Differences in the human gut microbiota with varying depressive symptom severity scores.

Depression is a prevalent mental health disorder, and its incidence has increased further because of the coronavirus disease 2019 (COVID-19) pandemic. The gut microbiome has been suggested as a potential target for mental health treatment because of the bidirectional communication system between the brain and gastrointestinal tract, known as the gut-brain axis. We aimed to investigate the relationship between the human gut microbiome and depression screening by analyzing the abundance and types of microbiomes among individuals living in Japan, where mental health awareness and support may differ from those in other countries owing to cultural factors. We used a data-driven approach to evaluate the gut microbiome of participants who underwent commercial gut microbiota testing services and completed a questionnaire survey that included a test for scoring depressive tendencies. Our data analysis results indicated that no significant differences in gut microbiome composition were found among the groups based on their depression screening scores. However, the results also indicated the potential existence of a few differentially abundant bacterial taxa. Specifically, the detected bacterial changes in abundance suggest that the Bifidobacteriaceae, Streptococcaceae, and Veillonellaceae families are candidates for differentially abundant bacteria. Our findings should contribute to the growing body of research on the relationship between gut microbiome and mental health, highlighting the potential of microbiome-based interventions for depression treatment. The limitations of this study include the lack of clear medical information on the participants' diagnoses. Future research could benefit from a larger sample size and more detailed clinical information.

Gut microbial stability in older Japanese populations: insights from the Mykinso cohort.

Gut microbiota imbalance plays an important role in the pathogenesis of various diseases. Here, we determined microbe-microbe interactions and gut microbiome stability in a Japanese population with varying body mass indices (BMIs) and enterotypes. Using 16S ribosomal RNA gene sequencing, we analyzed gut microbial data from fecal samples obtained from 3,365 older Japanese individuals. The individuals were divided into lean, normal, and obese groups based on their BMIs. They were further categorized according to their gut microbiota enterotypes: Bacteroides (enterotype B), Prevotella (enterotype P), and Ruminococcus (enterotype R). We obtained data on different host factors, such as age, BMI, and disease status, using a survey questionnaire evaluated by the Mykinso gut microbiome testing service. Subsequently, we evaluated the co-occurrence network. Individual differences in BMI were associated with differences in co-occurrence networks. By exploring the network topology based on BMI status, we observed that the network density was lower in the lean group than that in the normal group. Furthermore, a simulation-based stability analysis revealed a lower resistance index in the lean group than those in the other two groups. Our results provide insights into various microbe-microbe interactions and gut microbial stability and could aid in developing appropriate therapeutic strategies targeting gut microbiota modulation to manage frailty.

Epigenetic profile of Japanese supercentenarians: a cross-sectional study.

BACKGROUND: Centenarians and supercentenarians with exceptional longevity are excellent models for research towards improvements of healthy life expectancy. Extensive research regarding the maintenance and reduction of epigenetic age has provided insights into increasing healthy longevity. To this end, we explored the epigenetic signatures reflecting hallmarks of exceptional healthy longevity, including avoidance of age-related diseases and cognitive functional decline. METHODS: In this cross-sectional study, we enrolled Japanese non-centenarians (eligible participants aged 20-80 years) from the Tohoku Medical Megabank Community-Based Cohort Study and centenarians and supercentenarians (aged 101-115 years) from the Tokyo Centenarian Study and the Japanese Semi-supercentenarian Study. We assessed participants' whole-blood DNA methylation profiles and then developed sex-specific and non-specific first-generation epigenetic clocks by elastic net regression, calculated individuals' epigenetic ages, and assessed their age acceleration. We also screened for age-related CpG sites in non-centenarians by epigenome-wide linear regression analyses and ANOVA. We subsequently investigated which CpG sites in centenarians and supercentenarians had DNA methylation patterns following the age-related findings obtained from non-centenarians and which did not. We further characterised CpG sites with hypermethylation or hypomethylation in the centenarians and supercentenarians using enrichment and protein-protein interaction network analyses. FINDINGS: We enrolled 421 non-centenarians (231 [55%] women and 190 [45%] men; age range 20-78 years), recruited between May 20, 2013, and March 31, 2016, and 94 centenarians and supercentenarians (66 women [70%] and 28 [30%] men; age range 101-115 years), recruited between Jan 20, 2001, and April 17, 2018. Non-sex-specific epigenetic clock showed the highest accuracy (r=0·96) based on which centenarians and supercentenarians had negative epigenetic age acceleration. Epigenome-wide association analyses further showed that centenarians and supercentenarians had younger-than-expected epigenetic states (DNA methylation profiles similar to those of non-centenarians) for 557 CpG sites enriched in cancer-related and neuropsychiatric-related genes, whereas these individuals had advanced (or older) epigenetic states for 163 CpG sites represented by genes related to TGF-ß signalling, which is involved in anti-inflammatory responses and known to contribute to healthy ageing. INTERPRETATION: These results indicate that exceptionally healthy longevity depends not only on maintaining young epigenetic states but also on advanced states of specific epigenetic regions. FUNDING: The Japan Agency for Medical Research and Development, KDDI Research, and Keio University. TRANSLATION: For the Japanese translation of the abstract see Supplementary Materials section.

Prolonged gut microbial alterations in post-transplant survivors of allogeneic haematopoietic stem cell transplantation.

Dysbiosis of the gut microbiota has been reported to increase early complications after allogeneic haematopoietic stem cell transplantation (allo-HSCT). However, it remains unclear whether gut microbial alterations persist during late complications, such as chronic graft-versus-host disease (cGVHD) or secondary cancers. Here, we analysed the gut microbiota of 59 patients who survived for 1-21.7 years (median, 6.4 years) after allo-HSCT. Long-term survivors showed lower gut microbial diversity than the age- and sex-matched healthy controls. This decreased diversity was reflected in the reduced abundance of the butyrate-producing bacteria. Patients with a history of grade 3 acute graft-versus-host disease (aGVHD) exhibited higher Veillonella abundance than patients with a history of grade 1-2 or non-aGVHD cases. The abundance of Faecalibacterium showed no decrease only in limited cGVHD cases. Additionally, the microbial structure in the secondary cancer group was significantly different (p < 0.05) from that in the non-secondary cancer group. This study is the first to show that microbial dysbiosis is present over a 10-year lifetime after discharge following allo-HSCT. Our results suggest that these prolonged gut microbial alterations may be associated with the development and exacerbation of late complications in post-transplant survivors.

Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1-V2 and V3-V4 primer sets.

BACKGROUND: 16S rRNA gene amplicon sequencing (16S analysis) is widely used to analyze microbiota with next-generation sequencing technologies. Here, we compared fecal 16S analysis data from 192 Japanese volunteers using the modified V1-V2 (V12) and the standard V3-V4 primer (V34) sets to optimize the gut microbiota analysis protocol. RESULTS: QIIME1 and QIIME2 analysis revealed a higher number of unclassified representative sequences in the V34 data than in the V12 data. The comparison of bacterial composition demonstrated that at the phylum level, Actinobacteria and Verrucomicrobia were detected at higher levels with V34 than with V12. Among these phyla, we observed higher relative compositions of Bifidobacterium and Akkermansia with V34. To estimate the actual abundance, we performed quantitative real-time polymerase chain reaction (qPCR) assays for Akkermansia and Bifidobacterium. We found that the abundance of Akkermansia as detected by qPCR was close to that in V12 data, but was markedly lower than that in V34 data. The abundance of Bifidobacterium detected by qPCR was higher than that in V12 and V34 data. CONCLUSIONS: These results indicate that the bacterial composition derived from the V34 region might differ from the actual abundance for specific gut bacteria. We conclude that the use of the modified V12 primer set is more desirable in the 16S analysis of the Japanese gut microbiota.

A cross-sectional analysis from the Mykinso Cohort Study: establishing reference ranges for Japanese gut microbial indices.

The purpose of this study was to establish reference ranges for gut microbial indices by collecting real-world Japanese microbiome data from a Mykinso cohort. Although several large cohort studies have focused on the human gut microbiome, large cohort studies of the gut microbiome from Japanese populations are scarce, especially from healthy or non-diseased individuals. We collected stool samples and original survey lifestyle information from 5,843 Japanese individuals through the Mykinso gut microbiome testing service. From the obtained 16S rRNA sequence data derived from stool samples, the ratio and distribution of each taxon were analyzed. The relationship between different epidemiological attributes and gut microbial indicators were statistically analyzed. The qualitative and quantitative indicators of these common gut microbiota were confirmed to be strongly correlated with age, sex, constipation/diarrhea, and history of lifestyle-related diseases. Therefore, we set up a healthy sub-cohort that controlled for these attribute factors and defined reference ranges from the distribution of gut microbial index in that population. Taken together, these results show that the gut microbiota of Japanese people had high beta-diversity, with no single "typical" gut microbiota type. We believe that the reference ranges for the gut microbial indices obtained in this study can be new reference values for determining the balance and health of the gut microbiota of an individual. In the future, it is necessary to clarify the clinical validity of these reference values by comparing them with a clinical disease cohort.

Combined Application of Electrically Stimulated Antagonist Muscle Contraction and Volitional Muscle Contraction Prevents Muscle Strength Weakness and Promotes Physical Function Recovery After Total Knee Arthroplasty: A Randomized Controlled Trial.

BACKGROUND: Osteoarthritis of the knee (KOA) is the most common cause of disability in both the United States and in Japan. The Hybrid training system (HTS) has been developed as a resistance exercise method combining electrical stimulation with voluntary exercise. The purpose of the present study is to compare the effects of a conventional rehabilitation program with or without HTS on knee muscle strength and physical function after Total knee arthroplasty (TKA). METHODS: We conducted a 12-week randomized controlled trial, using standard rehabilitation (the control group, n = 27) or standard rehabilitation plus HTS (the HTS group, n= 26), in 53 female patients after TKA. The HTS group underwent HTS three times per week for twelve weeks after TKA. Muscle strength, thigh circumference, physical functional testing, QOL and knee pain were assessed before surgery, 6 and 12 weeks after TKA. RESULTS: There was a significant decrease in quadriceps strength and thigh circumference on the operative side in the control group, but not in the HTS group at 6 weeks. Hamstring strength on the operative side in the HTS group significantly increased and thigh circumference was bigger than in the control group at 12 weeks. Physical function improved at 6 weeks in the HTS group, but not in the control group. Knee pain significantly improved in both groups at 6 weeks. CONCLUSIONS: HTS was effective in preventing quadriceps weakness and in improving physical function and QOL after TKA.

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago.

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the "Ohanami Project", to obtain environmental DNA samples from petal surfaces of Cerasus × yedoensis 'Somei-yoshino' across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes.

The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.

Inter-Individual Differences in the Oral Bacteriome Are Greater than Intra-Day Fluctuations in Individuals.

Given the advent of massively parallel DNA sequencing, human microbiome is analyzed comprehensively by metagenomic approaches. However, the inter- and intra-individual variability and stability of the human microbiome remain poorly characterized, particularly at the intra-day level. This issue is of crucial importance for studies examining the effects of microbiome on human health. Here, we focused on bacteriome of oral plaques, for which repeated, time-controlled sampling is feasible. Eighty-one supragingival plaque subjects were collected from healthy individuals, examining multiple sites within the mouth at three time points (forenoon, evening, and night) over the course of 3 days. Bacterial composition was estimated by 16S rRNA sequencing and species-level profiling, resulting in identification of a total of 162 known bacterial species. We found that species compositions and their relative abundances were similar within individuals, and not between sampling time or tooth type. This suggests that species-level oral bacterial composition differs significantly between individuals, although the number of subjects is limited and the intra-individual variation also occurs. The majority of detected bacterial species (98.2%; 159/162), however, did not fluctuate over the course of the day, implying a largely stable oral microbiome on an intra-day time scale. In fact, the stability of this data set enabled us to estimate potential interactions between rare bacteria, with 40 co-occurrences supported by the existing literature. In summary, the present study provides a valuable basis for studies of the human microbiome, with significant implications in terms of biological and clinical outcomes.