Publications by authors named "Daisuke Motooka"

195 Publications

Longitudinal alterations of the gut mycobiota and microbiota on COVID-19 severity.

BMC Infect Dis 2022 Jun 24;22(1):572. Epub 2022 Jun 24.

Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, Osaka University, Suita, Japan.

Background: The impact of SARS-CoV-2 infection on the gut fungal (mycobiota) and bacterial (microbiota) communities has been elucidated individually. This study analyzed both gut mycobiota and microbiota and their correlation in the COVID-19 patients with severe and mild conditions and follow-up to monitor their alterations after recovery.

Methods: We analyzed the gut mycobiota and microbiota by bacterial 16S and fungal ITS1 metagenomic sequencing of 40 severe patients, 38 mild patients, and 30 healthy individuals and reanalyzed those of 10 patients with severe COVID-19 approximately 6 months after discharge.

Results: The mycobiota of the severe and mild groups showed lower diversity than the healthy group, and in some, characteristic patterns dominated by a single fungal species, Candida albicans, were detected. Lower microbial diversity in the severe group was observed, but no differences in its diversity or community structure were detected between the mild and healthy groups. The microbiota of the severe group was characterized by an increase in Enterococcus and Lactobacillus, and a decrease in Faecalibacterium and Bacteroides. The abundance of Candida was positively correlated with that of Enterococcus in patients with COVID-19. After the recovery of severe patients, alteration of the microbiota remained, but the mycobiota recovered its diversity comparable to that of mild and healthy groups.

Conclusion: In mild cases, the microbiota is stable during SARS-CoV-2 infection, but in severe cases, alterations persist for 6 months after recovery.
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http://dx.doi.org/10.1186/s12879-022-07358-7DOI Listing
June 2022

Chronic Pulmonary Disease Caused by Tsukamurella toyonakaense.

Emerg Infect Dis 2022 07;28(7):1437-1441

Unidentified Mycobacterium species are sometimes detected in respiratory specimens. We identified a novel Tsukamurella species (Tsukamurella sp. TY48, RIMD 2001001, CIP 111916), Tsukamurella toyonakaense, from a patient given a misdiagnosis of nontuberculous mycobacterial pulmonary disease caused by unidentified mycobacteria. Genomic identification of this Tsukamurella species helped clarify its clinical characteristics and epidemiology.
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http://dx.doi.org/10.3201/eid2807.212320DOI Listing
July 2022

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

Commun Biol 2022 05 30;5(1):516. Epub 2022 May 30.

Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.
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http://dx.doi.org/10.1038/s42003-022-03499-2DOI Listing
May 2022

Lysophosphatidylserines derived from microbiota in Crohn's disease elicit pathological Th1 response.

J Exp Med 2022 07 24;219(7). Epub 2022 May 24.

Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan.

Microbiota alteration and IFN-γ-producing CD4+ T cell overactivation are implicated in Crohn's disease (CD) pathogenesis. However, it remains unclear how dysbiosis enhances Th1 responses, leading to intestinal inflammation. Here, we identified key metabolites derived from dysbiotic microbiota that induce enhanced Th1 responses and exaggerate colitis in mouse models. Patients with CD showed elevated lysophosphatidylserine (LysoPS) concentration in their feces, accompanied by a higher relative abundance of microbiota possessing a gene encoding the phospholipid-hydrolyzing enzyme phospholipase A. LysoPS induced metabolic reprogramming, thereby eliciting aberrant effector responses in both human and mouse IFN-γ-producing CD4+ T cells. Administration of LysoPS into two mouse colitis models promoted large intestinal inflammation. LysoPS-induced aggravation of colitis was impaired in mice lacking P2ry10 and P2ry10b, and their CD4+ T cells were hyporesponsive to LysoPS. Thus, our findings elaborate on the mechanism by which metabolites elevated in patients with CD harboring dysbiotic microbiota promote Th1-mediated intestinal pathology.
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http://dx.doi.org/10.1084/jem.20211291DOI Listing
July 2022

Inhibition of nonhomologous end joining-mediated DNA repair enhances anti-HBV CRISPR therapy.

Hepatol Commun 2022 May 24. Epub 2022 May 24.

Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan.

Current anti-hepatitis B virus (HBV) therapies have little effect on covalently closed circular DNA (cccDNA) and fail to eliminate HBV. The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been reported to directly target cccDNA and exert antiviral effects. In this study, we hypothesized that the inhibition of the DNA repair machinery, which is important for the repair of CRISPR-induced double-strand breaks, may enhance the effect of CRISPR targeting cccDNA, and we investigated the antiviral effect of potential combination therapy. The antiviral effect of CRISPR targeting cccDNA (HBV-CRISPR) was evaluated in HBV-susceptible HepG2-hNTCP-C4 cells expressing Cas9 (HepG2-hNTCP-C4-iCas9) or primary human hepatocytes (PHHs) expressing Cas9. Following HBV infection, HBV-CRISPR reduced cccDNA levels, accompanied by decreases in pregenomic RNA (pgRNA) levels and supernatant HBV DNA, hepatitis B surface antigen and hepatitis B e antigen levels in HepG2-hNTCP-C4-iCas9 cells, and PHHs. HBV-CRISPR induced indel formation in cccDNA and up-regulated poly(adenosine diphosphate ribose) polymerase (PARP) activity in HBV-infected HepG2-hNTCP-C4-iCas9 cells. The suppression of PARP2-Histone PARylation factor 1 (HPF1) (involved in the initial step of DNA repair) with small interfering RNA (siRNA) targeting either PARP2 or HPF1 increased the reduction in pgRNA and cccDNA by HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells. The suppression of DNA Ligase 4 (LIG4) (essential for nonhomologous end joining [NHEJ]) but not breast cancer susceptibility gene (BRCA) (essential for homologous recombination) enhanced the antiviral effect of HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells. Finally, the clinically available PARP inhibitor olaparib increased the reductions in pgRNA and cccDNA levels induced by HBV-CRISPR in HBV-infected HepG2-hNTCP-C4-iCas9 cells and PHHs. Conclusion: The suppression of the NHEJ-mediated DNA repair machinery enhances the effect of CRISPR targeting cccDNA. The combination of CRISPR and olaparib may represent a therapy for HBV elimination.
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http://dx.doi.org/10.1002/hep4.2014DOI Listing
May 2022

sp. nov., a slowly growing, non-scotochromogenic species, isolated from sputum of an elderly man.

Int J Syst Evol Microbiol 2022 May;72(5)

Department of Respiratory Medicine, National Hospital Organization Osaka Toneyama Medical Center, 5-1-1 Toneyama Toyonaka, Osaka, Japan.

A slowly growing mycobacteria, identified as strain TY59, was isolated from sputum of an elderly man with pneumonia. Sequencing of the 16S rRNA gene indicated that this strain was similar to members of the complex and closely related species. Strain TY59 has highest 16S rRNA gene sequence similarities to the type strains of (99.80 % sequence similarity), (99.74 %), (99.54 %), subsp. (99.54 %) and subsp. (99.54 %). Analysis of the internal transcribed spacer (ITS) and DNA-directed RNA polymerase subunit beta () sequences gave similar results to the 16S rRNA gene analysis. The closest species to strain TY59 were and with 97.90-98.25 % identity in ITS and 96.4-96.6 % in . The strain's 65 kDa heat shock protein () gene was different from those of , and subsp. with 72.4-74.2 % identity. Average nucleotide identity results showed a 93.4 % match to as the maximum value. Phenotypically, the non-chromogenicity, rough colonies, growth at 42 °C, negative results for nitrate reduction, β-glucosidase and Tween 80 hydrolysis, and positive results for catalase activity set this strain apart from closely related species. We propose that sp. nov. is a novel species of slowly growing mycobacteria. The type strain is TY59 (RIMD 1371001=CIP 111917).
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http://dx.doi.org/10.1099/ijsem.0.005378DOI Listing
May 2022

Circulating extracellular vesicles carrying Firmicutes reflective of the local immune status may predict clinical response to pembrolizumab in urothelial carcinoma patients.

Cancer Immunol Immunother 2022 May 22. Epub 2022 May 22.

Department of Urology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

Bacterial flora has clinical significance for the host. The metabolic environment created by this flora influences immunotherapy in urothelial carcinoma. However, there are no reports on the clinical significance of bacterial flora in the host bloodstream. We aimed to clarify the correlation between extracellular vesicle (EV)-derived blood microflora information and tumor immunological status in urothelial carcinoma (UC) patients. Serum samples were collected from 20 healthy donors, 50 patients with localized UC, and 31 patients with metastatic UC (mUC) who had undergone pembrolizumab treatment. Bacterial DNA in EVs was extracted from each sample. Metagenomic sequencing was performed after amplification of the V1-V2 region of the bacterial 16S rRNA gene. Using the matched tumor tissue and serum samples, we revealed that the smaller amount of peripheral EVs carrying Firmicutes DNA was significantly correlated with the higher number of infiltrating T cells within tumor tissues (CD3; p = 0.015, CD4; p = 0.039, CD8; p = 0.0084) and the higher expression of activation markers on their surface (ICOS on both CD4; p = 0.0013 and CD8 T cells; p = 0.016 and 4-1BB on CD4 T cells; p = 0.016). In terms of circulating metabolic information, L-Ser and L-Pro levels, which play important roles in T cell expansion and proliferation, were significantly higher in the Firmicutes-low group (p = 0.010). All of the patients with higher Firmicutes abundance had disease progression without any clinical response (p = 0.026) and significantly inferior prognosis for pembrolizumab therapy (p = 0.035). This is the first study on the importance of peripheral bacterial EVs in cancer patients treated with cancer immunotherapy.
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http://dx.doi.org/10.1007/s00262-022-03213-5DOI Listing
May 2022

Multiomics identifies the link between intratumor steatosis and the exhausted tumor immune microenvironment in hepatocellular carcinoma.

Hepatology 2022 May 14. Epub 2022 May 14.

Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan.

Background And Aims: Immunotherapy has become the standard-of-care treatment for hepatocellular carcinoma (HCC), but its efficacy remains limited. To identify immunotherapy-susceptible HCC, we profiled the molecular abnormalities and tumor immune microenvironment (TIME) of rapidly increasing nonviral HCC.

Approaches And Results: We performed RNA-seq of tumor tissues in 113 patients with nonviral HCC and cancer genome sequencing of 69 genes with recurrent genetic alterations reported in HCC. Unsupervised hierarchical clustering classified nonviral HCCs into three molecular classes (Class I, II, III), which stratified patient prognosis. Class I, with the poorest prognosis, was associated with TP53 mutations, whereas class III, with the best prognosis, was associated with cadherin-associated protein beta 1 (CTNNB1) mutations. Thirty-eight percent of nonviral HCC was defined as an immune class characterized by a high frequency of intratumoral steatosis and a low frequency of CTNNB1 mutations. Steatotic HCC, which accounts for 23% of nonviral HCC cases, presented an immune-enriched but immune-exhausted TIME characterized by T cell exhaustion, M2 macrophage and cancer-associated fibroblast (CAF) infiltration, high PD-L1 expression, and TGF-β signaling activation. Spatial transcriptome analysis suggested that M2 macrophages and CAFs may be in close proximity to exhausted CD8+ T cells in steatotic HCC. An in vitro study showed that palmitic acid-induced lipid accumulation in HCC cells upregulated PD-L1 expression and promoted immunosuppressive phenotypes of cocultured macrophages and fibroblasts. Patients with steatotic HCC, confirmed by chemical-shift MR imaging, had significantly longer PFS with combined immunotherapy using anti-PD-L1 and anti-VEGF antibodies.

Conclusions: Multiomics stratified nonviral HCCs according to prognosis or TIME. We identified the link between intratumoral steatosis and immune-exhausted immunotherapy-susceptible TIME.
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http://dx.doi.org/10.1002/hep.32573DOI Listing
May 2022

An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.

Sci Transl Med 2022 Jun 22;14(650):eabn7737. Epub 2022 Jun 22.

Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.
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http://dx.doi.org/10.1126/scitranslmed.abn7737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9097879PMC
June 2022

Corrigendum: gen. nov., sp. nov., sp. nov., gen. nov. and sp. nov. isolated from human faeces.

Int J Syst Evol Microbiol 2022 04;72(4)

Benno Laboratory, RIKEN Baton Zone Program, RIKEN Cluster for Science Technology and Innovation Hab, Wako, Saitama 351-0198, Japan.

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http://dx.doi.org/10.1099/ijsem.0.005317DOI Listing
April 2022

Exacerbation of non-steroidal anti-inflammatory drug-induced enteropathy in C-C chemokine receptor type 7-deficient mice.

J Gastroenterol Hepatol 2022 Apr 18. Epub 2022 Apr 18.

Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Osaka, Japan.

Background And Aim: Non-steroidal anti-inflammatory drugs (NSAIDs) induce intestinal enteropathy and the pathophysiology is related to immune-mediated mechanisms. We aimed to investigate the role of C-C chemokine receptor type 7 (CCR7) which regulates immune cell migration in NSAID-induced enteropathy.

Methods: Injury of the small intestine was evaluated 24 h after the subcutaneous injection of indomethacin in CCR7-deficient (Ccr7 ) and wild-type (WT) mice. The cellular profile and cytokine production in intestinal cells were analyzed. Indomethacin-induced enteropathy was evaluated in mice adoptively transferred with CD103 dendritic cells (DCs) from Ccr7 or WT mice.

Results: Indomethacin induced more severe intestinal injury in Ccr7 mice than in WT mice. The major inflammatory cytokines were not increased and the proportion of regulatory T cells following indomethacin injection was not decreased in Ccr7 mice compared with WT mice. The expression of interleukin (IL)-22 binding protein (IL-22BP), which inhibits IL-22 activity, was significantly higher in CD103 DCs from Ccr7 mice than those from WT mice. Mice adoptively transferred with CD103 DCs isolated from Ccr7 mice exhibited more severe intestinal injury following indomethacin injection compared with those adoptively transferred with CD103 DCs of WT mice. Ccr7 mice injected with indomethacin showed a significant reduction in regenerating islet-derived 1 (Reg1) mRNA expression, which is regulated by IL-22, in intestinal epithelial cells.

Conclusions: C-C chemokine receptor type 7 deficiency exacerbated NSAID-induced enteropathy in association with an altered phenotype of CD103 DCs that produces IL-22BP. CCR7 contributes to protect the small intestine from NSAID-induced mucosal injury.
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http://dx.doi.org/10.1111/jgh.15868DOI Listing
April 2022

High-fat diet promotes prostate cancer growth through histamine signaling.

Int J Cancer 2022 Aug 26;151(4):623-636. Epub 2022 Apr 26.

Department of Urology, Osaka University, Graduate School of Medicine, Suita, Japan.

Western high-fat diets (HFD) are regarded as a major risk factor for prostate cancer (PCa). Using prostate-specific Pten-knockout mice as a PCa model, we previously reported that HFD promoted inflammatory PCa growth. The composition of the gut microbiota changes under the influence of diet exert various effects on the host through immunological mechanisms. Herein, we investigated the etiology of HFD-induced inflammatory cancer growth and the involvement of the gut microbiome. The expression of Hdc, the gene responsible for histamine biosynthesis, and histamine levels were upregulated in large prostate tumors of HFD-fed mice, and the number of mast cells increased around the tumor foci. Administration of fexofenadine, a histamine H1 receptor antagonist, suppressed tumor growth in HFD-fed mice by reducing the number of myeloid-derived suppressor cells and suppressing IL6/STAT3 signaling. HFD intake induced gut dysbiosis, resulting in the elevation of serum lipopolysaccharide (LPS) levels. Intraperitoneal injection of LPS increased Hdc expression in PCa. Inhibition of LPS/Toll-like receptor 4 signaling suppressed HFD-induced tumor growth. The number of mast cells increased around the cancer foci in total prostatectomy specimens of severely obese patients. In conclusion, HFD promotes PCa growth through histamine signaling via mast cells. Dietary high-fat induced gut dysbiosis might be involved in the inflammatory cancer growth.
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http://dx.doi.org/10.1002/ijc.34028DOI Listing
August 2022

Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids.

BMC Genomics 2022 Mar 23;23(1):226. Epub 2022 Mar 23.

Department of Bacterial Infection, Research Institute for Microbial Disease (RIMD), Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Background: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains.

Results: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1-3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains.

Conclusion: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.
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http://dx.doi.org/10.1186/s12864-022-08453-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8941779PMC
March 2022

Firmicutes in Gut Microbiota Correlate with Blood Testosterone Levels in Elderly Men.

World J Mens Health 2022 Feb 24. Epub 2022 Feb 24.

Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan.

Purpose: In males, testosterone levels have been implicated in various diseases. Recently, the influence of gut microbial-derived compounds on host metabolism has become evident, and it has been suggested that some gut bacteria may be involved in testosterone metabolism. In the present study, we examined the relationship between testosterone levels and gut microbiota in elderly Japanese men.

Materials And Methods: We collected samples from Japanese male subjects suspected of having prostate cancer and underwent prostate biopsies and excluded patients with positive biopsies to avoid the effect of prostate cancer on the gut microbiota. In total, 54 Japanese males with negative biopsy results were included in our study. The gut microbiota was analyzed by 16S rRNA gene sequencing of bacterial DNA extracted from rectal swabs. Gut microbiota compositions were compared between the two groups according to the level of serum testosterone (above or below 3.5 ng /mL).

Results: The median age of the cohort was 71 years, and the quartile range was 67 to 73 years. We observed no significant difference in alpha or beta diversity, but some bacteria belonging to the phylum Firmicutes (Clostridiales, , and ) were increased in the high testosterone group. Serum testosterone levels positively correlated with the relative amount of Firmicutes (=0.3323, p=0.0141), and the amount of Firmicutes affected serum testosterone levels independent of host factors (age, body mass index, triglyceride, and total cholesterol; β=0.770, p=0.0396).

Conclusions: Some intestinal bacteria belonging to the phylum Firmicutes were associated with testosterone levels in elderly males. Therefore, the gut microbiota could affect testosterone metabolism in elderly males.
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http://dx.doi.org/10.5534/wjmh.210190DOI Listing
February 2022

Osteoblast-derived vesicles induce a switch from bone-formation to bone-resorption in vivo.

Nat Commun 2022 02 24;13(1):1066. Epub 2022 Feb 24.

Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Suita, Osaka, Japan.

Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor β. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo.
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http://dx.doi.org/10.1038/s41467-022-28673-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8873258PMC
February 2022

Cell-Free DNA Derived From Neutrophils Triggers Type 1 Interferon Signature in Neuromyelitis Optica Spectrum Disorder.

Neurol Neuroimmunol Neuroinflamm 2022 05 24;9(3). Epub 2022 Feb 24.

From the Department of Neurology (Hisashi Murata, M.K., Y.Y., S.B., N.S., Y.S., K.K., S.T., T.K., T.O., Hideki Mochizuki), Graduate School of Medicine, Osaka University; Department of Experimental Immunology (Y.Y.), WPI Immunology Frontier Research Center, Osaka University; Integrated Frontier Research for Medical Science Division (Y.Y., D.M., A.K., Hideki Mochizuki), Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University; Genome Information Research Center (D.M.), Research Institute for Microbial Diseases, Osaka University; and Department of Respiratory Medicine and Clinical Immunology (H.K., H.T., A.K.), Graduate School of Medicine, Osaka University, Suita, Japan.

Background And Objectives: Recently accumulating evidence suggests the pivotal role of type 1 interferon (IFN-1) signature in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD). However, the mechanism of the initial trigger that augments IFN-1 pathway in the peripheral immune system of NMOSD has yet to be elucidated.

Methods: Clinical samples were obtained from 32 patients with aquaporin-4 antibody-positive NMOSD and 23 healthy subjects. IFN-1 induction in peripheral blood mononuclear cells (PBMCs) by serum-derived cell-free DNA (cfDNA) was assessed in combination with blockades of DNA sensors in vitro. CfDNA fraction was analyzed for DNA methylation profiles by bisulfite sequencing, elucidating the cellular origin of cfDNA. The induction of neutrophil extracellular trap related cell death (NETosis) was further analyzed in NMOSD and control groups, and the efficacy of pharmacologic intervention of NETosis was assessed.

Results: Enhanced IFN-1 induction by cfDNA derived from NMOSD was observed in PBMCs with cofactor of LL37 antimicrobial peptide. DNase treatment, cGAS inhibitor, and Toll-like receptor 9 antagonist efficiently inhibited IFN-1 production. DNA methylation pattern of cfDNA in patients with NMOSD demonstrated that the predominant cellular source of cfDNA was neutrophils. Whole blood transcriptome analysis also revealed neutrophil activation in NMOSD. In addition, enhanced NETosis induction was observed with NMOSD-derived sera, and efficient pharmacologic inhibition of NETosis with dipyridamole was observed.

Discussion: Our study highlights the previously unrevealed role of cfDNA predominantly released by neutrophil in the induction of IFN-1 signature in NMOSD and further indicate a novel pharmacologic target in NMOSD.
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http://dx.doi.org/10.1212/NXI.0000000000001149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8874356PMC
May 2022

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Sci Transl Med 2022 02 16;14(632):eaax7706. Epub 2022 Feb 16.

Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.
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http://dx.doi.org/10.1126/scitranslmed.aax7706DOI Listing
February 2022

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Proc Natl Acad Sci U S A 2022 02;119(7)

Department of Urology, Graduate School of Medicine, Osaka University, 565-0871 Osaka, Japan.

Foxp3-expressing CD25CD4 regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8 Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8 CCR8 tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8 effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.
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http://dx.doi.org/10.1073/pnas.2114282119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8851483PMC
February 2022

PIK3CA mutation correlates with mTOR pathway expression but not clinical and pathological features in Fibfibroipose vascular anomaly (FAVA).

Diagn Pathol 2022 Jan 30;17(1):19. Epub 2022 Jan 30.

Department of Pathology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, 565-0871, Suita, Osaka, Japan.

Background: Fibro-adipose vascular anomaly (FAVA) is a rare and new entity of vascular anomaly. Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene were identified at a frequency of 62.5% in FAVA cases. The PIK3CA mutations excessively activate mammalian target of rapamycin (mTOR) pathway, which promotes angiogenesis and lymphangiogenesis, implying that PIK3CA mutations may act as drivers of FAVAs. This study investigated the correlations between PIK3CA mutational status, clinicopathological features and immunohistochemical expression of the mTOR pathway in a series of FAVA.

Methods: We retrospectively evaluated the clinical and pathological findings of four FAVA cases. We performed next-generation sequencing (NGS) with a custom panel of genes associated with the mTOR pathway and genes responsible for other vascular anomalies; followed by direct sequencing and immunohistochemical analysis of the mTOR pathway.

Results: Two PIK3CA-mutation cases and two PIK3CA-wild-type (wt) cases exhibited similar typical clinical features of FAVA. Histological analysis revealed venous malformation, lymphatic malformation, nerves containing enlarged abnormal vessels and fibrofatty tissue were observed regardless of PIK3CA mutational status. In contrast to clinical and histological findings, the immunohistochemical expression of activated AKT and mTOR that are upstream of the mTOR pathway was detected in abnormal vessels of PIK3CA-mutation cases but not in those of PIK3CA-wt cases. However, activated eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase 1 (S6K1), both of which are downstream effectors of the mTOR pathway, were expressed in abnormal vessels of both PIK3CA-mutation and PIK3CA-wt cases. Furthermore, targeting NGS did not find any common genetic mutations involved in the mTOR pathway among PIK3CA-wt cases.

Conclusions: There was no significant association between the presence of PIK3CA mutations and the clinicopathological features of FAVA, suggesting that the PIK3CA gene is not necessarily involved in the onset of FAVA. FAVAs lacking PIK3CA mutations may be caused by other gene mutations that activate 4EBP1 and S6K1.
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http://dx.doi.org/10.1186/s13000-022-01199-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8802443PMC
January 2022

Epithelial miR-215 negatively modulates Th17-dominant inflammation by inhibiting CXCL12 production in the small intestine.

Genes Cells 2022 Apr 6;27(4):243-253. Epub 2022 Feb 6.

WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

MicroRNAs are a class of non-coding short-chained RNAs that control cellular functions by downregulating their target genes. Recent research indicates that microRNAs play a role in the maintenance of gut homeostasis. miR-215 was found to be highly expressed in epithelial cells of the small intestine; however, the involvement of miR-215 in gut immunity remains unknown. Here, we show that miR-215 negatively regulates inflammation in the small intestine by inhibiting CXCL12 production. Mice lacking miR-215 showed high susceptibility to inflammation induced by indomethacin, accompanied by an increased number of Th17 cells in the lamina propria of the small intestine. Our findings provide a rationale for targeting miR-215 as a therapeutic intervention for inflammatory conditions in the small intestine.
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http://dx.doi.org/10.1111/gtc.12922DOI Listing
April 2022

spp. have higher fitness for survival, in a pH-dependent manner, in pancreatic juice among duodenal bacterial flora.

JGH Open 2022 Jan 5;6(1):85-90. Epub 2022 Jan 5.

Department of Molecular Biochemistry and Clinical Investigation Osaka University Graduate School of Medicine Suita Osaka Japan.

Background And Aim: Bacterial infection is involved in the progression of many gastrointestinal diseases, including those of pancreas; however, how and which bacteria colonize in pancreatic juice and tissue have yet to be elucidated. Recently, we reported that exists in the pancreatic juice and tissues of patients with chronic pancreatic disease. Here, we investigated the survival of in duodenal juice with different pH conditions.

Methods: Pancreatic juice samples from 62 patients with cancers of the duodeno-pancreato-biliary region were evaluated for the presence of . 16S ribosomal RNA polymerase chain reaction and 16S-based metagenome analyses were performed to determine the bacterial composition. The survival of in various pancreatic juice conditions was evaluated.

Results: Of 62 samples, 27% (17/62) were positive for spp., among which 71% (12/17) contained . spp. showed the highest fitness for survival in alkaline pancreatic juice among various bacterial species. The microbiome of pancreatic juice from patients with pancreatic and bile duct cancer showed diversity, but spp. were enriched among duodenal tumors and intraductal papillary mucinous neoplasms.

Conclusions: Alkalinity is one of the important factors for the selective survival of among microbiota. can colonize the pancreatic duct when the pancreatic juice condition is altered.
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http://dx.doi.org/10.1002/jgh3.12703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8762619PMC
January 2022

Pleural Effusion Caused by in an Immunocompetent Host: A Case Report.

Front Med (Lausanne) 2021 24;8:797171. Epub 2021 Nov 24.

Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Osaka, Japan.

is a rare species among rapidly growing mycobacteria, and pleurisy is very rare. Here, we report for the first time, an immunocompetent patient with pleurisy caused by . The patient had no history of immunodeficiency and no recurrence of lung cancer after surgery. However, 8 months after surgery, he developed a new lung shadow and pleurisy. Although whole-genome analysis of the colony cultured from the patient's pleural fluid revealed , we could not identify it in time, resulting in a poor outcome. pleurisy in this case might have occurred via a bulla rupture of the lung lesion because computed tomography of the patient's chest showed pneumothorax and a lung lesion in contact with thoracic cavity. This case emphasized that nontuberculous mycobacterial pleurisy should be considered in the differential diagnoses of pleural effusion even in immunocompetent patients. Advancement of comprehensive and rapid analyses of genomic data from clinical specimens will lead to better treatment strategies.
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http://dx.doi.org/10.3389/fmed.2021.797171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8651622PMC
November 2021

Impact of sleep on the microbiome of oral biofilms.

PLoS One 2021 9;16(12):e0259850. Epub 2021 Dec 9.

Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.

Dysbiosis of the oral microbiome is associated with diseases such as periodontitis and dental caries. Because the bacterial counts in saliva increase markedly during sleep, it is broadly accepted that the mouth should be cleaned before sleep to help prevent these diseases. However, this practice does not consider oral biofilms, including the dental biofilm. This study aimed to investigate sleep-related changes in the microbiome of oral biofilms by using 16S rRNA gene sequence analysis. Two experimental schedules-post-sleep and pre-sleep biofilm collection-were applied to 10 healthy subjects. Subjects had their teeth and oral mucosa professionally cleaned 7 days and 24 h before sample collection. Samples were collected from several locations in the oral cavity: the buccal mucosa, hard palate, tongue dorsum, gingival mucosa, tooth surface, and saliva. Prevotella and Corynebacterium had higher relative abundance on awakening than before sleep in all locations of the oral cavity, whereas fluctuations in Rothia levels differed depending on location. The microbiome in different locations in the oral cavity is affected by sleep, and changes in the microbiome composition depend on characteristics of the surfaces on which oral biofilms form.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0259850PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8659294PMC
January 2022

Whole gut virome analysis of 476 Japanese revealed a link between phage and autoimmune disease.

Ann Rheum Dis 2022 02 8;81(2):278-288. Epub 2021 Dec 8.

Department of Statistical Genetics, Osaka University Graduate School of Medicine, Suita, Osaka, Japan

Objective: The relationship between autoimmune diseases and the gut microbiome has been intensively studied, and several autoimmunity-associated bacterial taxa have been identified. However, much less is known about the roles of the gut virome in autoimmune diseases.

Methods: Here, we performed a whole gut virome analysis based on the shotgun sequencing of 476 Japanese which included patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis and healthy control subjects.

Results: Our case-control comparison of the viral abundance revealed that crAss-like phages, which are one of the main components of a healthy gut virome, significantly decreased in the gut of the patients with autoimmune disease, specifically the patients with RA and SLE. In addition, significantly decreased in the gut of the patients with SLE. To understand how these viruses affected the bacteriome, we performed a quantitative virus-bacterium association analysis and clustered regularly interspaced short palindromic repeat-based virus-bacterium interaction analysis. We identified a symbiosis between e and . In addition, multiple bacterial targets of crAss-like phages were identified (eg, spp).

Conclusion: Our data suggest that the gut virome can affect our body either directly or via bacteria. Our analyses have elucidated a previously missing part of the autoimmunity-associated gut microbiome and presented new candidates that contribute to the development of autoimmune diseases.
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http://dx.doi.org/10.1136/annrheumdis-2021-221267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8761997PMC
February 2022

Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice.

Int Immunol 2022 03;34(4):207-223

Laboratory of Immune Regulation, Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan.

Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH-DH-JH and Vκ-Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.
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http://dx.doi.org/10.1093/intimm/dxab111DOI Listing
March 2022

Chronic pathophysiological changes in the normal brain parenchyma caused by radiotherapy accelerate glioma progression.

Sci Rep 2021 11 11;11(1):22110. Epub 2021 Nov 11.

Department of Neurosurgery, Osaka Medical and Pharmaceutical University, 2-7 Daigaku-Machi, Takatsuki, Osaka, 569-8686, Japan.

Radiation therapy is one of standard treatment for malignant glioma after surgery. The microenvironment after irradiation is considered not to be suitable for the survival of tumor cells (tumor bed effect). This study investigated whether the effect of changes in the microenvironment of parenchymal brain tissue caused by radiotherapy affect the recurrence and progression of glioma. 65-Gy irradiation had been applied to the right hemisphere of Fisher rats. After 3 months from irradiation, we extracted RNA and protein from the irradiated rat brain. To study effects of proteins extracted from the brains, we performed WST-8 assay and tube formation assay in vitro. Cytokine production were investigated for qPCR. Additionally, we transplanted glioma cell into the irradiated and sham animals and the median survival time of F98 transplanted rats was also examined in vivo. Immunohistochemical analyses and invasiveness of implanted tumor were evaluated. X-ray irradiation promoted the secretion of cytokines such as CXCL12, VEGF-A, TGF-β1 and TNFα from the irradiated brain. Proteins extracted from the irradiated brain promoted the proliferation and angiogenic activity of F98 glioma cells. Glioma cells implanted in the irradiated brains showed significantly high proliferation, angiogenesis and invasive ability, and the post-irradiation F98 tumor-implanted rats showed a shorter median survival time compared to the Sham-irradiation group. The current study suggests that the microenvironment around the brain tissue in the chronic phase after exposure to X-ray radiation becomes suitable for glioma cell growth and invasion.
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http://dx.doi.org/10.1038/s41598-021-01475-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8585920PMC
November 2021

Direct RNA Sequencing Unfolds the Complex Transcriptome of Vibrio parahaemolyticus.

mSystems 2021 Dec 9;6(6):e0099621. Epub 2021 Nov 9.

Single Cell Genomics, Human Immunology, WPI Immunology Frontier Research Center, Osaka Universitygrid.136593.b, Osaka, Japan.

Conventional bacterial genome annotation provides information about coding sequences but ignores untranslated regions and operons. However, untranslated regions contain important regulatory elements as well as targets for many regulatory factors, such as small RNAs. Operon maps are also essential for functional gene analysis. In the last decade, considerable progress has been made in the study of bacterial transcriptomes through transcriptome sequencing (RNA-seq). Given the compact nature of bacterial genomes, many challenges still cannot be resolved through short reads generated using classical RNA-seq because of fragmentation and loss of the full-length information. Direct RNA sequencing is a technology that sequences the native RNA directly without information loss or bias. Here, we employed direct RNA sequencing to annotate the Vibrio parahaemolyticus transcriptome with its full features, including transcription start sites (TSSs), transcription termination sites, and operon maps. A total of 4,103 TSSs were identified. In comparison to short-read sequencing, full-length information provided a deeper view of TSS classification, showing that most internal and antisense TSSs were actually a result of gene overlap. Sequencing the transcriptome of V. parahaemolyticus grown with bile allowed us to study the landscape of pathogenicity island Vp-PAI. Some genes in this region were reannotated, providing more accurate annotation to increase precision in their characterization. Quantitative detection of operons in V. parahaemolyticus showed high complexity in some operons, shedding light on a greater extent of regulation within the same operon. Our study using direct RNA sequencing provides a quantitative and high-resolution landscape of the V. parahaemolyticus transcriptome. Vibrio parahaemolyticus is a halophilic bacterium found in the marine environment. Outbreaks of gastroenteritis resulting from seafood poisoning by these pathogens have risen over the past 2 decades. Upon ingestion by humans-often through the consumption of raw or undercooked seafood-V. parahaemolyticus senses the host environment and expresses numerous genes, the products of which synergize to synthesize and secrete toxins that can cause acute gastroenteritis. To understand the regulation of such adaptive response, mRNA transcripts must be mapped accurately. However, due to the limitations of common sequencing methods, not all features of bacterial transcriptomes are always reported. We applied direct RNA sequencing to analyze the V. parahaemolyticus transcriptome. Mapping the full features of the transcriptome is anticipated to enhance our understanding of gene regulation in this bacterium and provides a data set for future work. Additionally, this study reveals a deeper view of a complicated transcriptome landscape, demonstrating the importance of applying such methods to other bacterial models.
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http://dx.doi.org/10.1128/mSystems.00996-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577284PMC
December 2021
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