The transcription factor ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory.

Immunological memory is thought to be mediated exclusively by lymphocytes. However, enhanced innate immune responses caused by a previous infection increase protection against reinfection, which suggests the presence of innate immunological memory. Here we identified an important role for the stress-response transcription factor ATF7 in innate immunological memory. ATF7 suppressed a group of genes encoding factors involved in innate immunity in macrophages by recruiting the histone H3K9 dimethyltransferase G9a. Treatment with lipopolysaccharide, which mimics bacterial infection, induced phosphorylation of ATF7 via the kinase p38, which led to the release of ATF7 from chromatin and a decrease in repressive histone H3K9me2 marks. A partially disrupted chromatin structure and increased basal expression of target genes were maintained for long periods, which enhanced resistance to pathogens. ATF7 might therefore be important in controlling memory in cells of the innate immune system.

QNetDiff: a quantitative measurement of network rewiring.

Bacteria in the human body, particularly in the large intestine, are known to be associated with various diseases. To identify disease-associated bacteria (markers), a typical method is to statistically compare the relative abundance of bacteria between healthy subjects and diseased patients. However, since bacteria do not necessarily cause diseases in isolation, it is also important to focus on the interactions and relationships among bacteria when examining their association with diseases. In fact, although there are common approaches to represent and analyze bacterial interaction relationships as networks, there are limited methods to find bacteria associated with diseases through network-driven analysis. In this paper, we focus on rewiring of the bacterial network and propose a new method for quantifying the rewiring. We then apply the proposed method to a group of colorectal cancer patients. We show that it can identify and detect bacteria that cannot be detected by conventional methods such as abundance comparison. Furthermore, the proposed method is implemented as a general-purpose tool and made available to the general public.

Intestinal Bacteria Fluctuating in Early-Stage Colorectal Cancer Carcinogenesis are Associated with Diet in Healthy Adults.

This hospital-based, cross-sectional study aimed to explore the association between diet and fluctuating intestinal bacteria in early-stage colorectal cancer (CRC) (Atopobium parvulum, Actinomyces odontolyticus, Solobacterium moorei, and Bifidobacterium longum). Healthy participants (n = 212) who underwent total colonoscopy at National Cancer Center Hospital (Tokyo, Japan) were divided into two groups according to the relative abundance of bacteria in their feces: those in the top 25% of relative bacterial abundance as cases and the bottom 25% as controls. The participants were divided into three groups (low, medium, and high) according to their intake of food groups associated with CRC. Multivariable logistic regression analysis was conducted to estimate the association between dietary intake and higher relative abundance of bacteria. Dairy products were inversely associated with a higher relative abundance of A. parvulum, A. odontolyticus, and S. moorei, with odds ratios (high vs. low) and 95% confidence interval as follows: 0.16 (0.06-0.44), 0.25 (0.08-0.82), and 0.29 (0.11-0.78), respectively. Additionally, dietary fiber was inversely associated with a higher relative abundance of S.moorei (0.29 [0.11-0.78]). No association was observed between diet and B.longum. In conclusion, healthy adults with a higher intake of dairy products and fiber had lower odds of having a higher relative abundance of CRC-associated microbiota.

Mediation effect of intestinal microbiota on the relationship between fiber intake and colorectal cancer.

Higher fiber intake has been associated with a lower risk of colorectal cancer (CRC) and has been shown to protect against CRC based on probable evidence. Recent studies revealed a possible mechanism whereby the interaction between intestinal microbiota and fiber intake mediates CRC risk. However, the specific intestinal bacteria and the amount of these bacteria involved in this mechanism are not fully known. Therefore, this single-center study aimed to determine whether specific intestinal bacteria mediated the relationship between fiber intake and CRC risk. We enrolled patients who received colonoscopy at National Cancer Center Hospital. This cross-sectional study included 180 patients with clinically diagnosed CRC and 242 controls. We conducted a causal mediation analysis to assess the natural indirect effect and natural direct effect of specific intestinal bacteria on association between fiber intake and CRC risk. The median age was 64 (interquartile range, 54-70) years, and 58% of the participants were males. We used metagenomics for profiling gut microbiomes. The relative abundance of each species in each sample was calculated. Among the candidate, Fusobacterium nucleatum and Gemella morbillorum had a significant natural indirect effect based on their highest fiber intake compared to the lowest fiber intake, with a risk difference (95% confidence interval, proportion of mediation effect) of -0.06 [-0.09 to -0.03, 23%] and -0.03 [-0.06 to -0.01, 10.5%], respectively. Other bacteria did not display natural indirect effects. In conclusion, Fusobacterium nucleatum and Gemella morbillorum were found to mediate the relationship between fiber intake and CRC risk.

Analysis of genomic characteristics and their influence on metabolism in Aspergillus luchuensis albino mutants using genome sequencing.

Black Aspergillus luchuensis and its white albino mutant are essential fungi for making alcoholic beverages in Japan. A large number of industrial strains have been created using novel isolation or gene/genome mutation techniques. Such mutations influence metabolic and phenotypic characteristics in industrial strains, but few comparative studies of inter-strain mutation have been conducted. We carried out comparative genome analyses of 8 industrial strains of A. luchuensis and A. kawachii IFO 4308, the latter being the first albino strain to be isolated. Phylogenetic analysis based on 8938 concatenated genes exposed the diversity of black koji strains and uniformity among albino industrial strains, suggesting that passaged industrial albino strains have more genetic mutations compared with strain IFO 4308 and black koji strains. Comparative analysis showed that the albino strains had mutations in genes not only for conidial pigmentation but also in those that encode N-terminal acetyltransferase A and annexin XIV-like protein. The results also suggest that some mutations may have emerged through subculturing of albino strains. For example, mutations in the genes for isocitrate lyase and sugar transporters were observed only in industrial albino strains. This implies that selective pressure for increasing enzyme activity or secondary metabolites may have influenced the mutation of genes associated with environmental stress responses in A. luchuensis albino strains. Our study clarifies hitherto unknown genetic and metabolic characteristics of A. luchuensis industrial strains and provides potential applications for comparative genome analysis for breeding koji strains.

Evolution of biomolecular networks: lessons from metabolic and protein interactions.

Despite only becoming popular at the beginning of this decade, biomolecular networks are now frameworks that facilitate many discoveries in molecular biology. The nodes of these networks are usually proteins (specifically enzymes in metabolic networks), whereas the links (or edges) are their interactions with other molecules. These networks are made up of protein-protein interactions or enzyme-enzyme interactions through shared metabolites in the case of metabolic networks. Evolutionary analysis has revealed that changes in the nodes and links in protein-protein interaction and metabolic networks are subject to different selection pressures owing to distinct topological features. However, many evolutionary constraints can be uncovered only if temporal and spatial aspects are included in the network analysis.

Influence of gastrectomy for gastric cancer treatment on faecal microbiome and metabolome profiles.

OBJECTIVE: Recent evidence points to the gut microbiome's involvement in postoperative outcomes, including after gastrectomy. Here, we investigated the influence of gastrectomy for gastric cancer on the gut microbiome and metabolome, and how it related to postgastrectomy conditions. DESIGN: We performed shotgun metagenomics sequencing and capillary electrophoresis time-of-flight mass spectrometry-based metabolomics analyses on faecal samples collected from participants with a history of gastrectomy for gastric cancer (n=50) and compared them with control participants (n=56). RESULTS: The gut microbiota in the gastrectomy group showed higher species diversity and richness (p<0.05), together with greater abundance of aerobes, facultative anaerobes and oral microbes. Moreover, bile acids such as genotoxic deoxycholic acid and branched-chain amino acids were differentially abundant between the two groups (linear discriminant analysis (LDA) effect size (LEfSe): p<0.05, q<0.1, LDA>2.0), as were also Kyoto Encyclopedia of Genes and Genomes modules involved in nutrient transport and organic compounds biosynthesis (LEfSe: p<0.05, q<0.1, LDA>2.0). CONCLUSION: Our results reveal alterations of gut microbiota after gastrectomy, suggesting its association with postoperative comorbidities. The multi-omic approach applied in this study could complement the follow-up of patients after gastrectomy.

Significance of the gut microbiome in multistep colorectal carcinogenesis.

Colorectal cancer (CRC) is highly prevalent worldwide. In 2018, there were over 1.8 million new cases. Most sporadic CRC develop from polypoid adenomas and are preceded by intramucosal carcinoma (stage 0), which can progress into more malignant forms. This developmental process is known as the adenoma-carcinoma sequence. Early detection and endoscopic removal are crucial for CRC management. Accumulating evidence suggests that the gut microbiota is associated with CRC development in humans. Comprehensive characterization of this microbiota is of great importance to assess its potential as a diagnostic marker in the very early stages of CRC. In this review, we summarized recent studies on CRC-associated bacteria and their carcinogenic mechanisms in animal models, human cell lines and human cohorts. High-throughput technologies have facilitated the identification of CRC-associated bacteria in human samples. We have presented our metagenome and metabolome studies on fecal samples collected from a large Japanese cohort that revealed stage-specific phenotypes of the microbiota in CRC. Furthermore, we have discussed the potential carcinogenic mechanisms of the gut microbiota, from which we can infer whether changes in the gut microbiota are a cause or effect in the multi-step process of CRC carcinogenesis.

FuncTree2: an interactive radial tree for functional hierarchies and omics data visualization.

SUMMARY: Functional annotations and their hierarchical classification are widely used in omics workflows to build novel insight upon existing biological knowledge. Currently, a plethora of tools is available to explore omics datasets at the level of functional annotations, but there is a lack of feature rich and user-friendly tools that help scientists take advantage of their hierarchical classification for additional and often invaluable insights. Here, we present FuncTree2, a user-friendly web application that turns hierarchical classifications into interactive and highly customizable radial trees, and enables researchers to visualize their data simultaneously on all its levels. FuncTree2 features mapping of data from multiple samples and several navigation features like zooming, panning, re-rooting and collapsing of nodes or levels. AVAILABILITY AND IMPLEMENTATION: FuncTree2 is freely available at https://bioviz.tokyo/functree2/ as a web application and a REST API. Source code is available on GitHub https://github.com/yamada-lab/functree-ng. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

iPath3.0: interactive pathways explorer v3.

iPath3.0 (http://pathways.embl.de) is a web-application for the visualization and analysis of cellular pathways. It is freely available and open to everyone. Currently it is based on four KEGG global maps, which summarize up to 158 traditional KEGG pathway maps, 192 KEGG modules and other metabolic elements into one connected and manually curated metabolic network. Users can fully customize these networks and interactively explore them through its redesigned, fast and lightweight interface, which highlights general metabolic trends in multi-omics data. It also offers navigation at various levels of details to help users further investigate those trends and ultimately uncover novel biological insights. Support for multiple experimental conditions and time-series datasets, tools for generation of customization data, programmatic access, and a free user accounts system were introduced in this version to further streamline its workflow.

Bacteroides vulgatus and Bacteroides dorei Reduce Gut Microbial Lipopolysaccharide Production and Inhibit Atherosclerosis.

BACKGROUND: It is increasingly recognized that gut microbiota play a pivotal role in the development of atherosclerotic cardiovascular disease. Previously, we have reported that the abundance of genus Bacteroides is lower in patients with coronary artery disease (CAD) than in patients without CAD with coronary risk factors or in healthy volunteers. However, it remains unclear which and how specific gut bacteria contribute to the progression of atherosclerosis. METHODS: We recruited patients with CAD patients and controls without CAD with coronary risk factors. We then compared gut microbial composition using 16S ribosomal RNA gene sequencing in fecal samples to detect species with differential abundance between 2 groups. Subsequently, we used atherosclerosis-prone mice to study the mechanisms underlying the relationship between such species and atherosclerosis. RESULTS: Human fecal 16S ribosomal RNA gene sequencing revealed a significantly lower abundance of Bacteroides vulgatus and Bacteroides dorei in patients with CAD. This significant differential abundance was confirmed by quantitative polymerase chain reaction. Gavage with live B. vulgatus and B. dorei attenuated atherosclerotic lesion formation in atherosclerosis-prone mice, markedly ameliorating endotoxemia followed by decreasing gut microbial lipopolysaccharide production, effectively suppressing proinflammatory immune responses. Furthermore, fecal lipopolysaccharide levels in patients with CAD were significantly higher and negatively correlated with the abundance of B. vulgatus and B. dorei. CONCLUSIONS: Our translational research findings identify a previously unknown link between specific gut bacteria and atherosclerosis. Treatment with live B. vulgatus and B. dorei may help prevent CAD. CLINICAL TRIAL REGISTRATION: URL: https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000018051 . Unique identifier: UMIN000015703.

Staging of gastric cancer with the Clinical Stage Prediction score.

BACKGROUND: Chemotherapy with or without surgery is the first-line treatment for stage III/IV gastric cancer, while surgery is the first-line treatment for stage I/II gastric cancer. Accordingly, it is important to distinguish between stage III/IV and stage I/II gastric cancer, but clinical staging is less accurate than pathological staging. This study was performed to develop a clinical score that could distinguish stage III/IV gastric cancer from stage I/II gastric cancer. METHODS: We reviewed 2722 patients who underwent gastrectomy at our hospital from January 1996 to December 2015. As pretreatment factors potentially related to tumor stage, we assessed age, sex, tumor markers, tumor diameter, tumor location, tumor histology, and macroscopic type. Factors showing significance on multivariate analysis were used to develop the Clinical Stage Prediction score (CSP score), and a cutoff value for the score was determined by receiver operating characteristics analysis. RESULTS: According to multivariate analysis, clinical factors associated with stage III/IV disease were elevation of the carcinoembryonic antigen level, tumor diameter ≥ 60 mm, circumferential gastric involvement, esophageal infiltration, mucinous adenocarcinoma, and macroscopic types 2-4. The CSP score was obtained by weighting these factors according to the non-standardized ß-coefficient. Receiver operating characteristics analysis indicated that the optimum cutoff value of the CSP score was 17 points. Among 1042 patients with a CSP score ≥ 17 points, 820 patients (78.7%) had stage III/IV gastric cancer. Conversely, among 1680 patients with a CSP score < 17 points, 1547 patients (92.1%) had stage I/II gastric cancer. When discrimination of stage III/IV gastric cancer from stage I/II gastric cancer by the CSP score was assessed, the sensitivity was 78.7%, specificity was 92.1%, positive predictive value was 86.0%, and negative predictive value was 87.5%. CONCLUSIONS: The CSP score can be helpful for differentiating stage III/IV gastric cancer from stage I/II gastric cancer based on pretreatment clinical factors.

Gut Microbiome and Plasma Microbiome-Related Metabolites in Patients With Decompensated and Compensated Heart Failure.

BACKGROUND: Gut microbiome composition or circulating microbiome-related metabolites in patients with heart failure (HF) have not been investigated at different time points (i.e., in the decompensated (Decomp) and compensated (Comp) phases). Methods and Results: We prospectively enrolled 22 patients admitted for HF and 11 age-, sex-, and comorbidity-matched hospitalized control subjects without a history of HF. Gut flora and plasma microbiome-related metabolites were evaluated by amplicon sequencing of the bacterial 16S ribosomal RNA gene and capillary electrophoresis time-of-flight mass spectrometry, respectively. HF patients were evaluated in both the Decomp and Comp phases during hospitalization. The phylum Actinobacteria was enriched in HF patients compared with control subjects. At the genus level, Bifiodobacterium was abundant while Megamonas was depleted in HF patients. Meanwhile, plasma concentration of trimethylamine N-oxide (TMAO), a gut microbiome-derived metabolite, was increased in HF patients (Decomp HF vs. control, P=0.003; Comp HF vs. control, P=0.004). A correlation analysis revealed positive correlations between the abundance of the genus Escherichia/Shigella and levels of TMAO and indoxyl sulfate (IS, a microbe-dependent uremic toxin) in Comp HF (TMAO: r=0.62, P=0.002; IS: r=0.63, P=0.002). Escherichia/Shigella was more abundant in Decomp than in Comp HF (P=0.030). CONCLUSIONS: Our results suggest that gut microbiome composition and microbiome-related metabolites are altered in HF patients.

Comprehensive microbiome analysis of tonsillar crypts in IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most prevalent primary chronic glomerular disease, in which the mucosal immune response elicited particularly in the tonsils or intestine has been estimated to be involved in the development of the disease. To explore the relationship between IgAN and bacterial flora in the tonsils, we conducted a comprehensive microbiome analysis. METHODS: We enrolled 48 IgAN patients, 21 recurrent tonsillitis (RT) patients without urine abnormalities and 30 children with tonsillar hyperplasia (TH) who had undergone tonsillectomy previously. Genomic DNA from tonsillar crypts of each patient was extracted, and V4 regions of the 16S ribosomal RNA gene were amplified and analysed using a high-throughput multiplexed sequencing approach. Differences in genus composition among the three study groups were statistically analysed by permutational multivariate analysis of variance and visualized by principal component analysis (PCA). RESULTS: Substantial diversity in bacterial composition was detected in each sample. Prevotella spp., Fusobacterium spp., Sphingomonas spp. and Treponema spp. were predominant in IgAN patients. The percentage of abundance of Prevotella spp., Haemophilus spp., Porphyromonas spp. and Treponema spp. in IgAN patients was significantly different from that in TH patients. However, there was no significant difference in the percentage of abundance of any bacterial genus between IgAN and RT patients. PCA did not distinguish IgAN from RT, although it discriminated TH. No significant differences in microbiome composition among the groups of IgAN patients according to clinicopathological parameters were observed. CONCLUSIONS: Similar patterns of bacteria are present in tonsillar crypts of both IgAN and RT patients, suggesting that the host response to these bacteria might be important in the development of IgAN.

Enterotypes of the human gut microbiome.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.

The true distribution volume and bioavailability of mizoribine in children with chronic kidney disease.

BACKGROUND: Mizoribine (MZR) is used kidney transplant and various kidney diseases. However, few studies reported the association between pharmacokinetics and pharmacodynamics. The Pharmacokinetics Study Group for Pediatric Kidney Disease (PSPKD) used population pharmacokinetics (PPK) analysis and Bayesian analysis to investigate the usefulness of MZR. In this study, the fact that almost all MZR are excreted unchanged in urine was used to calculate its bioavailability (F) and true distribution volume (V d), and analyzed these correlation with age. METHODS: Ishida et al. reported a PPK analysis by the PSPKD. In the present study, 71 samples extracted from their study population of 105 pediatric chronic kidney disease patients aged between 1 and 20 years were investigated. The bioavailability was calculated by measuring total excreted MZR in 24 h urine samples, and this was compared to the oral dosage. The apparent distribution volume (V d/F) obtained from Bayesian analysis was then used to calculate true distribution volume (V d), and the correlation of each parameter with age was investigated. RESULTS: The median dose of MZR per weight was 5.17 mg/kg/day. Median bioavailability was 32.02%. Median V d per weight was 0.46 L/kg. There was a significant, weakly positive correlation between bioavailability and age (p = 0.026). There was also a significant, weakly negative correlation between V d per weight and age (p = 0.003). CONCLUSION: Bioavailability and V d per weight tended to decrease depending on age. The younger patient required larger dose required to obtain the maximum effect from MZR, and this is important for immunosuppressive therapy.

Identification of Enzyme Genes Using Chemical Structure Alignments of Substrate-Product Pairs.

Although there are several databases that contain data on many metabolites and reactions in biochemical pathways, there is still a big gap in the numbers between experimentally identified enzymes and metabolites. It is supposed that many catalytic enzyme genes are still unknown. Although there are previous studies that estimate the number of candidate enzyme genes, these studies required some additional information aside from the structures of metabolites such as gene expression and order in the genome. In this study, we developed a novel method to identify a candidate enzyme gene of a reaction using the chemical structures of the substrate-product pair (reactant pair). The proposed method is based on a search for similar reactant pairs in a reference database and offers ortholog groups that possibly mediate the given reaction. We applied the proposed method to two experimentally validated reactions. As a result, we confirmed that the histidine transaminase was correctly identified. Although our method could not directly identify the asparagine oxo-acid transaminase, we successfully found the paralog gene most similar to the correct enzyme gene. We also applied our method to infer candidate enzyme genes in the mesaconate pathway. The advantage of our method lies in the prediction of possible genes for orphan enzyme reactions where any associated gene sequences are not determined yet. We believe that this approach will facilitate experimental identification of genes for orphan enzymes.

A human gut microbial gene catalogue established by metagenomic sequencing.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Population pharmacokinetics of mizoribine in pediatric patients with kidney disease.

BACKGROUND: The present study aimed to obtain information enabling optimisation of the clinical effect of mizoribine (MZR) in pediatric patients with kidney disease. METHODS: A total of 105 pediatric patients with kidney disease treated at our institutions were enrolled. Kidney transplant patients were excluded. Population pharmacokinetic analysis of MZR was performed based on serum concentration data. Area under the curve from time zero to infinity (AUC∞) and maximal concentration (C max) were calculated by Bayesian analysis. RESULTS: In children, the appearance of MZR in the blood tended to be slower and the subsequent rise in blood concentration tended to be more sluggish, compared to healthy adults. Apparent volume of distribution and oral clearance were also higher in children compared to adults. A significant positive correlation was observed between patient age and AUC∞. There were significant differences of AUC∞ and C max by age group. No relationship was observed between the administration method of MZR and serum concentration. CONCLUSION: The pharmacokinetics of MZR was different in children compared to adults. To obtain the expected clinical efficacy, the regular MZR dosage schedule (2-3 mg/kg/day) might be insufficient for pediatric patients. In particular, younger patients might require a higher dosage of MZR per unit body weight.