Blautia parvula sp. nov., isolated from Japanese faecal samples.

Two Gram-positive, anaerobic, non-spore-forming and coccoid or oval-shaped bacterial strains, namely, DN0138T and DN0266, were isolated from faecal samples of healthy Japanese people. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain DN0138T clustered with a species of the genus Blautia and was closely related to Blautia producta JCM 1471T, Blautia coccoides JCM 1395T, Blautia hominis KB1T and 'Blautia marasmi' Marseille-P2377, with sequence similarities of 98.6, 98.5, 98.8 and 98.2 %, respectively. The average nucleotide identity values were 85.3 % for B. producta JCM 1471T, 85.0 % for B. coccoides NCTC 11035T, 84.3 % for B. hominis KB1T and 84.3 % for 'B. marasmi' Marseille-P2377. The major end products of glucose metabolism were acetic acid, lactic acid and succinic acid. The genome length of strain DN0138T was 6 247 046 bp with 46.7 mol% G+C content of genome sequence. Based on their phenotypic, cellular fatty acid and phylogenetic characteristics, the three isolates represent a novel species within the genus Blautia, for which the name Blautia parvula sp. nov. is proposed. The type strain is DN0138T (=NBRC 113351T=BCRC 81349T).

Kikuchi Disease After SARS-CoV-2 Vaccination: A Case Report With Immunohistochemical Analyses.

SARS-CoV-2 vaccines have been administered in many countries after the COVID-19 pandemic. Lymphadenopathy is a side effect of SARS-CoV-2 vaccine. We report a rare example of Kikuchi disease in the cervical lymph nodes after SARS-CoV-2 vaccination. A 41-year-old man complained of a swollen neck and fever 9 days after the first dose of SARS-CoV-2 mRNA-1273 vaccine. Computed tomography revealed enlarged cervical lymph nodes. Fine needle aspiration and resection were performed, and the clinicopathological diagnosis was consistent with Kikuchi disease. Histologically, the resected lymph nodes lost their polarity, and many histiocytes were aggregated with karyorrhectic nuclear debris and apoptosis. SARS-CoV-2 positive cells were small lymphocytes detected by immunohistochemistry. This is the first report that demonstrated SARS-CoV-2 expression in Kikuchi disease post-SARS-CoV-2 vaccination.

Chromatin conformational changes at human satellite II contribute to the senescence phenotype in the tumor microenvironment.

Cellular senescence and senescence-associated secretory phenotype (SASP) in stromal cells within the tumor microenvironment promote cancer progression. Although cellular senescence has been shown to induce changes in the higher-order chromatin structure and abnormal transcription of repetitive elements in the genome, the functional significance of these changes is unclear. In this study, we examined the human satellite II (hSATII) loci in the pericentromere to understand these changes and their functional significance. Our results indicated that the hSATII loci decompact during senescence induction, resulting in new DNA-DNA interactions in distinct genomic regions, which we refer to as DRISR (Distinctive Regions Interacted with Satellite II in Replicative senescent Fibroblasts). Interestingly, decompaction occurs before the expression of hSATII RNA. The DRISR with altered chromatin accessibility was enriched for motifs associated with cellular senescence and inflammatory SASP genes. Moreover, DNA-fluorescence in situ hybridization analysis of the breast cancer tissues revealed hSATII decompaction in cancer and stromal cells. Furthermore, we reanalyzed the single-cell assay for transposase-accessible chromatin with sequencing data and found increased SASP-related gene expression in fibroblasts exhibiting hSATII decompaction in breast cancer tissues. These findings suggest that changes in the higher-order chromatin structure of the pericentromeric repetitive sequences during cellular senescence might directly contribute to the cellular senescence phenotype and cancer progression via inflammatory gene expression.

North-to-South diversity of lipomycetaceous yeasts in soils evaluated with a cultivation-based approach from 11 locations in Japan.

To understand the species distribution, diversity, and density of lipomycetaceous yeasts in soil based on their north-to-south location in Japan, 1146 strains were isolated from soil samples at 11 locations from Hokkaido to Okinawa Prefecture and taxonomically characterized. Lipomycetaceous yeast strains were isolated efficiently from soil by selecting watery mucoid-like colonies on agar plates with nitrogen-depleted medium. Twenty-four (80%) of the 30 known species of the genus Lipomyces were isolated from the soil samples collected in Japan, including species recently proposed. Among the species isolated, L. starkeyi was the most predominant in Japan, except on Iriomote Island, Okinawa, and accounted for 60-98% of the isolated strains. Lipomyces yarrowii was the dominant species on Iriomote Island (64%). The second most dominant species were L. chichibuensis in Saitama Prefecture and L. doorenjongii from Yamaguchi to Okinawa Prefecture. The species diversity of lipomycetaceous yeasts was in Japan and the significant correlation with the latitude of the sampling sites was revealed.

Identification of Novel Senescent Markers in Small Extracellular Vesicles.

Senescent cells exhibit several typical features, including the senescence-associated secretory phenotype (SASP), promoting the secretion of various inflammatory proteins and small extracellular vesicles (EVs). SASP factors cause chronic inflammation, leading to age-related diseases. Recently, therapeutic strategies targeting senescent cells, known as senolytics, have gained attention; however, noninvasive methods to detect senescent cells in living organisms have not been established. Therefore, the goal of this study was to identify novel senescent markers using small EVs (sEVs). sEVs were isolated from young and senescent fibroblasts using three different methods, including size-exclusion chromatography, affinity column for phosphatidylserine, and immunoprecipitation using antibodies against tetraspanin proteins, followed by mass spectrometry. Principal component analysis revealed that the protein composition of sEVs released from senescent cells was significantly different from that of young cells. Importantly, we identified ATP6V0D1 and RTN4 as novel markers that are frequently upregulated in sEVs from senescent and progeria cells derived from patients with Werner syndrome. Furthermore, these two proteins were significantly enriched in sEVs from the serum of aged mice. This study supports the potential use of senescent markers from sEVs to detect the presence of senescent cells in vivo.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

Hepatocyte growth factor derived from senescent cells attenuates cell competition-induced apical elimination of oncogenic cells.

Cellular senescence and cell competition are important tumor suppression mechanisms that restrain cells with oncogenic mutations at the initial stage of cancer development. However, the link between cellular senescence and cell competition remains unclear. Senescent cells accumulated during the in vivo aging process contribute toward age-related cancers via the development of senescence-associated secretory phenotype (SASP). Here, we report that hepatocyte growth factor (HGF), a SASP factor, inhibits apical extrusion and promotes basal protrusion of Ras-mutated cells in the cell competition assay. Additionally, cellular senescence induced by a high-fat diet promotes the survival of cells with oncogenic mutations, whereas crizotinib, an inhibitor of HGF signaling, provokes the removal of mutated cells from mouse livers and intestines. Our study provides evidence that cellular senescence inhibits cell competition-mediated elimination of oncogenic cells through HGF signaling, suggesting that it may lead to cancer incidence during aging.

Characterization and Demonstration of Mock Communities as Control Reagents for Accurate Human Microbiome Community Measurements.

Standardization and quality assurance of microbiome community analysis by high-throughput DNA sequencing require widely accessible and well-characterized reference materials. Here, we report on newly developed DNA and whole-cell mock communities to serve as control reagents for human gut microbiota measurements by shotgun metagenomics and 16S rRNA gene amplicon sequencing. The mock communities were formulated as near-even blends of up to 20 bacterial species prevalent in the human gut, span a wide range of genomic guanine-cytosine (GC) contents, and include multiple strains with Gram-positive type cell walls. Through a collaborative study, we carefully characterized the mock communities by shotgun metagenomics, using previously developed standardized protocols for DNA extraction and sequencing library construction. Further, we validated fitness of the mock communities for revealing technically meaningful differences among protocols for DNA extraction and metagenome/16S rRNA gene amplicon library construction. Finally, we used the mock communities to reveal varying performance of metagenome-based taxonomic profilers and the impact of trimming and filtering of sequencing reads on observed species profiles. The latter showed that aggressive preprocessing of reads may result in substantial GC-dependent bias and should thus be carefully evaluated to minimize unintended effects on species abundances. Taken together, the mock communities are expected to support a myriad of applications that rely on well-characterized control reagents, ranging from evaluation and optimization of methods to assessment of reproducibility in interlaboratory studies and routine quality control. IMPORTANCE Application of high-throughput DNA sequencing has greatly accelerated human microbiome research and its translation into new therapeutic and diagnostic capabilities. Microbiome community analyses results can, however, vary considerably across studies or laboratories, and establishment of measurement standards to improve accuracy and reproducibility has become a priority. The here-developed mock communities, which are available from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE, Japan), provide well-characterized control reagents that allow users to judge the accuracy of their measurement results. Widespread and consistent adoption of the mock communities will improve reproducibility and comparability of microbiome community analyses, thereby supporting and accelerating human microbiome research and development.

Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum.

OBJECTIVE: Although immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism. DESIGN: We generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging. RESULTS: We obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgAtm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA-/-). IgA-/- exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+ T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+ biosensor showed that IgA-/- had elevated Ca2+ signalling in Peyer's patch B cells. On the other hand, IgAtm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder. CONCLUSION: IgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Progress toward improving outcomes in patients with cholangiocarcinoma.

PURPOSE OF REVIEW: To provide an update on latest advances in treatment of cholangiocarcinoma. RECENT FINDINGS: Incidence of cholangiocarcinoma has been increasing over the past decade. A better understanding of the genetic landscape of cholangiocarcinoma and its risk factors resulted in earlier diagnosis and treatment option expansion to targeted therapy with FGFR inhibitors, and liver transplantation for early perihilar cholangiocarcinoma and early intrahepatic cholangiocarcinoma. IDH1/2 inhibition for intrahepatic cholangiocarcinoma is an emerging targeted therapy approach. Data supports benefits of adjuvant therapy for a subset of patients undergoing surgical resection. Approaches combining different treatment modalities such as chemotherapy, surgery, radiation therapy appear promising. SUMMARY: Earlier diagnosis and genetic characterization provided additional treatment options for patients with previously incurable cholangiocarcinoma. A precision medicine approach with a focus on actionable genetic alterations and combination of treatment modalities are actively being explored and will further improve outcomes in our patients with cholangiocarcinoma.

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

BACKGROUND: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. RESULTS: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. CONCLUSIONS: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

An exploratory MALDI-TOF MS library based on SARAMIS superspectra for rapid identification of Aspergillus section Nigri.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a broadly used technique for identification and typing of microorganisms. However, its application to filamentous fungi has been delayed. The objective of this study was to establish a data library for rapid identification of the genus Aspergillus sect. Nigri using MALDI-TOF MS. With respect to sample preparation, we compared the utility of using mature mycelia, including conidial structures, to accumulate a wider range of proteins versus the conventional method relying on young hyphae. Mass spectral datasets obtained for 61 strains of 17 species were subjected to cluster analysis and compared with a phylogenetic tree based on calmodulin gene sequences. Specific and frequent mass spectral peaks corresponding to each phylogenetic group were selected (superspectra for the SARAMIS system). Fifteen superspectra representing nine species were ultimately created. The percentage of correct identification for 217 spectra was improved from 36.41% to 86.64% using the revised library. Additionally, 2.76% of the spectra were assigned to candidates that comprised several related species, including the correct species.

gcType: a high-quality type strain genome database for microbial phylogenetic and functional research.

Taxonomic and functional research of microorganisms has increasingly relied upon genome-based data and methods. As the depository of the Global Catalogue of Microorganisms (GCM) 10K prokaryotic type strain sequencing project, Global Catalogue of Type Strain (gcType) has published 1049 type strain genomes sequenced by the GCM 10K project which are preserved in global culture collections with a valid published status. Additionally, the information provided through gcType includes >12 000 publicly available type strain genome sequences from GenBank incorporated using quality control criteria and standard data annotation pipelines to form a high-quality reference database. This database integrates type strain sequences with their phenotypic information to facilitate phenotypic and genotypic analyses. Multiple formats of cross-genome searches and interactive interfaces have allowed extensive exploration of the database's resources. In this study, we describe web-based data analysis pipelines for genomic analyses and genome-based taxonomy, which could serve as a one-stop platform for the identification of prokaryotic species. The number of type strain genomes that are published will continue to increase as the GCM 10K project increases its collaboration with culture collections worldwide. Data of this project is shared with the International Nucleotide Sequence Database Collaboration. Access to gcType is free at http://gctype.wdcm.org/.

Complete Genome Sequence of Collinsella aerofaciens JCM 10188T.

We report a complete genome sequence of Collinsella aerofaciens JCM 10188T (=VPI 1003T). The genome consists of a circular chromosome (2,428,218 bp with 60.6% G+C content) and two extrachromosomal elements. The genome was predicted to contain 5 sets of rRNA genes, 58 tRNA genes, and 2,079 protein-encoding sequences.

Complete Genome Sequence of Megamonas funiformis JCM 14723T.

We announce the complete genome sequence of Megamonas funiformis JCM 14723T (YIT 11815T). The genome consists of a circular chromosome (2,522,577 bp, 31.5% G+C content) and a plasmid of 46,189 bp (29.4% G+C content). The genome was predicted to contain 6 rRNA operons, 53 tRNA genes, and 2,440 protein-coding sequences.

Complete Genome Sequence of Flavonifractor plautii JCM 32125T.

We report the complete genome sequence of Flavonifractor plautii JCM 32125T (=VPI 0310T). The genome consists of a single circular chromosome of 3,985,392 bp (G+C content, 60.9%) and was predicted to contain 3 complete sets of rRNA genes, 63 tRNA genes, and 3,764 protein-coding sequences.

Complete Genome Sequence of Blautia producta JCM 1471T.

We report a complete genome sequence of Blautia producta JCM 1471T The genome consists of a single circular chromosome of 6,197,116 bp with a G+C content of 45.7%. The genome was annotated as containing 5 complete sets of rRNA genes, 70 tRNA genes, and 5,516 protein-coding sequences.

Fourteen novel lipomycetaceous yeast species isolated from soil in Japan and transfer of Dipodascopsis anomala to the genus Babjevia based on ascospore production phenotype.

Fourteen novel lipomycetaceous yeasts species were isolated from soil samples collected from the Hokkaido, Chiba and Okinawa prefectures of Japan. Phylogenetic analyses of the D1/D2 domains of the large subunit rRNAs and translation elongation factor 1 alpha genes (TEF1-α) revealed that five strains of two species from the soil in Furano-shi, Hokkaido were related to Dipodascopsis anomala and 29 strains representing 12 species from soils in Kamogawa-shi, Chiba and Iriomote Island, Okinawa were in the Myxozyma clade. The two species of Dipodascopsis form globose or ellipsoid ascospores in their sac-like ascus and pseudohyphae. Furthermore, these species produce ascospores in their pseudohyphae and do not produce an acicular ascus, which is common among the three species including D. anomala. Therefore, we propose transferring D. anomala to the genus Babjevia and amending Babjevia. Two novel species were described and included in the genus Babjevia: Babjevia hyphoforaminiformans sp. nov. (holotype NBRC 111233; MycoBank no. MB 829051) and Babjevia hyphasca sp. nov. (holotype NBRC 112965; MycoBank no. MB 829053). The 12 species in the Myxozyma clade produce neither ascospores nor pseudohyphae and have different characteristics in assimilating several carbon sources from each other. Thus, we propose that the novel species of Lipomyces be classified as forma asexualis (f.a.). From Kamogawa-shi, Chiba (19 strains representing five species): Lipomyces melibiosiraffinosiphilus f.a., sp. nov. (holotype NBRC 111411; MycoBank no. MB 829034), Lipomyces kiyosumicus f.a., sp. nov. (holotype NBRC 111424; MycoBank no. MB 829035), Lipomyces chibensis f.a., sp. nov. (holotype NBRC 111413; MycoBank no. MB 829036), Lipomyces kamogawensis f.a., sp. nov. (holotype NBRC 112967; MycoBank no. MB 829037), Lipomyces amatsuensis f.a., sp. nov. (holotype NBRC 111420; MycoBank no. MB 829041). From Iriomote island, Okinawa (10 strains representing seven species): Lipomyces taketomicus f.a., sp. nov. (holotype NBRC 112966; MycoBank no. MB 829042), Lipomyces yaeyamensis f.a., sp. nov. (holotype NBRC 110433; MycoBank no. MB 829050), Lipomyces iriomotensis f.a., sp. nov. (holotype NBRC 110436; MycoBank no. MB 829045), Lipomyces haiminakanus f.a., sp. nov. (holotype NBRC 110435; MycoBank no. MB 829046), Lipomyces komiensis f.a., sp. nov. (holotype NBRC 110440; MycoBank no. MB 829047), Lipomyces nakamensis f.a., sp. nov. (holotype NBRC 110434; MycoBank no. MB 829048), Lipomyces sakishimensis f.a., sp. nov. (holotype NBRC 110439; MycoBank no. MB 829049).

Gene Mutations in Ganoderma lucidum During Long-Term Preservation by Repeated Subculturing.

Subculturing is frequently used for the preservation of basidiomycetes. In this study, to assess and verify the risks of repeated subculturing on the long-term preservation of strains of culture collections, we performed single nucleotide polymorphism (SNP) analysis in genes encoding enzymes of the mevalonate pathway, 1,3-ß-glucan synthesis, lignin degradation, and the tricarboxylic acid (TCA) cycle of mycelia before and after preserving for a 4-year period by the subculturing 30 times every 45 days of Ganoderma lucidum NBRC 8346. As a result of analyzing 60 genes of the strain, SNPs were found in 18 genes, and 14 of them were found in the exon region. Nonsynonymous coding SNPs were found in two genes (atoB_2, hmgr) encoding enzymes of mevalonate pathway and five genes (lcc1_9, lcc1_11, lcc1_13, dslcc6, aa5_1_9) encoding enzymes of lignin degradation after subculturing of G. lucidum NBRC 8346.

Detection of DNA damage response in nonalcoholic fatty liver disease via p53-binding protein 1 nuclear expression.

Nonalcoholic fatty liver disease is a major liver disease that leads to cirrhosis and/or hepatocellular carcinoma in a subset of patients. The mechanism underlying disease progression is largely unknown. p53-binding protein 1 (53BP1) is a DNA damage response protein that rapidly localizes at the site of DNA double-strand breaks. In this study, we investigated nuclear 53BP1-positive foci formation as an indicator of DNA double-strand breaks in human nonalcoholic fatty liver disease liver tissues by immunofluorescence microscopy. A total of 52 liver tissue samples, including 43 nonalcoholic fatty liver disease samples and 9 controls, were studied. Our results show that the number of abnormal 53BP1-positive foci in hepatocytes (defined as three or more discrete nuclear foci and/or large foci greater than 1 µM) was significantly increased in nonalcoholic fatty liver disease patients compared to that in controls, both in nonalcoholic fatty liver (p < 0.01) and nonalcoholic steatohepatitis patients (p < 0.01). The number of large foci was significantly increased in the nonalcoholic steatohepatitis cases compared to that in the nonalcoholic fatty liver cases (p < 0.05) and correlated with increased stage of fibrosis. The number of large-foci-expressing hepatocytes was positively correlated with increased age (p < 0.01) and negatively correlated with serum platelet count (p < 0.05). In addition, we performed an in vitro assay using rat hepatocytes treated with the saturated free fatty acid palmitate. Treatment appeared to augment the number of abnormal foci, indicating an induction of double-strand breaks in the hepatocytes through free fatty acid treatment in a caspase-dependent manner. This study demonstrates that 53BP1-positive nuclear foci formation is associated with disease progression in nonalcoholic fatty liver disease patients. Analysis of 53BP1 expression might be a feasible technique to estimate genomic instability in nonalcoholic fatty liver disease.