DDBJ update in 2023: the MetaboBank for metabolomics data and associated metadata.

The Bioinformation and DNA Data Bank of Japan (DDBJ) Center (https://www.ddbj.nig.ac.jp) provides database archives that cover a wide range of fields in life sciences. As a founding member of the International Nucleotide Sequence Database Collaboration (INSDC), DDBJ accepts and distributes nucleotide sequence data as well as their study and sample information along with the National Center for Biotechnology Information in the United States and the European Bioinformatics Institute (EBI). Besides INSDC databases, the DDBJ Center provides databases for functional genomics (GEA: Genomic Expression Archive), metabolomics (MetaboBank) and human genetic and phenotypic data (JGA: Japanese Genotype-phenotype Archive). These database systems have been built on the National Institute of Genetics (NIG) supercomputer, which is also open for domestic life science researchers to analyze large-scale sequence data. This paper reports recent updates on the archival databases and the services of the DDBJ Center, highlighting the newly redesigned MetaboBank. MetaboBank uses BioProject and BioSample in its metadata description making it suitable for multi-omics large studies. Its collaboration with MetaboLights at EBI brings synergy in locating and reusing public data.

DDBJ update: streamlining submission and access of human data.

The Bioinformation and DDBJ Center (DDBJ Center, https://www.ddbj.nig.ac.jp) provides databases that capture, preserve and disseminate diverse biological data to support research in the life sciences. This center collects nucleotide sequences with annotations, raw sequencing data, and alignment information from high-throughput sequencing platforms, and study and sample information, in collaboration with the National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI). This collaborative framework is known as the International Nucleotide Sequence Database Collaboration (INSDC). In collaboration with the National Bioscience Database Center (NBDC), the DDBJ Center also provides a controlled-access database, the Japanese Genotype-phenotype Archive (JGA), which archives and distributes human genotype and phenotype data, requiring authorized access. The NBDC formulates guidelines and policies for sharing human data and reviews data submission and use applications. To streamline all of the processes at NBDC and JGA, we have integrated the two systems by introducing a unified login platform with a group structure in September 2020. In addition to the public databases, the DDBJ Center provides a computer resource, the NIG supercomputer, for domestic researchers to analyze large-scale genomic data. This report describes updates to the services of the DDBJ Center, focusing on the NBDC and JGA system enhancements.

Cluster analysis identifies a pathophysiologically distinct subpopulation with increased serum leptin levels and severe obstructive sleep apnea.

PURPOSE: To investigate the different pathophysiologies of obstructive sleep apnea (OSA) phenotypes using cluster analysis. Differences between leptin/adiponectin levels in the resulting OSA phenotypes were also examined. METHODS: In total, 1057 OSA patients were selected, and a retrospective survey of clinical records, polysomnography results, and blood gas data was conducted. Patients were grouped into four clusters by their OSA severity, PaCO2, body mass index (BMI), and sleepiness. A k-means cluster analysis was performed, resulting in a division into four subpopulations. The Tukey or Games-Howell tests were used for intergroup comparisons. RESULTS: Among the 20 clinical OSA items, four common factors (Epworth Sleepiness Scale [ESS], BMI, Apnea-Hypopnea Index [AHI], and PaCO2) were extracted by principal component analysis, and a cluster analysis was performed using the k-means method, resulting in four distinct phenotypes. The Clusters 1 (middle age, symptomatic severe OSA) and 4 (young, obese, symptomatic very severe OSA) exhibited high leptin levels. C-reactive protein levels were also elevated in Cluster 4, indicating a different pathophysiological background. No apparent differences between clusters were observed regarding adiponectin/leptin ratios and adiponectin levels. Classification into groups based on phenotype showed that Epworth Sleepiness Scale [ESS] score and disease severity were not correlated, suggesting that sleepiness is affected by multiple elements. CONCLUSIONS: The existence of multiple clinical phenotypes suggests that different pathophysiological backgrounds exist such as systemic inflammation and metabolic disorder. This classification may be used to determine the efficacy of continuous positive airway pressure treatment that cannot be determined by the AHI.

Increased extracellular vesicle miRNA-466 family in the bronchoalveolar lavage fluid as a precipitating factor of ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening disease; however, its treatment has not yet been fully established. The progression of ARDS is considered to be mediated by altered intercellular communication between immune and structural cells in the lung. One of several factors involved in intercellular communication is the extracellular vesicle (EV). They act as carriers of functional content such as RNA molecules, proteins, and lipids and deliver cargo from donor to recipient cells. EVs have been reported to regulate the nucleotide-binding oligomerization like receptor 3 (NLRP3) inflammasome. This has been identified as the cellular machinery responsible for activating inflammatory processes, a key component responsible for the pathogenesis of ARDS. METHODS: Here, we provide comprehensive genetic analysis of microRNAs (miRNAs) in EVs, demonstrating increased expression of the miRNA-466 family in the bronchoalveolar lavage fluid of a mouse ARDS model. RESULTS: Transfection of bone marrow-derived macrophages (BMDMs) with miRNA-466 g and 466 m-5p resulted in increased interleukin-1 beta (IL-1ß) release after LPS and ATP treatment, which is an established in vitro model of NLRP3 inflammasome activation. Moreover, LPS-induced pro-IL-1ß expression was accelerated by miRNA-466 g and 466 m-5p in BMDMs. CONCLUSIONS: These findings imply that miRNA-466 family molecules are secreted via EVs into the airways in an ARDS model, and this exacerbates inflammation through the NLRP3 inflammasome. Our results suggest that the NLRP3 inflammasome pathway, regulated by extracellular vesicle miRNA, could act as a therapeutic target for ARDS.

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome.

Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes malignant and inflammatory diseases in ∼10% of infected people. A typical host has between 10(4) and 10(5) clones of HTLV-1-infected T lymphocytes, each clone distinguished by the genomic integration site of the single-copy HTLV-1 provirus. The HTLV-1 bZIP (HBZ) factor gene is constitutively expressed from the minus strand of the provirus, whereas plus-strand expression, required for viral propagation to uninfected cells, is suppressed or intermittent in vivo, allowing escape from host immune surveillance. It remains unknown what regulates this pattern of proviral transcription and latency. Here, we show that CTCF, a key regulator of chromatin structure and function, binds to the provirus at a sharp border in epigenetic modifications in the pX region of the HTLV-1 provirus in T cells naturally infected with HTLV-1. CTCF is a zinc-finger protein that binds to an insulator region in genomic DNA and plays a fundamental role in controlling higher order chromatin structure and gene expression in vertebrate cells. We show that CTCF bound to HTLV-1 acts as an enhancer blocker, regulates HTLV-1 mRNA splicing, and forms long-distance interactions with flanking host chromatin. CTCF-binding sites (CTCF-BSs) have been propagated throughout the genome by transposons in certain primate lineages, but CTCF binding has not previously been described in present-day exogenous retroviruses. The presence of an ectopic CTCF-BS introduced by the retrovirus in tens of thousands of genomic locations has the potential to cause widespread abnormalities in host cell chromatin structure and gene expression.

Validity study of the Japanese version of the Nijmegen Questionnaire for verifying dysfunctional breathing in Japanese asthma patients.

Background: Dysfunctional breathing (DB) is a clinical condition characterized by irregular breathing patterns presenting a sensation of dyspnea and a feeling of chest tightness. DB is a known comorbidity of asthma that is difficult to control, leading to poor quality of life, so early diagnosis and therapeutic intervention are essential to improve the clinical condition of asthma. The Nijmegen Questionnaire (NQ), developed to screen for DB and translated into various languages, is used worldwide. However, a Japanese NQ (JNQ) is unavailable, so DB has not been clinically verified in people with asthma in Japan. Objective: This study aimed to prepare a JNQ, verify its reliability and validity, and demonstrate its clinical benefits in asthma treatment. Methods: The JNQ was prepared by back-translating the NQ with the author's consent. The answers to self-administered questionnaires, including the JNQ, Asthma Control Test (ACT), Asthma Control Questionnaire (ACQ), Mini Asthma Quality of Life Questionnaire (Mini-AQLQ), and Patient Health Questionnaire 9 (PHQ-9), were obtained with the consent of 68 people with asthma (average age ± SD, 52.04 ± 12.43 years) who visited Nihon University Itabashi Hospital. The reliability of the JNQ was analyzed by the Cronbach alpha coefficient. A comparative test was conducted for each questionnaire (ACT, ACQ, Mini-AQLQ, PHQ-9), considering a JNQ score of 23 as the cutoff value. Patients with a score of 23 or more were assigned to the DB group, whereas patients with a score of less than 23 were assigned to the non-DB group. We analyzed the correlation between the JNQ and each questionnaire. Results: The JNQ showed sufficient reliability (Cronbach alpha = 0.875). Correlation analysis between the JNQ score and each questionnaire revealed negative correlations with the ACT score (r = 0.262) and Mini-AQLQ score (r = -0.453) and positive correlations with the ACQ score (r = 0.337) and PHQ-9 score (r = 0.539). All of these correlations were statistically significant. As a result of the comparative test, the DB and non-DB groups showed a significant difference in Mini-AQLQ (P = .023) and PHQ-9 (P = .003) scores. No significant difference was observed between ACT (P = .294) and ACQ (P = .177) scores. Conclusions: The JNQ validates DB in Japanese people with asthma and reflects the deterioration of asthma control, decreased quality of life, and depression. Using the JNQ, early diagnosis and therapeutic intervention (eg, breathing exercises and a psychosomatic approach) for DB in people with asthma may help suppress the severity of asthma in Japan.

The Relationship between Leptin Levels and Continuous Positive Airway Pressure Treatment: A Cluster Analysis.

Objective Leptin is an appetite-suppressing hormone released by adipose tissue that plays an important role in severe obstructive sleep apnea syndrome (OSAS). However, it is unclear whether leptin levels are a useful biomarker for this syndrome. The present study aimed to assess the effect of continuous positive airway pressure (CPAP) treatment on the syndrome according to leptin levels, using a cluster classification based on clinical features of the syndrome. Materials and Methods We performed a hierarchical cluster analysis of data from 97 OSAS patients diagnosed via polysomnography. We also evaluated the effect after 6 months of CPAP administration. Results Clusters 1 (49 patients; 50.5%) and 2 (6 patients; 6.2%) presented normal leptin levels, and clusters 3 (11 patients; 11.3%) and 4 (31 patients; 32%) presented high leptin levels. Clusters 3 and 4 presented different leptin levels, but the same degree of obesity. After treatment, the levels of excessive daytime sleepiness improved in all clusters. In Cluster 3, leptin levels were significantly reduced after treatment. Conclusion Using the conventional diagnostic method of the apnea-hypopnea index, it was not clear whether leptin is a useful biomarker for the CPAP treatment. However, it may be helpful for particular clusters, including obese women, and where particular populations require CPAP treatment.

Actinoallomurus radicium sp. nov., isolated from the roots of two plant species.

Three strains, K08-0182(T), K08-0178 and K08-0195 were isolated from the paste of ground plant roots collected in Kanagawa Prefecture, Japan. These strains contained meso-diaminopimelic acid and lysine as the diamino acids in cell-wall peptidoglycan, and MK-9(H(6)) and MK-9(H(8)) as the predominant menaquinones. The G+C contents of the DNA were 72-73 mol%. Taken together, these characteristics combined with 16S rRNA gene sequence analyses revealed that the isolated strains belong to the genus Actinoallomurus. DNA-DNA hybridization values showed that the three strains belonged to a novel species of the genus Actinoallomurus. Therefore strains K08-0182(T), K08-0178 and K08-0195 are proposed as representatives of a novel species, Actinoallomurus radicium sp. nov. The type strain is K08-0182(T) ( = DSM 45523(T)  = NBRC 107678(T)  = JCM 17294(T)).

The NBDC-DDBJ imputation server facilitates the use of controlled access reference panel datasets in Japan.

Accurate genotype imputation requires large-scale reference panel datasets. When conducting genotype imputation on the Japanese population, researchers can use such datasets under collaborative studies or controlled access conditions in public databases. We developed the NBDC-DDBJ imputation server, which securely provides users with a web user interface to execute genotype imputation on the server. Our benchmarking analysis showed that the accuracy of genotype imputation was improved by leveraging controlled access datasets to increase the number of haplotypes available for analysis compared to using publicly available reference panels such as the 1000 Genomes Project. The NBDC-DDBJ imputation server facilitates the use of controlled access datasets for accurate genotype imputation.

Iron-Dependent Mitochondrial Dysfunction Contributes to the Pathogenesis of Pulmonary Fibrosis.

Although the pathogenesis of pulmonary fibrosis remains unclear, it is known to involve epithelial injury and epithelial-mesenchymal transformation (EMT) as a consequence of cigarette smoke (CS) exposure. Moreover, smoking deposits iron in the mitochondria of alveolar epithelial cells. Iron overload in mitochondria causes the Fenton reaction, leading to reactive oxygen species (ROS) production, and ROS leakage from the mitochondria induces cell injury and inflammation in the lungs. Nevertheless, the mechanisms underlying iron metabolism and pulmonary fibrosis are yet to be elucidated. In this study, we aimed to determine whether iron metabolism and mitochondrial dysfunction are involved in the pathogenesis of pulmonary fibrosis. We demonstrated that administration of the iron chelator deferoxamine (DFO) reduced CS-induced pulmonary epithelial cell death, mitochondrial ROS production, and mitochondrial DNA release. Notably, CS-induced cell death was reduced by the administration of an inhibitor targeting ferroptosis, a unique iron-dependent form of non-apoptotic cell death. Transforming growth factor-ß-induced EMT of pulmonary epithelial cells was also reduced by DFO. The preservation of mitochondrial function reduced Transforming growth factor-ß-induced EMT. Furthermore, transbronchial iron chelation ameliorated bleomycin-induced pulmonary fibrosis and leukocyte migration in a murine model. Our findings indicate that iron metabolism and mitochondrial dysfunction are involved in the pathogenesis of pulmonary fibrosis. Thus, they may be leveraged as new therapeutic targets for pulmonary fibrosis.

DsRNA induction of microRNA-155 disrupt tight junction barrier by modulating claudins.

BACKGROUND: The impaired barrier function of the airway epithelium due to RNA virus infection is closely related to the development and exacerbation of allergic airway inflammation. OBJECTIVE: In this study, we investigated the roles of microRNAs on the mechanisms of double-stranded RNA (dsRNA)-induced epithelial barrier dysfunction. METHODS: 16HBE14o- human bronchial epithelial cells were grown to confluence on Transwell inserts and exposed to poly-I:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of tight junctions by immunofluorescence microscopy. RESULTS: Poly-I:C treated 16HBE14o- cells increased paracellular permeability. Knockdown of Toll-like receptor 3 and TRIF abrogated these effects. The expression of microRNA-155 (miR-155) was increased by poly-I:C in dose-dependent manner. Transfection of mir155 mimics into 16HBE14o- cells increased permeability and inhibited tight junction formation. Transfection of miR-155 inhibitor suppressed poly-I:C-induced barrier disruption. Poly-I:C treatment significantly decreased the expression of claudin members-claudin-1, -3, -4, -5, -9, -11, -16, -18 and -19. Transfection of miR-155 mimics showed similar changing expression pattern of claudin members with those of poly-I:C treatment. CONCLUSION: These results suggest that RNA virus infection can impair the epithelial barrier disruption mechanism by down-regulation of claudin members through the induction of miR-155.

Total HIV-1 DNA Dynamics and Influencing Factors in Long-Term ART-Treated Japanese Adults: A Retrospective Longitudinal Analysis.

BACKGROUND: Understanding HIV persistence in treated patients is an important milestone toward drug-free control. We aimed at analyzing total HIV DNA dynamics and influencing factors in Japanese patients who received more than a decade of suppressive antiretroviral treatment (ART). METHODS: A retrospective study including clinical records and 840 peripheral blood mononuclear cells samples (mean 14 samples/patient) for 59 patients (92% male) was performed. Subjects were divided into 2 groups: with and without hematological comorbidity (mainly hemophilia) plus hepatitis C virus coinfection. Total HIV DNA was measured in peripheral blood mononuclear cells by quantitative polymerase chain reaction. The dynamics, regression over time, and influence of antiretrovirals by group were estimated using a novel regression model (R software v 3.2.3). RESULTS: Total HIV DNA decreased on ART initiation, and subsequently, its dynamics varied between groups with previously undescribed fluctuations. If calculated by on-treatment, the mean total HIV DNA levels were similar. The comorbidity group had unstable levels showing different regression over time (P = 0.088/0.094 in year 1/after year 8 of ART) and significantly different treatment responses as shown by antiretroviral group switching estimates. Furthermore, curing hepatitis C virus in hemophiliacs did not significantly alter total HIV DNA levels or regression. CONCLUSIONS: Our data identified some effects of the long-term treatment on total HIV DNA levels and highlighted the partial influence of comorbidities and coinfections. Total HIV DNA monitoring contributed to therapy response estimates and HIV reservoir quantification. The results suggest that HIV DNA monitoring during ART might be useful as a persistence marker in both HIV-monoinfected patients and those with comorbidities and coinfections.

Author Correction: Dynamics and mechanisms of clonal expansion of HIV-1-infected cells in a humanized mouse model.

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Dynamics and mechanisms of clonal expansion of HIV-1-infected cells in a humanized mouse model.

Combination anti-retroviral therapy (cART) has drastically improved the clinical outcome of HIV-1 infection. Nonetheless, despite effective cART, HIV-1 persists indefinitely in infected individuals. Clonal expansion of HIV-1-infected cells in peripheral blood has been reported recently. cART is effective in stopping the retroviral replication cycle, but not in inhibiting clonal expansion of the infected host cells. Thus, the proliferation of HIV-1-infected cells may play a role in viral persistence, but little is known about the kinetics of the generation, the tissue distribution or the underlying mechanism of clonal expansion in vivo. Here we analyzed the clonality of HIV-1-infected cells using high-throughput integration site analysis in a hematopoietic stem cell-transplanted humanized mouse model. Clonally expanded, HIV-1-infected cells were detectable at two weeks post infection, their abundance increased with time, and certain clones were present in multiple organs. Expansion of HIV-1-infected clones was significantly more frequent when the provirus was integrated near host genes in specific gene ontological classes, including cell activation and chromatin regulation. These results identify potential drivers of clonal expansion of HIV-1-infected cells in vivo.

Chronic Thromboembolic Pulmonary Hypertension Associated with Chronic Inflammation.

Chronic thromboembolic pulmonary hypertension (CTEPH) is one of the leading causes of severe pulmonary hypertension. According to previously reported studies in the pertinent literature, chronic inflammatory conditions may be implicated in the development of CTEPH. We herein describe the case of a 56-year-old woman who was diagnosed with CTEPH in association with chronic infection. The patient had experienced five episodes of pneumonia in the five years prior to the diagnosis of CTEPH. Blood tests from the previous five years of outpatient follow-up demonstrated that the C-reactive protein level was slightly elevated. This case suggests that a relationship exists between chronic inflammation and CTEPH, and furthermore, may contribute towards elucidating the pathophysiology of CTEPH.

Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.

The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.