High-throughput identification and quantification of bacterial cells in the microbiota based on 16S rRNA sequencing with single-base accuracy using BarBIQ.

Bacteria often function as a community, called the microbiota, consisting of many different bacterial species. The accurate identification of bacterial types and the simultaneous quantification of the cells of each bacterial type will advance our understanding of microbiota; however, this cannot be performed by conventional 16S rRNA sequencing methods as they only identify and quantify genes, which do not always represent cells. Here, we present a protocol for our developed method, barcoding bacteria for identification and quantification (BarBIQ). In BarBIQ, the 16S rRNA genes of single bacterial cells are amplified and attached to a unique cellular barcode in a droplet. Sequencing the tandemly linked cellular barcodes and 16S rRNA genes from many droplets (representing many cells with unique cellular barcodes) and clustering the sequences using the barcodes determines both the bacterial type for each cell based on 16S rRNA gene and the number of cells for each bacterial type based on the quantity of barcode types sequenced. Single-base accuracy for 16S rRNA sequencing is achieved via the barcodes and by avoiding chimera formation from 16S rRNA genes of different bacteria using droplets. For data processing, an easy-to-use bioinformatic pipeline is available ( https://github.com/Shiroguchi-Lab/BarBIQ_Pipeline_V1_2_0 ). This protocol allows researchers with experience in molecular biology but without bioinformatics experience to perform the process in ~2 weeks. We show the application of BarBIQ in mouse gut microbiota analysis as an example; however, this method is also applicable to other microbiota samples, including those from the mouth and skin, marine environments, soil and plants, as well as those from other terrestrial environments.

High-throughput identification and quantification of single bacterial cells in the microbiota.

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. To fully understand the function of bacterial microbiota, individual cells must be identified; however, it is difficult with current techniques. Here, we develop a method, Barcoding Bacteria for Identification and Quantification (BarBIQ), which classifies single bacterial cells into taxa-named herein cell-based operational taxonomy units (cOTUs)-based on cellularly barcoded 16S rRNA sequences with single-base accuracy, and quantifies the cell number for each cOTU in the microbiota in a high-throughput manner. We apply BarBIQ to murine cecal microbiotas and quantify in total 3.4 × 105 bacterial cells containing 810 cOTUs. Interestingly, we find location-dependent global differences in the cecal microbiota depending on the dietary vitamin A deficiency, and more differentially abundant cOTUs at the proximal location than the distal location. Importantly, these location differences are not clearly shown by conventional 16S rRNA gene-amplicon sequencing methods, which quantify the 16S rRNA genes, not the cells. Thus, BarBIQ enables microbiota characterization with the identification and quantification of individual constituent bacteria, which is a cornerstone for microbiota studies.

Changes in the microcirculation in periodontal tissue due to experimental peri-implantitis.

OBJECTIVES: Peri-implantitis causes dislodgement of dental implants due to inflammation in the peri-implant tissue. The microcirculation in the periodontal tissue undergoes morphological and physiological changes due to inflammation. The immune mechanism of peri-implantitis differs from that of periodontitis. In this study, we examined the changes in the microcirculation in the peri-implant tissue with experimentally induced inflammation, using morphological and physiological techniques. METHODS: Six beagle dogs were used in the experiment. After extracting both mandibular premolars, three titanium screw implants were inserted on each side of the mandibular jaw. Dental floss was placed on the right side for 90 days in the study group but not in the control group. Microvascular resin cast models were created, and morphological changes were observed using scanning electron microscopy. Periodontal blood flow was measured using laser Doppler flowmetry. RESULTS: Ninety days after induction of inflammation, bone resorption was observed around the implant body. Osseointegration was impaired, and a gap at the implant-bone interface was observed. The resin cast models showed that inflamed gingival blood vessels had invaded the bone marrow through the resorbed apical margin of the alveolar bone. Analysis of the physiological data obtained using laser Doppler flowmetry showed a significant increase in blood flow around the implants with experimentally induced inflammation. CONCLUSIONS: Significant morphological and physiological changes occur in the gingival microcirculation of peri-implant tissue due to inflammation. Evaluating the vasculature and blood flow in the tissue surrounding the site of peri-implantitis may be helpful for pathologic analysis in clinical settings.

Prefrontal Inter-brain Synchronization Reflects Convergence and Divergence of Flow Dynamics in Collaborative Learning: A Pilot Study.

Flow is a highly motivated and affectively positive state in which a person is deeply engaged in an activity and feeling enjoyment from it. In collaborative activities, it would be optimal if all participants were in a state of flow. However, flow states fluctuate amongst individuals due to differences in the dynamics of motivation and cognition. To explore the possibility that inter-brain synchronization can provide a quantitative measure of the convergence and divergence of collective motivational dynamics, we conducted a pilot study to investigate the relationship between inter-brain synchronization and the interpersonal similarity of flow state dynamics during the collaborative learning process. In two English as a Foreign Language (EFL) classes, students were divided into groups of three-four and seated at desks facing each other while conducting a 60-min group work. In both classes, two groups with four members were randomly selected, and their medial prefrontal neural activities were measured simultaneously using wireless functional near-infrared spectroscopy (fNIRS) devices. Later the participants observed their own activities on recorded videos and retrospectively rated their subjective degree of flow state on a seven-point scale for each 2-min period. For the pairs of students whose neural activities were measured, the similarity of their flow experience dynamics was evaluated by the temporal correlation between their flow ratings. Prefrontal inter-brain synchronization of the same student pairs during group work was evaluated using wavelet transform coherence. Statistical analyses revealed that: (1) flow dynamics were significantly more similar for the student pairs within the same group compared to the pairs of students assigned across different groups; (2) prefrontal inter-brain synchronization in the relatively short time scale (9.3-13.9 s) was significantly higher for the within-group pairs than for the cross-group pairs; and (3) the prefrontal inter-brain synchronization at the same short time scale was significantly and positively correlated with the similarity of flow dynamics, even after controlling for the effects of within- vs. cross-group pair types from the two variables. These suggest that inter-brain synchronization can indeed provide a quantitative measure for converging and diverging collective motivational dynamics during collaborative learning, with higher inter-brain synchronization corresponding to a more convergent flow experience.

Orally Administered Polysaccharide Derived from Blackcurrants (Ribes nigrum L.) Improves Skin Hydration in Ultraviolet-Irradiated Hairless Mice.

Blackcurrants (Ribes nigrum L.) have various benefits for human health. In particular, a polysaccharide derived from blackcurrant was found to be an immunostimulating food ingredient in a mouse model. We named a polysaccharide derived from blackcurrant cassis polysaccharide (CAPS). In a previous clinical study, we reported that CAPS affects skin dehydration, demonstrating its effectiveness against skin inflammation was related to atopic dermatitis; skin inflammation caused skin dehydration. However, there are no studies regarding CAPS effectiveness against skin dehydration. The current study aimed to investigate CAPS effectiveness against skin dehydration. We further demonstrate the effect of oral administration of CAPS on skin dehydration caused by ultraviolet (UV) irradiation-induced inflammation in mice. We found that CAPS administration suppresses skin dehydration caused by UV irradiation. We also found that CAPS decreases interleukin-6 and matrix metalloproteinase transcription levels in the mouse skin. These results show that CAPS improves skin hydration in UV-irradiated mice.

Effect of administrating polysaccharide from black currant (Ribes nigrum L.) on atopic dermatitis in NC/Nga mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that causes dry skin and functional disruption of the skin barrier. AD is often accompanied by allergic inflammation. AD patient suffer from heavy itching, and their quality of life is severely affected. Some pharmaceuticals for AD have some side effects such as skin atrophy. So it is necessary to develop mild solutions such as food ingredients without side effects. There are various causes of AD. It is especially induced by immunological imbalances such as IFN-γ reduction. IFN-γ has an important role in regulating IgE, which can cause an allergy reaction. NC/Nga mice develop AD and IgE hyperproduction. In a previous study, we revealed that administration of polysaccharide from black currant (R. nigrum) has an effect on immunomodulation. It induces IFN-γ production from myeloid dendritic cells. We named this polysaccharide cassis polysaccharide (CAPS). In this report, we studied the effect of administering CAPS on atopic dermatitis in NC/Nga mice. Thirty NC/Nga mice that developed symptoms of atopic dermatitis were used. We divided them into three groups (control, CAPS administration 12 mg/kg/day, CAPS administration 60 mg/kg/day). For 4 weeks, we evaluated clinical score, serum IgE levels, gene expression of spleen, and skin pathology. We revealed that CAPS administration improves atopic dermatitis symptoms. We also found that CAPS administration suppresses IgE hyperproduction and induces IFN-γ gene transcription in the spleen. Finally, we confirmed that CAPS administration suppresses mast cell migration to epidermal skin. These results indicated that CAPS has an effect on AD.

Polysaccharide from black currant (Ribes nigrum L.) stimulates dendritic cells through TLR4 signaling.

Black currant (Ribes nigrum) has various beneficial properties for human health. In particular, polysaccharide from black currant was found to be an immunostimulating food ingredient and was reported to have antitumor activity in a mouse model. We named it cassis polysaccharide (CAPS). In a previous study, CAPS administration caused tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) production in vitro and in vivo, but the immunological mechanism of CAPS was not demonstrated. In this study, we revealed the CAPS immunostimulating mechanism in vitro. First, we found that CAPS activated dendritic cells (DCs). Second, we investigated whether it depends on Toll-like receptor 4 (TLR4) and myeloid differentiation primary response (Myd). We concluded that CAPS stimulates DCs through Myd88 depending TLR4 signaling and activates Th1-type cytokine release.

Aggravation of collagen-induced arthritis by orally administered Porphyromonas gingivalis through modulation of the gut microbiota and gut immune system.

Porhyromonas gingivalis, a causative bacterium of periodontitis, is implicated in the etiology of rheumatoid arthritis (RA), mainly because of expressing peptidyl arginine deiminase (PAD) that generates RA-related autoantigens. However, compared with other periodontopathic bacteria, the precise role of P. gingivalis in RA is largely unknown. We found that orally administered P. gingivalis changed the gut microbiome with concomitant elevation of serum endotoxin and inflammatory markers, and impairment of the gut barrier function. Based on findings showing a relationship between gut microbiota and RA, we investigated whether the change of gut microbiota induced by P. gingivalis and Prevotella intermedia, another periodontopathic bacterium without PAD, is associated with collagen-induced arthritis (CIA). DBA/1J mice were orally administered with or without bacteria followed by induction of CIA. P. gingivalis, but not P. intermedia, administration significantly aggravated arthritis with increased interleukin-17 levels in sera and culture supernatants, increased Th17 cell proportions among mesenteric lymphocytes, and a significant change in the gut microbiome. However, P. gingivalis administration did not elevate the level of anti-citrullinated protein antibody. These results suggest a unique role of P. gingivalis in the link between periodontitis and RA by affecting the gut immune system and the gut microbiota composition.

Systems pharmacology of adiposity reveals inhibition of EP300 as a common therapeutic mechanism of caloric restriction and resveratrol for obesity.

Both caloric restriction (CR) and resveratrol (RSV) have beneficial effects on obesity. However, the biochemical pathways that mediate these beneficial effects might be complex and interconnected and have not been fully elucidated. To reveal the common therapeutic mechanism of CR and RSV, we performed a comparative transcriptome analysis of adipose tissues from diet-induced obese (DIO) zebrafish and obese humans. We identified nine genes in DIO zebrafish and seven genes in obese humans whose expressions were regulated by CR and RSV. Although the gene lists did not overlap except for one gene, the gene ontologies enriched in the gene lists were highly overlapped, and included genes involved in adipocyte differentiation, lipid storage and lipid metabolism. Bioinformatic analysis of cis-regulatory sequences of these genes revealed that their transcriptional regulators also overlapped, including EP300, HDAC2, CEBPB, CEBPD, FOXA1, and FOXA2. We also identified 15 and 46 genes that were dysregulated in the adipose tissue of DIO zebrafish and obese humans, respectively. Bioinformatics analysis identified EP300, HDAC2, and CEBPB as common transcriptional regulators for these genes. EP300 is a histone and lysyl acetyltransferase that modulates the function of histone and various proteins including CEBPB, CEBPD, FOXA1, and FOXA2. We demonstrated that adiposity in larval zebrafish was significantly reduced by C646, an inhibitor of EP300 that antagonizes acetyl-CoA. The reduction of adiposity by C646 was not significantly different from that induced by RSV or co-treatment of C646 and RSV. These results indicate that the inhibition of EP300 might be a common therapeutic mechanism between CR and RSV in adipose tissues of obese individuals.

Inhibition of polysaccharide synthesis by the sinR orthologue PGN_0088 is indirectly associated with the penetration of Porphyromonas gingivalis biofilms by macrolide antibiotics.

Microbes commonly adhere to surfaces, aggregate in self-produced extracellular polymeric substances (EPS) and live in biofilms. Periodontitis is a serious oral infection that is initiated by the formation of biofilms by Porphyromonas gingivalis. EPS act as a barrier that protects biofilm-forming cells against sources of stress, including those induced by host immune cells and antimicrobial agents. Therefore, drugs intended to kill such micro-organisms cannot be used for the treatment of biofilm infections. Our previous studies revealed that subminimal inhibitory concentrations (subMIC) of two macrolide antibiotics (azithromycin, AZM and erythromycin, ERY) reduced P. gingivalis biofilms. Furthermore, we demonstrated that the Bacillus subtilis sinR orthologue (PGN_0088) inhibits the synthesis of carbohydrates that are components of EPS in P. gingivalis biofilms. Here, we constructed a novel sinR mutant from P. gingivalis ATCC 33277 and reveal that the increased abundance of carbohydrate in EPS of the mutant led to a reduced infiltration rate of AZM and ERY through EPS, and consequently elevated biofilm resistance to these macrolides. Detailed elucidation of the interaction between the product of the sinR gene and EPS will assist in the development of novel approaches that target EPS to prevent and inhibit the formation of biofilms.

Promotion of endodontic lesions in rats by a novel extraradicular biofilm model using obturation materials.

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (P < 0.05 by Welch's t test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.

Up-regulation of endothelial nitric oxide synthase (eNOS), silent mating type information regulation 2 homologue 1 (SIRT1) and autophagy-related genes by repeated treatments with resveratrol in human umbilical vein endothelial cells.

Resveratrol, a polyphenolic phytoalexin found in red wine and various plants, has been reported to up-regulate the expression of endothelial NO synthase (eNOS) in human umbilical vein endothelial cells (HUVEC). However, this effect was neither long term in nature nor physiologically relevant at the concentration of resveratrol studied. In the present study, we investigated the effects of repeated treatments with a lower concentration of resveratrol on the expression of genes in HUVEC. The expression levels of eNOS and silent mating type information regulation 2 homologue 1 (SIRT1) were up-regulated in HUVEC by repeated treatments with 1 µM-resveratrol for 6 d, but not with fenofibrate. Moreover, resveratrol treatment increased the expression of autophagy-regulated genes such as γ-aminobutyric acid A receptor-associated protein (GABARAP), microtubule-associated protein 1 light chain 3B (LC3B) and autophagy-related protein 3 (ATG3), the radical scavenger activity-related metallothionein-1X (MT1X) gene and the anti-inflammatory activity-related annexin A2 (ANXA) gene. In addition, resveratrol treatment down-regulated the expression of the cell-cycle checkpoint control RAD9 homologue B (RAD9B) gene. These results indicate the beneficial effects of resveratrol on the cardiovascular system.

Porphyromonas gingivalis biofilms persist after chlorhexidine treatment.

Chlorhexidine (CHX) gluconate effectively reduces the viability of biofilm-forming bacteria, such as Porphyromonas gingivalis. However, it is impossible to completely remove biofilms. The goal of the present study was to assess the potential pathogenicity of residual P. gingivalis biofilms in vitro after treatment with CHX gluconate. Scanning and transmission electron microscopy and confocal laser imaging revealed that treatment with CHX gluconate disrupted individual biofilm-forming P. gingivalis cells but did not destroy the biofilms. The volumes of the protein and carbohydrate constituents in the residual biofilms were not significantly different from those of the controls. The physical resistance of the residual biofilms to ultrasonication was significantly higher than that of controls. The volume of P. gingivalis adherent to the residual biofilms was higher than that to saliva-coated wells. These findings suggest that although CHX gluconate caused disruption of biofilm-forming cells, the constituents derived from disrupted cells were maintained in the biofilms, which sustained their external structures. Moreover, the residual biofilms could serve as a scaffold for the formation of new biofilms.

The sinR ortholog PGN_0088 encodes a transcriptional regulator that inhibits polysaccharide synthesis in Porphyromonas gingivalis ATCC 33277 biofilms.

Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.

Bone marrow-derived HipOP cell population is markedly enriched in osteoprogenitors.

We recently succeeded in purifying a novel multipotential progenitor or stem cell population from bone marrow stromal cells (BMSCs). This population exhibited a very high frequency of colony forming units-osteoblast (CFU-O; 100 times higher than in BMSCs) and high expression levels of osteoblast differentiation markers. Furthermore, large masses of mineralized tissue were observed in in vivo transplants with this new population, designated highly purified osteoprogenitors (HipOPs). We now report the detailed presence and localization of HipOPs and recipient cells in transplants, and demonstrate that there is a strong relationship between the mineralized tissue volume formed and the transplanted number of HipOPs.

Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation.

Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.

Overexpression of Ycg1 or Ydr520c confers resistance to cadmium in Saccharomyces cerevisiae.

We introduced a yeast open reading frame library into Saccharomyces cerevisiae strain BY4742 to search for genes whose overexpression conferred cadmium resistance to yeast, toward the goal of elucidating the mechanism of cadmium toxicity. As a result, we found that the overexpression of two newly identified genes, Ycg1 and Ydr520c, each conferred strong cadmium resistance to yeast.

Effects of polysaccharide derived from black currant on relieving clinical symptoms of Japanese cedar pollinosis: a randomized double-blind, placebo-controlled trial.

We investigated the efficacy of the polysaccharide derived from black currant, named cassis polysaccharide (CAPS), for inhibiting Japanese cedar pollinosis symptoms and improving quality of life by a randomized double-blind, placebo-controlled trial in 2006. A total of 28 subjects were enrolled in the study, and 10 subjects in each group completed the trial. Although there was no significant difference between the CAPS and placebo group in the weekly mean value of any symptom in the daily symptom diary at any time, a smaller degree of final symptom aggravation was found in the CAPS group. Significant aggravation of the score was finally observed in the placebo group with inferior conch swelling and with sneezing, itchy nose, itchy eye and watery eye in the Japan rhino-conjunctivitis quality of life questionnaire assessment, while the changes observed in the CAPS group were not significant. In conclusion, our findings clearly indicate that CAPS would be useful as a food supplement in assisting the treatment of Japanese cedar pollinosis.

Improvement of the antitumor activity of black currant polysaccharide by an enzymatic treatment.

A polysaccharide-rich substance isolated from black currant, named cassis polysaccharide (CAPS), was partially digested with beta-galactosidase from Aspergillus oryzae and its immunostimulatory activity was investigated. The in vitro cytokine-inducing effect of CAPS on RAW264 cells was gradually decreased along with lowering of the average MW of CAPS. In vivo, partially digested CAPS with a mean MW of approximately 20,000 showed the most potent antitumor activity against Ehrlich carcinoma in mice.