.

Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinal disorders involving the progressive dysfunction of photoreceptors and the retinal pigment epithelium, for which there is currently no treatment. The rd6 mouse is a natural model of autosomal recessive retinal degeneration. Given the known contributions of oxidative stress caused by reactive oxygen species (ROS) and selective inhibition of potent ROS peroxynitrite and OH·by H2 gas we have previously demonstrated, we hypothesized that ingestion of H2 water may delay the progression of photoreceptor death in rd6 mice. H2 mice showed significantly higher retinal thickness as compared to controls on optical coherence tomography. Histopathological and morphometric analyses revealed higher thickness of the outer nuclear layer for H2 mice than controls, as well as higher counts of opsin red/green-positive cells. RNA sequencing (RNA-seq) analysis of differentially expressed genes in the H2 group versus control group revealed 1996 genes with significantly different expressions. Gene and pathway ontology analysis showed substantial upregulation of genes responsible for phototransduction in H2 mice. Our results show that drinking water high in H2 (1.2-1.6 ppm) had neuroprotective effects and inhibited photoreceptor death in mice, and suggest the potential of H2 for the treatment of RP.

Lactiplantibacillus plantarum LOC1 Isolated from Fresh Tea Leaves Modulates Macrophage Response to TLR4 Activation.

Previously, we demonstrated that Lactiplantibacillus plantarum LOC1, originally isolated from fresh tea leaves, was able to improve epithelial barrier integrity in in vitro models, suggesting that this strain is an interesting probiotic candidate. In this work, we aimed to continue characterizing the potential probiotic properties of the LOC1 strain, focusing on its immunomodulatory properties in the context of innate immunity triggered by Toll-like receptor 4 (TLR4) activation. These studies were complemented by comparative and functional genomics analysis to characterize the bacterial genes involved in the immunomodulatory capacity. We carried out a transcriptomic study to evaluate the effect of L. plantarum LOC1 on the response of murine macrophages (RAW264.7 cells) to the activation of TLR4. We demonstrated that L. plantarum LOC1 exerts a modulatory effect on lipopolysaccharide (LPS)-induced inflammation, resulting in a differential regulation of immune factor expression in macrophages. The LOC1 strain markedly reduced the LPS-induced expression of some inflammatory cytokines (IL-1ß, IL-12, and CSF2) and chemokines (CCL17, CCL28, CXCL3, CXCL13, CXCL1, and CX3CL1), while it significantly increased the expression of other cytokines (TNF-α, IL-6, IL-18, IFN-ß, IFN-γ, and CSF3), chemokines (IL-15 and CXCL9), and activation markers (H2-k1, H2-M3, CD80, and CD86) in RAW macrophages. Our results show that L. plantarum LOC1 would enhance the intrinsic functions of macrophages, promoting their protective effects mediated by the stimulation of the Th1 response without affecting the regulatory mechanisms that help control inflammation. In addition, we sequenced the LOC1 genome and performed a genomic characterization. Genomic comparative analysis with the well-known immunomodulatory strains WCSF1 and CRL1506 demonstrated that L. plantarum LOC1 possess a set of adhesion factors and genes involved in the biosynthesis of teichoic acids and lipoproteins that could be involved in its immunomodulatory capacity. The results of this work can contribute to the development of immune-related functional foods containing L. plantarum LOC1.

Isolation, identification, and impact on intestinal barrier integrity of Lactiplantibacillus plantarum from fresh tea leaves (Camellia sinensis).

Lactic acid bacteria (LAB) are safe microorganisms that have been used in the processing of fermented food for centuries. The aim of this study was to isolate Lactobacillus from fresh tea leaves and examine the impact of an isolated strain on intestinal barrier integrity. First, the presence of Lactobacillus strains was investigated in fresh tea leaves from Kagoshima, Japan. Strains were isolated by growing on De Man, Rogosa and Sharpe (MRS) agar medium containing sodium carbonate, followed by the identification of one strain by polymerase chain reaction (PCR) and pheS sequence analysis, with the strain identified as Lactiplantibacillus plantarum and named L. plantarum LOC1. Second, the impact of strain LOC1 in its heat-inactivated form on intestinal barrier integrity was investigated. Strain LOC1, but not L. plantarum ATCC 14917T or L. plantarum ATCC 8014, significantly suppressed dextran sulfate sodium (DSS)-induced decreases in transepithelial electrical resistance values of Caco-2:HT29-MTX 100:0 and 90:10 co-cultures. Moreover, in Caco-2:HT29-MTX co-cultures (90:10 and 75:25), levels of occludin mRNA were significantly increased by strain LOC1 compared with untreated co-cultures, and strain LOC1 had higher mRNA levels of MUC2 and MUC4 mucins than L. plantarum ATCC 14917T and L. plantarum YT9. These results indicate that L. plantarum LOC1 may be used as a safe probiotic with beneficial effects on the intestinal barrier, suggesting that fresh tea leaves could be utilized as a safe source for isolating probiotics.

Identification of genes encoding a novel ABC transporter in Lactobacillus delbrueckii for inulin polymers uptake.

Lactobacillus delbrueckii JCM 1002T grows on highly polymerized inulin-type fructans as its sole carbon source. When it was grown on inulin, a > 10 kb long gene cluster inuABCDEF (Ldb1381-1386) encoding a plausible ABC transporter was suggested to be induced, since a transcriptome analysis revealed that the fourth gene inuD (Ldb1384) was up-regulated most prominently. Although Bacillus subtilis 168 is originally unable to utilize inulin, it became to grow on inulin upon heterologous expression of inuABCDEF. When freshly cultured cells of the recombinant B. subtilis were then densely suspended in buffer containing inulin polymers and incubated, inulin gradually disappeared from the buffer and accumulated in the cells without being degraded, whereas levan-type fructans did not disappear. The results imply that inuABCDEF might encode a novel ABC transporter in L. delbrueckii to "monopolize" inulin polymers selectively, thereby, providing a possible advantage in competition with other concomitant inulin-utilizing bacteria.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis.

Development of a selective and sensitive analytical method to detect isomerized aspartic acid residues in crystallin using a combination of derivatization and liquid chromatography mass spectrometry.

The isomerization of amino acids in peptides and proteins induces structural changes and aggregation. The isomerization rate of aspartic acid (Asp) is high and causes various serious diseases including Alzheimer's disease and cataract. Herein, a method for the comprehensive separation and sensitive detection of isomerized crystallin containing Asp (l-α-Asp, l-ß-Asp, d-α-Asp, and d-ß-Asp) was developed using chiral derivatization and reversed-phase UHPLC separation. Of three candidate derivatization reagents tested for the separation of peptides containing isomerized aspartic acid, 2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazine-2-yl) pyrrolidine-2-carboxylate (DMT-(R)-Pro-OSu) was the most suitable reagent for separating isomerized peptides and improved the sensitivity of mass spectrometry by 50-fold. This method was applied to analyze heat-denatured crystallin. Asp58 and Asp151 residues in αA-crystallin (AAC) exhibited the highest isomerization rate in heated crystallin. Furthermore, the analysis of α-crystallin extracted from bovine eye lens identified isomerized Asp residues (Asp24/35, Asp58, and Asp151 in AAC and Asp140 in αB-crystallin (ABC)). These results indicate that the newly developed method using chiral derivatization provides selective and sensitive analysis of isomerized Asp sites in α-crystallin protein. This novel method will allow for the identification and quantification of isomerized amino acids in crystallin proteins.

Oral Administration of Fucoidan Can Exert Anti-Allergic Activity after Allergen Sensitization by Enhancement of Galectin-9 Secretion in Blood.

A previous study revealed that fucoidan inhibited mast cell degranulation through the upregulation of galectin-9 in blood. The purpose of this study is to elucidate its mechanism using ovalbumin (OVA) induced anaphylaxis model mice (BALB/c, Female, 5-week-old) and mast cell line (RBL-2H3 cells). Oral administration of fucoidan after sensitization with OVA/Al(OH)3 inhibited reduction of rectal temperature induced by activation of mast cells. Fucoidan increased galectin-9 mRNA expression only in colonic epithelial cells. These results suggested that fucoidan could suppress the allergic symptoms in sensitized mice by inducing galectin-9 production from colonic epithelial cells. In addition, to check the influence of galectin 9 on the degranulation of mast cells, RBL-2H3 cell lines were treated directly with recombinant galectin-9. As expected, galectin-9 inhibited degranulation of RBL-2H3 cells pre-bound with IgE. Moreover, the residual amounts of IgE on RBL-2H3 cells were decreased by an addition of galectin-9. It was demonstrated that galectin-9 could remove IgE even if IgE was already bound to mast cells and suppress the mast cells degranulation induced by antigen. This study shows that fucoidan might become an effective therapeutic agent for patients already developed type I allergic diseases.

Administration of hydrogen-rich water prevents vascular aging of the aorta in LDL receptor-deficient mice.

The main cause of arteriosclerosis is atherosclerosis in the aorta. Atherosclerosis is recognized as a chronic inflammatory condition that begins with the dysfunction or activation of arterial endothelium. Low-density lipoprotein (LDL) and especially its oxidized form play a key role in endothelial dysfunction and atherogenesis. Recent studies showed that senescent cells are involved in the development and progression of atherosclerosis, and eliminating senescent cells suppresses the senescence-associated secretory phenotype. We previously reported that molecular hydrogen-rich water (HW) has antioxidant and anti-inflammatory effects in numerous diseases. Here, we used LDL receptor-deficient mice fed a high-fat diet (HFD) for 13 weeks as a model for atherosclerosis and evaluated the effects of continuous administration of HW. The numbers of endothelial cells in the atheroma expressing the senescence factors p16INK4a and p21 decreased in HFD-fed mice given HW compared with HFD-fed mice given control water. Furthermore, macrophage infiltration and Tnfα expression in the atheroma were also suppressed. These results suggest that vascular aging can be suppressed by HW.

Direct metabolomics for plant cells by live single-cell mass spectrometry.

Live single-cell mass spectrometry (live MS) provides a mass spectrum that shows thousands of metabolite peaks from a single live plant cell within minutes. By using an optical microscope, a cell is chosen for analysis and a metal-coated nanospray microcapillary tip is used to remove the cell's contents. After adding a microliter of ionization solvent to the opposite end of the tip, the trapped contents are directly fed into the mass spectrometer by applying a high voltage between the tip and the inlet port of the spectrometer to induce nanospray ionization. Proteins are not detected because of insufficient sensitivity. Metabolite peaks are identified by exact mass or tandem mass spectrometry (MS/MS) analysis, and isomers can be separated by combining live MS with ion-mobility separation. By using this approach, spectra can be acquired in 10 min. In combination with metabolic maps and/or molecular databases, the data can be annotated into metabolic pathways; the data analysis takes 30 min to 4 h, depending on the MS/MS data availability from databases. This method enables the analysis of a number of metabolites from a single cell with rapid sampling at sub-attomolar-level sensitivity.

Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes.

In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 µM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity. (-)-Epicatechin (EC) and (-)-epigallocatechin (EGC), nongallate-type catechins, increased glucose uptake activity in the dose- and time-dependent manner, whereas (-)-catechin 3-gallate (Cg) and (-)-epigallocatechin 3-gallate (EGCg), gallate-type catechins, decreased insulin-induced glucose uptake activity in the dose- and time-dependent manner. When the cells were treated with 50 µM catechins for 30 min, EC and EGC promoted GLUT4 translocation, whereas Cg and EGCg decreased the insulin-induced translocation in the cells. EC and EGC increased phosphorylation of PKCλ/ζ without phosphorylation of insulin receptor (IR) and Akt. Wortmannin and LY294002, inhibitors for phosphatidylinositol 3'-kinase (PI3K), decreased EC- and EGC-induced glucose uptake activity in the cells. Cg and EGCg decreased phosphorylation of PKCλ/ζ in the presence of insulin without affecting insulin-induced phosphorylation of IR, and Akt. Therefore, EC and EGC promote the translocation of GLUT4 through activation of PI3K, and Cg and EGCg inhibit insulin-induced translocation of GLUT4 by the insulin signaling pathway in 3T3-L1 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.

Determination of mechanism of flock sediment formation in tea beverages.

The mechanism of sediment formation during the storage of green tea beverage was investigated. Green tea extract was separated by Diaion HP-20 column chromatography, and a sediment-formation test was performed. Results showed that at least one compound of the substance causing flock sediment was contained in each of the HP-20 nonadsorbed and adsorbed fractions. From the following fractionations and structure analyses, the substance in the HP-20 adsorbed fraction was determined to be 1-O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-beta-D-glucose (strictinin), which is one of the ellagitannins. Strictinin was hydrolyzed to ellagic acid by heat-sterilization processes such as retort sterilization or the ultra-high temperature processing used during the manufacturing of tea beverages. Ellagic acid combined with proteins in the HP-20 nonadsorbed fraction to form an irreversible sediment of green tea beverage; ellagic acid and proteins were confirmed to be present in that sediment. The HP-20 adsorbed fraction contained little strictinin and formed hardly any sediment, suggesting that control of the strictinin content is significant in avoiding sediment formation during the manufacturing process of tea beverages.

Black tea theaflavins suppress dioxin-induced transformation of the aryl hydrocarbon receptor.

Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC(50) values of theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 muM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.

Pigments in green tea leaves (Camellia sinensis) suppress transformation of the aryl hydrocarbon receptor induced by dioxin.

Environmental contaminants such as dioxins enter the body mainly through diet and cause various toxicities through transformation of the aryl hydrocarbon receptor (AhR). We previously reported that certain natural flavonoids at the dietary level suppress the AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we identified lutein and chlorophyll a and b from green tea leaves as the novel antagonists for AhR. These active compounds suppressed AhR transformation dose-dependently with the 50% inhibitory concentration (IC(50)) values against 0.1 nM TCDD-induced AhR transformation at 3.2, 5.0, and 5.9 microM, respectively. (-)-Epigallocatechin gallate, which is the most abundant flavonoid in green tea leaves, also showed stronger suppressive effects than did other major tea components, with the IC(50) value of 1.7 microM. Thus, these pigments of green tea leaves have the potential to protect from dioxin toxicity through the suppression of AhR transformation.

Black tea extract suppresses transformation of aryl hydrocarbon receptor induced by dioxin.

Dioxins cause various adverse effects through binding to an aryl hydrocarbon receptor (AhR) and transformation of the receptor. In this study, we investigated whether black tea extract suppresses AhR transformation. Dried black tea leaves were extracted with 75% ethanol, and the extract was pretreated to the rat liver cytosol fraction 10 min prior to addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transformed AhR was detected by electrophoretic gel mobility shift assay. Black tea extract suppressed AhR transformation in a dose-dependent manner, and the IC50 value against 1 nM TCDD-induced AhR transformation was 8.9 microg/ml. The result suggests that intake of black tea has a potential to suppress the AhR transformation, leading protection from dioxin toxicity.

[Isolation and quantitative analysis of the alpha-amylase inhibitor in Lagerstroemia speciosa (L.) Pers. (Banaba)].

Banaba [Lagerstroemia speciosa (L.) Pers.] has been used as a folk medicine for diabetes in the Philippines. Using bioassay-guided separation, valoneaic acid dilactone (1) was isolated from the leaves as a potent alpha-amylase inhibitor. A simple and efficient method for the quantitative determination of valoneaic acid and its derivatives in Banaba extract was established. Valoneaic acid exists as the structural part of the polyphenols, which like flosin A, reginin A, and lagerstroemin, are characteristic constituents of Banaba. These derivatives were hydrolyzed to valoneaic acid by HCl and extracted with 2-butanone. This extract was subjected to HPLC analysis, and the contents of valoneaic acid determined as the whole valoneaic acid contents. Using this method, the whole valoneaic acid contents were measured in eight Banaba leaf decoctions. The alpha-amylase-inhibiting activities of the decoctions were dependent on the whole valoneaic acid contents. In addition, a strong linear correlation was observed between the whole valoneaic acid contents and total polyphenol contents. This analytical procedure is applicable to the chemical evaluation of Banaba.