Design and Progress of Oral Health Examinations in the Tohoku Medical Megabank Project.

In order to assess the long-term impact of the Great East Japan Earthquake on the oral health of disaster victims and to evaluate gene-environmental interactions in the development of major oral diseases and oral-systemic associations, the oral part of two large-scale genome cohort studies by the Tohoku Medical Megabank Organization (ToMMo), including the Community-based cohort (CommCohort) study and the Birth and Three-Generation cohort (BirThree) study, have been conducted. The study population comprised 32,185 subjects, including 16,886 participants in the CommCohort study and 15,299 participants in the BirThree cohort study, recruited from 2013 to 2017. The oral studies consist of a questionnaire regarding oral hygiene behavior, clinical examinations by dentists, and oral plaque and saliva sampling for microbiome analyses, which were carried out at seven community support centers in Miyagi prefecture. The median age of all participants was 55.0 years, and 66.1% of participants were women. Almost all participants reported that they brushed their teeth more than once a day. The median number of present teeth was 27.0, and the decayed, missing and filled tooth number was 16.0, with a significant difference according to age and sex. The median periodontal pocket and clinical attachment level was 2.48 mm and 4.00 mm, respectively. Periodontal parameters increased significantly according to age, except for the accumulation of dental calculus. The oral part of these extensive cross-sectional studies provides a unique and important platform for future studies on oral health and diseases that elicit through interactions with systemic diseases, lifestyles, life events and genetic backgrounds, and contributes to researches clarifying the long-term effects of disasters on oral health.

Sequential Sampling of the Gastrointestinal Tract to Characterize the Entire Digestive Microbiome in Japanese Subjects.

The gastrointestinal (GI) tract harbors trillions of microorganisms known to influence human health and disease, and next-generation sequencing (NGS) now enables the in-depth analysis of their diversity and functions. Although a significant amount of research has been conducted on the GI microbiome, comprehensive metagenomic datasets covering the entire tract are scarce due to cost and technical challenges. Despite the widespread use of fecal samples, integrated datasets encompassing the entire digestive process, beginning at the mouth and ending with feces, are lacking. With this study, we aimed to fill this gap by analyzing the complete metagenome of the GI tract, providing insights into the dynamics of the microbiota and potential therapeutic avenues. In this study, we delved into the complex world of the GI microbiota, which we examined in five healthy Japanese subjects. While samples from the whole GI flora and fecal samples provided sufficient bacteria, samples obtained from the stomach and duodenum posed a challenge. Using a principal coordinate analysis (PCoA), clear clustering patterns were identified; these revealed significant diversity in the duodenum. Although this study was limited by its small sample size, the flora in the overall GI tract showed unwavering consistency, while the duodenum exhibited unprecedented phylogenetic diversity. A visual heat map illustrates the discrepancy in abundance, with Fusobacteria and Bacilli dominating the upper GI tract and Clostridia and Bacteroidia dominating the fecal samples. Negativicutes and Actinobacteria were found throughout the digestive tract. This study demonstrates that it is possible to continuously collect microbiome samples throughout the human digestive tract. These findings not only shed light on the complexity of GI microbiota but also provide a basis for future research.

Oral Microbiome Analysis in Prospective Genome Cohort Studies of the Tohoku Medical Megabank Project.

A baseline oral microbiome study of the Tohoku Medical Megabank Organization (TMM) was planned to characterize the profile of the oral microbiome in the Japanese population. The study also aimed to clarify risk factors for multifactorial diseases by integrated analysis of the oral microbiome and host genome/omics information. From 2013 to 2016, we collected three types of oral biospecimens, saliva, supragingival plaque, and tongue swab, from a total of 25,101 participants who had a dental examination in TMM. In this study, we used two independent cohorts; the Community-Based Cohort and Birth and Three-Generation Cohort as discovery and validation cohorts, respectively, and we selected participants examined by a single dentist. We found through the 16S ribosomal RNA gene sequencing analysis of 834 participants of the Community-Based Cohort Study that there are differences in the microbial composition and community structure between saliva and plaque. The species diversities in both saliva and plaque were increased in correlation with the severity of periodontal disease. These results were nicely reproduced in the analysis of 455 participants of the Birth and Three-Generation Cohort Study. In addition, strong positive and negative associations of microbial taxa in both plaque and saliva with periodontitis-associated biofilm formation were detected by co-occurrence network analysis. The classes Actinobacteria and Bacilli, including oral health-associated bacterial species, showed a positive correlation in saliva. These results revealed differences in microbial composition and community structure between saliva and plaque and a correlation between microbial species and the severity of periodontal disease. We expect that the large database of the oral microbiome in the TMM biobank will help in the discovery of novel targets for the treatment and prevention of oral diseases, as well as for the discovery of therapeutic and/or preventive targets of systemic diseases.

Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis.

Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.

Identification of laticifer-specific genes and their promoter regions from a natural rubber producing plant Hevea brasiliensis.

Latex, the milky cytoplasm of highly differentiated cells called laticifers, from Hevea brasiliensis is a key source of commercial natural rubber production. One way to enhance natural rubber production would be to express genes involved in natural rubber biosynthesis by a laticifer-specific overexpression system. As a first step to identify promoters which could regulate the laticifer-specific expression, we identified random clones from a cDNA library of H. brasiliensis latex, resulting in 4325 expressed sequence tags (ESTs) assembled into 1308 unigenes (692 contigs and 617 singletons). Quantitative analyses of the transcription levels of high redundancy clones in the ESTs revealed genes highly and predominantly expressed in laticifers, such as Rubber Elongation Factor (REF), Small Rubber Particle Protein and putative protease inhibitor proteins. HRT1 and HRT2, cis-prenyltransferases involved in rubber biosynthesis, was also expressed predominantly in laticifers, although these transcript levels were 80-fold lower than that of REF. The 5'-upstream regions of these laticifer-specific genes were cloned and analyzed in silico, revealing seven common motifs consisting of eight bases. Furthermore, transcription factors specifically expressed in laticifers were also identified. The common motifs in the laticifer-specific genes and the laticifer-specific transcription factors are potentially involved in the regulation of gene expression in laticifers.