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Progress in genomic analysis has resulted in the proposal that the intestinal microbiota is a crucial environmental factor in the development of multifactorial diseases, such as obesity, diabetes, rheumatoid arthritis, and inflammatory bowel diseases represented by Crohn’s disease and ulcerative colitis. Dysregulated gut microbiome contributes to the pathogenesis of such disorders; however, there are few effective treatments for controlling only disease-mediating bacteria. Here, we review current knowledge about the intestinal microbiome in health and disease, and discuss a regulatory strategy using a parenteral vaccine with emulsified curdlan and CpG oligodeoxynucleotides, which we have recently developed. Unlike other conventional injectable immunizations, our vaccine contributes to the induction of antigen-specific systemic and mucosal immunity. This vaccine strategy can prevent infectious diseases such as Streptococcus pneumoniae infection, and control metabolic symptoms mediated by intestinal bacteria (e.g. Clostridium ramosum) by induction of high titers of antigen-specific IgA at target mucosal sites. In the future, our vaccination approach could be an effective therapy for common infectious diseases and dysbiosis-related disorders that have been difficult to control so far.

MotivationBacteriophages/Phages are the viruses that infect and replicate within bacteria and archaea, and rich in human body. To investigate the relationship between phages and microbial communities, the identification of phages from metagenome sequences is the first step. Currently, there are two main methods for identifying phages: database-based (alignment-based) methods and alignment-free methods. Database-based methods typically use a large number of sequences as references; alignment-free methods usually learn the features of the sequences with machine learning and deep learning models.ResultsWe propose INHERIT which uses a deep representation learning model to integrate both database-based and alignment-free methods, combining the strengths of both. Pre-training is used as an alternative way of acquiring knowledge representations from existing databases, while the BERT-style deep learning framework retains the advantage of alignment-free methods. We compare INHERIT with four existing methods on a third-party benchmark dataset. Our experiments show that INHERIT achieves a better performance with the F1-score of 0.9932. In addition, we find that pre-training two species separately helps the non-alignment deep learning model make more accurate predictions.AvailabilityThe codes of INHERIT are now available in: https://github.com/Celestial-Bai/INHERIT.Contactyaozhong@ims.u-tokyo.ac.jp and imoto@hgc.jpSupplementary informationSupplementary data are available at BioRxiv online.

Our bodies are constantly exposed to a wide variety of pathogenic micro-organisms through mucosal sites. Therefore, effective vaccines that can protect at the mucosa are vital; however, only a few clinically established mucosal vaccines are available. Although conventional injectable vaccines can induce antigen-specific serum immunoglobulin G (IgG) and prevent severe infection, it is difficult to efficiently inhibit the invasion of pathogens at mucosal surfaces because of the inadequate ability to induce antigen-specific IgA. Recently, we have developed a parenteral vaccine with emulsified curdlan and CpG oligodeoxynucleotides and reported its application. Unlike other conventional injectable vaccines, this immunization contributes to the induction of antigen-specific mucosal and systemic immune responses. Even if antigen-specific IgA at the mucosa disappears, this immunization can induce high-titer IgA after boosting with a small amount of antigen on the target mucosal surface. Indeed, vaccination with Streptococcus pneumoniae antigen effectively prevented lung infection induced by this bacterium. In addition, vaccination with Clostridium ramosum, which is a representative pathobiont associated with obesity and diabetes in humans, reduced obesity in mice colonized with this microorganism. This immunization approach might be an effective treatment for intestinal bacteria-mediated diseases that have been difficult to regulate so far, as well as common infectious diseases.

The bacterial species living in the gut mediate many aspects of biological processes such as nutrition and activation of adaptive immunity. In addition, commensal fungi residing in the intestine also influence host health. Although the interaction of bacterium and fungus has been shown, its precise mechanism during colonization of the human intestine remains largely unknown. Here, we show interaction between bacterial and fungal species for utilization of dietary components driving their efficient growth in the intestine. Next generation sequencing of fecal samples from Japanese and Indian adults revealed differential patterns of bacterial and fungal composition. In particular, Indians, who consume more plant polysaccharides than Japanese, harbored increased numbers of Prevotella and Candida. Candida spp. showed strong growth responses to the plant polysaccharide arabinoxylan in vitro. Furthermore, the culture supernatants of Candida spp. grown with arabinoxylan promoted rapid proliferation of Prevotella copri. Arabinose was identified as a potential growth-inducing factor in the Candida culture supernatants. Candida spp. exhibited a growth response to xylose, but not to arabinose, whereas P. copri proliferated in response to both xylose and arabinose. Candida spp., but not P. copri, colonized the intestine of germ-free mice. However, P. copri successfully colonized mouse intestine already harboring Candida. These findings demonstrate a proof of concept that fungal members of gut microbiota can facilitate a colonization of the intestine by their bacterial counterparts, potentially mediated by a dietary metabolite.

The receptor activator of nuclear factor κB ligand (RANKL) is an important factor for osteoclastogenesis and contributes to the pathology of rheumatoid arthritis (RA); thus, the anti-RANKL antibody (Ab) has been expected to protect joint destruction in RA patients. IL-8 also has osteoclastogenic activity; however, the role of IL-8 in the bone pathology of RA as well as the relation between IL-8 and RANKL remain unclear. In the present study, clinical observation revealed serum IL-8 levels of 611 pg ml−1 in RA patients with anti-RANKL Ab and 266 pg ml−1 in the same patients without anti-RANKL Ab. In vitro assay showed that anti-RANKL Ab induced production of IL-8 from pre-osteoclast-like cells (OCLs), and IL-8 promoted the formation of OCLs from peripheral monocytes even without RANKL activity. We further showed that treatment with FK506 (tacrolimus) possibly inhibits the increase in IL-8 levels in RA patients with anti-RANKL Ab, and in vitro assay confirmed that FK506 suppressed IL-8 production in pre-OCLs. These results suggest that inhibition of RANKL induces the change in osteoclastogenesis-promoting factor from RANKL to IL-8, and FK506 may be a valuable combination drug to support the use of anti-RANKL Ab in treatment of RA., A therapeutic anti-RANKL Ab increases IL-8 production in RA

Worldwide prevalence of obesity is associated with the increase of lifestyle-related diseases. The accumulation of intermuscular adipose tissue (IMAT) is considered a major problem whereby obesity leads to sarcopenia and metabolic disorders and thus is a promising target for treating these pathological conditions. However, whereas obesity-associated IMAT is suggested to originate from PDGFRα+ mesenchymal progenitors, the processes underlying this adipogenesis remain largely unexplored. Here, we comprehensively investigated intra- and extracellular changes associated with these processes using single-cell RNA sequencing and mass spectrometry. Our single-cell RNA sequencing analysis identified a small PDGFRα+ cell population in obese mice directed strongly toward adipogenesis. Proteomic analysis showed that the appearance of this cell population is accompanied by an increase in galectin-3 in interstitial environments, which was found to activate adipogenic PPARγ signals in PDGFRα+ cells. Moreover, IMAT formation during muscle regeneration was significantly suppressed in galectin-3 knockout mice. Our findings, together with these multi-omics datasets, could unravel microenvironmental networks during muscle regeneration highlighting possible therapeutic targets against IMAT formation in obesity.

The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.

Genome-wide association studies and subsequent deep sequencing analysis have identified susceptible loci for inflammatory bowel diseases (IBDs) including ulcerative colitis (UC). A gene encoding RING finger protein 186 (RNF186) is located within UC-susceptible loci. However, it is unclear whether RNF186 is involved in IBD pathogenesis. Here, we show that RNF186 controls protein homeostasis in colonic epithelia and regulates intestinal inflammation. RNF186, which was highly expressed in colonic epithelia, acted as an E3 ligase mediating polyubiquitination of its substrates. Permeability of small organic molecules was augmented in the intestine of Rnf186-/- mice. Increased expression of several RNF186 substrates, such as occludin, was found in Rnf186-/- colonic epithelia. The disturbed protein homeostasis in Rnf186-/- mice correlated with enhanced endoplasmic reticulum (ER) stress in colonic epithelia and increased sensitivity to intestinal inflammation after dextran sulfate sodium (DSS) treatment. Introduction of an UC-associated Rnf186 mutation led to impaired E3 ligase activity and increased sensitivity to DSS-induced intestinal inflammation in mice. Thus, RNF186 maintains gut homeostasis by controlling ER stress in colonic epithelia.

Summary The application of bacteriophages (phages) is proposed as a highly specific therapy for intestinal pathobiont elimination. However, the infectious associations between phages and bacteria in the human intestine, which is essential information for the development of phage therapies, have yet to be fully elucidated. Here, we report the intestinal viral microbiomes (viromes), together with bacterial microbiomes (bacteriomes), in 101 healthy Japanese individuals. Based on the genomic sequences of bacteriomes and viromes from the same fecal samples, the host bacteria-phage associations are illustrated for both temperate and virulent phages. To verify the usefulness of the comprehensive host bacteria-phage information, we screened Clostridioides difficile-specific phages and identified antibacterial enzymes whose activity is confirmed both in vitro and in vivo. These comprehensive metagenome analyses reveal not only host bacteria-phage associations in the human intestine but also provide vital information for the development of phage therapies against intestinal pathobionts.

Non-steroidal anti-inflammatory drugs (NSAIDs) cause damage in the small intestine in a bacteria-dependent manner. As high-fat diet (HFD) is a potent inducer of gut dysbiosis, we investigated the effects of HFD on bacterial flora in the small intestine and NSAID-induced enteropathy. 16S rRNA gene analysis revealed that the population of Bifidobacterium spp. significantly decreased by fold change of individual operational taxonomic units in the small intestine of mice fed HFD for 8 weeks. HFD increased intestinal permeability, as indicated by fluorescein isothiocyanate-dextran absorption and serum lipopolysaccharide levels, accompanied by a decrease in the protein expressions of ZO-1 and occludin and elevated mRNA expression of interleukin (IL)-17A in the small intestine. HFD-fed mice exhibited increased susceptibility to indomethacin-induced damage in the small intestine; this phenotype was observed in normal diet-fed mice that received small intestinal microbiota from HFD-fed mice. Administration of neutralizing antibodies against IL-17A to HFD-fed mice reduced intestinal permeability and prevented exacerbation of indomethacin-induced damage. Thus, HFD-induced microbial dysbiosis in small intestine caused microinflammation through the induction of IL-17A and increase in intestinal permeability, resulting in the aggravation of NSAID-induced small intestinal damage.

Background & Aims Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. Methods The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria–phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. Results Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. Conclusions The coordinated action of phages and their host bacteria restored the recipients' intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

Background Intestinal permeability and psychological stress are considered the key mechanism(s) in functional dyspepsia (FD). Although proton pump inhibitors (PPIs) are commonly used for the treatment of FD, the effect of PPIs on intestinal permeability has not been elucidated. This study investigated the effect of PPI on intestinal permeability under stressed conditions. Methods C57BL/6J mice were subjected to water avoidance stress (WAS) and administered rabeprazole (40 mg/kg) or vehicle treatment (VT). We then evaluated intestinal permeability both in vivo and ex vivo using plasma fluorescein isothiocyanate-dextran and by assessing the paracellular permeability and transepithelial electrical resistance (TEER) in an Ussing chamber, respectively. Furthermore, we evaluated the effect of PPI-treated fecal microbiota transplant (FMT) on intestinal permeability in vivo. Microbiota profiles of donor feces were assessed by 16S rRNA gene analysis using MiSeq and QIIME2. Key results In the WAS treatment, PPI significantly enhanced intestinal permeability in vivo compared to that in VT. Moreover, PPI significantly increased paracellular permeability and decreased TEER in the duodenum and jejunum, respectively, compared to those in VT under stressed conditions. Moreover, both vasoactive intestinal peptide (VIP) receptor antagonist and ketotifen significantly reversed the effect of PPI on intestinal permeability. Furthermore, PPI-treated FMT significantly increased the intestinal permeability in vivo compared to that in vehicle-treated FMT. Proton pump inhibitors treatment altered the gut microbiota composition, indicating that PPI induced dysbiosis. Conclusions and inferences Under stressed conditions, PPI enhances intestinal permeability via dysbiosis of gut microbiota. Vasoactive intestinal peptide and mast cells are also implicated in the underlying mechanisms.

Background & Aims Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. Methods C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. Results Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. Conclusions Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine.

Purpose: Image-guided cryotherapy of renal cancer is an emerging alternative to surgical nephrectomy, particularly for those who cannot sustain the physical burden of surgery. It is well known that the outcome of this therapy depends on the accurate placement of the cryotherapy probe. Therefore, a robotic instrument guide may help physicians aim the cryotherapy probe precisely to maximize the efficacy of the treatment and avoid damage to critical surrounding structures. The objective of this paper was to propose a robotic instrument guide for orienting cryotherapy probes in image-guided cryotherapy of renal cancers. The authors propose a body-mounted robotic guide that is expected to be less susceptible to guidance errors caused by the patient’s whole body motion. Methods: Keeping the device’s minimal footprint in mind, the authors developed and validated a body-mounted, robotic instrument guide that can maintain the geometrical relationship between the device and the patient’s body, even in the presence of the patient’s frequent body motions. The guide can orient the cryotherapy probe with the skin incision point as the remote-center-of-motion. The authors’ validation studies included an evaluation of the mechanical accuracy and position repeatability of the robotic instrument guide. The authors also performed a mock MRI-guided cryotherapy procedure with a phantom to compare the advantage of robotically assisted probe replacements over a free-hand approach, by introducing organmotions to investigate their effects on the accurate placement of the cryotherapy probe. Measurements collected for performance analysis included accuracy and time taken for probe placements. Multivariate analysis was performed to assess if either or both organmotion and the robotic guide impacted these measurements. Results: The mechanical accuracy and position repeatability of the probe placement using the robotic instrument guide were 0.3 and 0.1 mm, respectively, at a depth of 80 mm. The phantom test indicated that the accuracy of probe placement was significantly better with the robotic instrument guide (4.1 mm) than without the guide (6.3 mm, p

The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those for bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio’s circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

As glucose-dependent insulinotropic polypeptide (GIP) possesses pro-adipogenic action, the suppression of the GIP hypersecretion seen in obesity might represent a novel therapeutic approach to the treatment of obesity. However, the mechanism of GIP hypersecretion remains largely unknown. In the present study, we investigated GIP secretion in two mouse models of obesity: High-fat diet-induced obese (DIO) mice and leptin-deficient Lepob/ob mice. In DIO mice, plasma GIP was increased along with an increase in GIP mRNA expression in the lower small intestine. Despite the robust alteration in the gut microbiome in DIO mice, co-administration of maltose and the &alpha, glucosidase inhibitor (&alpha, GI) miglitol induced the microbiome-mediated suppression of GIP secretion. The plasma GIP levels of Lepob/ob mice were also elevated and were suppressed by fat transplantation. The GIP mRNA expression in fat tissue was not increased in Lepob/ob mice, while the expression of an interleukin-1 receptor antagonist (IL-1Ra) was increased. Fat transplantation suppressed the expression of IL-1Ra. The plasma IL-1Ra levels were positively correlated with the plasma GIP levels. Accordingly, although circulating GIP levels are increased in both DIO and Lepob/ob mice, the underlying mechanisms differ, and the anti-obesity actions of &alpha, GIs and leptin sensitizers may be mediated partly by the suppression of GIP secretion.

Extracellular adenosine 5'-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In Enpp3-/- mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer's patches. Intestinal pDCs of Enpp3-/- mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in P2rx7-/- pDCs. Accordingly, the number of intestinal pDCs was restored in Enpp3-/- P2rx7-/- mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.

The innate immune system provides the first line of host defense against invading pathogens. Innate immune responses are initiated by germline-encoded PRR, which recognize specific structures expressed by microorganisms. TLR are a family of PRR which sense a wide range of microorganisms, including bacteria, fungi, protozoa and viruses. TLR are also expressed in the intestine and are critical for intestinal homeostasis. Recently, cytoplasmic PRR, such as NLR and RLR, have been shown to detect pathogens that have invaded the cytosol. One of the NLR, NOD2, is thought to be involved in the pathogenesis of Crohn's disease. This review focuses on the innate immune responses triggered by PRR in the intestine.

The invention relates to a vibration wave driving apparatus which comprises a vibration member in which an electro-mechanical energy conversion element is fixed to the elastic member, and a contact member pressed against a sliding part provided in the vibration member, so as to be kept in contact therewith, and which is configured to supply alternating signals in a predetermined driving frequency band to the electro-mechanical energy conversion element, thereby generating a predetermined natural vibration mode in the elastic member to drive the contact member. In the apparatus, the natural vibration mode is generated in the elastic member, a natural frequency of another vibration mode different from the natural vibration mode is detected, and a stiffness of the support member is altered if the natural frequency of the other vibration mode detected is included in the driving frequency band.