Antibiotic effects on gut microbiota modulate diet-induced metabolic dysfunction-associated steatohepatitis development in C57BL/6 mice.

The potential involvement of the gut microbiota in metabolic dysfunction-associated steatohepatitis (MASH) pathogenesis has garnered increasing attention. In this study, we elucidated the link between high-fat/cholesterol/cholate-based (iHFC)#2 diet-induced MASH progression and gut microbiota in C57BL/6 mice using antibiotic treatments. Treatment with vancomycin (VCM), which targets gram-positive bacteria, exacerbated the progression of liver damage, steatosis, and fibrosis in iHFC#2-fed C57BL/6 mice. The expression levels of inflammation- and fibrosis-related genes in the liver significantly increased after VCM treatment for 8 weeks. F4/80+ macrophage abundance increased in the livers of VCM-treated mice. These changes were rarely observed in the iHFC#2-fed C57BL/6 mice treated with metronidazole, which targets anaerobic bacteria. A16S rRNA sequence analysis revealed a significant decrease in α-diversity in VCM-treated mice compared with that in placebo-treated mice, with Bacteroidetes and Firmicutes significantly decreased, while Proteobacteria and Verrucomicrobia increased markedly. Finally, VCM treatment dramatically altered the level and balance of bile acid (BA) composition in iHFC#2-fed C57BL/6 mice. Thus, the VCM-mediated exacerbation of MASH progression depends on the interaction between the gut microbiota, BA metabolism, and inflammatory responses in the livers of iHFC#2-fed C57BL/6 mice.

Impacts of liver macrophages, gut microbiota, and bile acid metabolism on the differences in iHFC diet-induced MASH progression between TSNO and TSOD mice.

BACKGROUND: Tsumura-Suzuki non-obese (TSNO) mice exhibit a severe form of metabolic dysfunction-associated steatohepatitis (MASH) with advanced liver fibrosis upon feeding a high-fat/cholesterol/cholate-based (iHFC) diet. Another ddY strain, Tsumura-Suzuki diabetes obese (TSOD) mice, are impaired in the progression of iHFC diet-induced MASH. AIM: To elucidate the underlying mechanisms contributing to the differences in MASH progression between TSNO and TSOD mice. METHODS: We analyzed differences in the immune system, gut microbiota, and bile acid metabolism in TSNO and TSOD mice fed with a normal diet (ND) or an iHFC diet. RESULTS: TSOD mice had more anti-inflammatory macrophages in the liver than TSNO mice under ND feeding, and were impaired in the iHFC diet-induced accumulation of fibrosis-associated macrophages and formation of histological hepatic crown-like structures in the liver. The gut microbiota of TSOD mice also exhibited a distinct community composition with lower diversity and higher abundance of Akkermansia muciniphila compared with that in TSNO mice. Finally, TSOD mice had lower levels of bile acids linked to intestinal barrier disruption under iHFC feeding. CONCLUSIONS: The dynamics of liver macrophage subsets, and the compositions of the gut microbiota and bile acids at steady state and post-onset of MASH, had major impacts on MASH development.

Dynamics of Liver Macrophage Subsets in a Novel Mouse Model of Non-Alcoholic Steatohepatitis Using C57BL/6 Mice.

Macrophages are critical for the development of non-alcoholic steatohepatitis (NASH). Our previous findings in TSNO mouse livers showed that an iHFC (high-fat/cholesterol/cholate) diet induced liver fibrosis similar to human NASH and led to the accumulation of distinct subsets of macrophage: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+/Ly6C- cells were associated with the promotion of advanced liver fibrosis in NASH. On the other hand, CD11c-/Ly6C+ cells exhibited an anti-inflammatory effect and were involved in tissue remodeling processes. This study aimed to elucidate whether an iHFC diet with reduced cholic acid (iHFC#2 diet) induces NASH in C57BL/6 mice and examine the macrophage subsets accumulating in the liver. Histological and quantitative real-time PCR analyses revealed that the iHFC#2 diet promoted inflammation and fibrosis indicative of NASH in the livers of C57BL/6 mice. Cell numbers of Kupffer cells decreased and recruited macrophages were accumulated in the livers of iHFC#2 diet-fed C57BL/6 mice. Notably, the iHFC#2 diet resulted in the accumulation of three macrophage subsets in the livers of C57BL/6 mice: CD11c+/Ly6C-, CD11c-/Ly6C+, and CD11c+/Ly6C+ cells. However, CD11c+/Ly6C+ cells were not distinct populations in the iHFC-fed TSNO mice. Thus, differences in cholic acid content and mouse strain affect the macrophage subsets that accumulate in the liver.

Malignant testicular unclassified sex cord stromal tumor: a case report.

BACKGROUND: Most testicular tumors are germ cell tumors; sex cord stromal tumors are infrequent, accounting for only 3-5% of testicular tumors. Unclassified sex cord stromal tumors are extremely rare. Generally, 10% of sex cord stromal tumors are malignant. We report a case of malignant unclassified sex cord stromal tumor with retroperitoneal lymph node metastasis at first visit and a corresponding literature review. CASE PRESENTATION: A 72-year-old Japanese man visited our department primarily for indolent right scrotum enlargement in September 2020. Blood biochemistry examination, urinalysis, and tumor markers (alpha-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase) showed no abnormal findings. Contrast-enhanced computed tomography showed enlarged para-aortic lymph node (18 × 16 and 10 × 102 mm); a 50 × 452 mm mass with uneven contents was found in the right testicle. The patient underwent inguinal orchiectomy in September 2020. As per immunohistochemistry, the tumor cells were diffusely positive for SF-1 and Ki-67, partially positive for inhibin, and negative for CAM5.2, CK7, CK20, C-KIT, CD30, LCA, GATA-3, TTF-1, and PAX8. Calretinin was expressed in approximately 5% of tumor cells; thus, sex cord/gonadal stroma components were considered to be involved. The final pathological diagnosis was unclassified malignant sex cord stromal tumor. The patient was diagnosed with pT1, N1, M0, S0, and tumor-node-metastasis stage IIA disease. The patient received postoperative chemotherapy with four courses of etoposide and cisplatin therapy from November 2020. Post-chemotherapeutic computed tomography showed new metastatic lesions including lung, liver, pancreas, and para-aortic lymphadenopathy, which increased in size. Disease progression was observed. Cancer genome research was performed using the OncoGuide National Cancer Center oncopanel system; however, no gene mutation for which the drug could be expected to be effective was found. The patient opted for best supportive care at a nearby hospital and died from cancer progression in January 2022. CONCLUSION: We encountered a case of malignant testicular unclassified sex cord stromal tumor pathologically diagnosed as testicular tumor with retroperitoneal lymph node metastasis in a patient who underwent inguinal orchiectomy. Future data collection is necessary to establish multimodality therapy for malignant testicular unclassified sex cord stromal tumor.

Cnm of Streptococcus mutans is important for cell surface structure and membrane permeability.

Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is a major pathogen of dental caries. The protein Cnm of S. mutans is involved in collagen binding, but its other biological functions are unknown. In this study, a Cnm-deficient isogenic mutant and a complementation strain were generated from a Cnm-positive S. mutans strain to help determine the properties of Cnm. Initially, comparison of the cell surface structure was performed by electron microscopy, which demonstrated that Cnm appears to be localized on the cell surface and associated with a protruding cell surface structure. Deep RNA sequencing of the strains revealed that the defect in Cnm caused upregulated expression of many genes related to ABC transporters and cell-surface proteins, while a few genes were downregulated. The amount of biofilm formed by the Cnm-defective strain increased compared with the parental and complemented strains, but the biofilm structure was thinner because of elevated expression of genes encoding glucan synthesis enzymes, leading to increased production of extracellular polysaccharides. Particular antibiotics, including bacitracin and chloramphenicol, had a lower minimum inhibitory concentration for the Cnm-defective strain than particular antibiotics, including bacitracin and chloramphenicol, compared with the parental and complemented strains. Our results suggest that S. mutans Cnm is located on the cell surface, gives rise to the observed protruding cell surface, and is associated with several biological properties related to membrane permeability.

Pneumococcal BgaA Promotes Host Organ Bleeding and Coagulation in a Mouse Sepsis Model.

Streptococcus pneumoniae is a major cause of invasive diseases such as pneumonia, meningitis, and sepsis, with high associated mortality. Our previous molecular evolutionary analysis revealed that the S. pneumoniae gene bgaA, encoding the enzyme ß-galactosidase (BgaA), had a high proportion of codons under negative selection among the examined pneumococcal genes and that deletion of bgaA significantly reduced host mortality in a mouse intravenous infection assay. BgaA is a multifunctional protein that plays a role in cleaving terminal galactose in N-linked glycans, resistance to human neutrophil-mediated opsonophagocytic killing, and bacterial adherence to human epithelial cells. In this study, we performed in vitro and in vivo assays to evaluate the precise role of bgaA as a virulence factor in sepsis. Our in vitro assays showed that the deletion of bgaA significantly reduced the bacterial association with human lung epithelial and vascular endothelial cells. The deletion of bgaA also reduced pneumococcal survival in human blood by promoting neutrophil-mediated killing, but did not affect pneumococcal survival in mouse blood. In a mouse sepsis model, mice infected with an S. pneumoniae bgaA-deleted mutant strain exhibited upregulated host innate immunity pathways, suppressed tissue damage, and blood coagulation compared with mice infected with the wild-type strain. These results suggest that BgaA functions as a multifunctional virulence factor whereby it induces host tissue damage and blood coagulation. Taken together, our results suggest that BgaA could be an attractive target for drug design and vaccine development to control pneumococcal infection.

Characterization of Plasmodium falciparum Pantothenate Kinase and Identification of Its Inhibitors From Natural Products.

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum, the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases (PfPanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant PfPanK1 for the first time. PfPanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant PfPanK1 and identified four PfPanK inhibitors from natural compounds.

Role of BgaA as a Pneumococcal Virulence Factor Elucidated by Molecular Evolutionary Analysis.

Streptococcus pneumoniae is a major cause of pneumonia, sepsis, and meningitis. Previously, we identified a novel virulence factor by investigating evolutionary selective pressure exerted on pneumococcal choline-binding cell surface proteins. Herein, we focus on another pneumococcal cell surface protein. Cell wall-anchoring proteins containing the LPXTG motif are conserved in Gram-positive bacteria. Our evolutionary analysis showed that among the examined genes, nanA and bgaA had high proportions of codon that were under significant negative selection. Both nanA and bgaA encode a multi-functional glycosidase that aids nutrient acquisition in a glucose-poor environment, pneumococcal adherence to host cells, and evasion from host immunity. However, several studies have shown that the role of BgaA is limited in a mouse pneumonia model, and it remains unclear if BgaA affects pneumococcal pathogenesis in a mouse sepsis model. To evaluate the distribution and pathogenicity of bgaA, we performed phylogenetic analysis and intravenous infection assay. In both Bayesian and maximum likelihood phylogenetic trees, the genetic distances between pneumococcal bgaA was small, and the cluster of pneumococcal bgaA did not contain other bacterial orthologs except for a Streptococcus gwangjuense gene. Evolutionary analysis and BgaA structure indicated BgaA active site was not allowed to change. The mouse infection assay showed that the deletion of bgaA significantly reduced host mortality. These results indicated that both nanA and bgaA encode evolutionally conserved pneumococcal virulence factors and that molecular evolutionary analysis could be a useful alternative strategy for identification of virulence factors.

Identification of evolutionarily conserved virulence factor by selective pressure analysis of Streptococcus pneumoniae.

Evolutionarily conserved virulence factors can be candidate therapeutic targets or vaccine antigens. Here, we investigated the evolutionary selective pressures on 16 pneumococcal choline-binding cell-surface proteins since Streptococcus pneumoniae is one of the pathogens posing the greatest threats to human health. Phylogenetic and molecular analyses revealed that cbpJ had the highest codon rates to total numbers of codons under considerable negative selection among those examined. Our in vitro and in vivo assays indicated that CbpJ functions as a virulence factor in pneumococcal pneumonia by contributing to evasion of neutrophil killing. Deficiency of cbpL under relaxed selective pressure also caused a similar tendency but showed no significant difference in mouse intranasal infection. Thus, molecular evolutionary analysis is a powerful tool that reveals the importance of virulence factors in real-world infection and transmission, since calculations are performed based on bacterial genome diversity following transmission of infection in an uncontrolled population.

Competence-induced protein Ccs4 facilitates pneumococcal invasion into brain tissue and virulence in meningitis.

Streptococcus pneumoniae is a major pathogen that causes pneumonia, sepsis, and meningitis. The candidate combox site 4 (ccs4) gene has been reported to be a pneumococcal competence-induced gene. Such genes are involved in development of S. pneumoniae competence and virulence, though the functions of ccs4 remain unknown. In the present study, the role of Ccs4 in the pathogenesis of pneumococcal meningitis was examined. We initially constructed a ccs4 deletion mutant and complement strains, then examined their association with and invasion into human brain microvascular endothelial cells. Wild-type and Ccs4-complemented strains exhibited significantly higher rates of association and invasion as compared to the ccs4 mutant strain. Deletion of ccs4 did not change bacterial growth activity or expression of NanA and CbpA, known brain endothelial pneumococcal adhesins. Next, mice were infected either intravenously or intranasally with pneumococcal strains. In the intranasal infection model, survival rates were comparable between wild-type strain-infected and ccs4 mutant strain-infected mice, while the ccs4 mutant strain exhibited a lower level of virulence in the intravenous infection model. In addition, at 24 hours after intravenous infection, the bacterial burden in blood was comparable between the wild-type and ccs4 mutant strain-infected mice, whereas the wild-type strain-infected mice showed a significantly higher bacterial burden in the brain. These results suggest that Ccs4 contributes to pneumococcal invasion of host brain tissues and functions as a virulence factor.

Evolutionary inactivation of a sialidase in group B Streptococcus.

Group B Streptococcus (GBS) is a leading cause of bacterial sepsis and meningitis in newborns. GBS possesses a protein with homology to the pneumococcal virulence factor, NanA, which has neuraminidase (sialidase) activity and promotes blood-brain barrier penetration. However, phylogenetic sequence and enzymatic analyses indicate the GBS NanA ortholog has lost sialidase function - and for this distinction we designate the gene and encoded protein nonA/NonA. Here we analyze NonA function in GBS pathogenesis, and through heterologous expression of active pneumococcal NanA in GBS, potential costs of maintaining sialidase function. GBS wild-type and ΔnonA strains lack sialidase activity, but forced expression of pneumococcal NanA in GBS induced degradation of the terminal sialic acid on its exopolysaccharide capsule. Deletion of nonA did not change GBS-whole blood survival or brain microvascular cell invasion. However, forced expression of pneumococcal NanA in GBS removed terminal sialic acid residues from the bacterial capsule, restricting bacterial proliferation in human blood and in vivo upon mouse infection. GBS expressing pneumococcal NanA had increased invasion of human brain microvascular endothelial cells. Thus, we hypothesize that nonA lost enzyme activity allowing the preservation of an effective survival factor, the sialylated exopolysaccharide capsule.

CD4 and CD8 homologues in Japanese flounder, Paralichthys olivaceus: Differences in the expressions and localizations of CD4-1, CD4-2, CD8α and CD8ß.

CD4 and CD8 molecules are co-receptors of T cell receptors which interact specifically with MHC class II and I, respectively, during antigen presentation. Here we investigated CD4 and CD8 expression patterns in a fish, Japanese flounder, Paralichthys olivaceus in response to infection and tuberculin injection. The CD4-1 mRNA level was gradually and weakly increased in trunk kidney after infection with Streptococcus iniae, Edwardsiella tarda and viral hemorrhagic septicemia virus (VHSV), while the CD4-2 mRNA level was dramatically increased after E. tarda and VHSV infection, but not increased after S. iniae infection. CD4-2 mRNA but not CD4-1mRNA increased in the kidney during tuberculin response which is mediated by memory Th1 cells. The patterns for the change of mRNA level in CD8α and CD8ß were similar to those of the CD4-2 during the infections and tuberculin response. Fluorescent in situ hybridization detected CD4-1 mRNAs on melano-macrophage centers and CD4-2 mRNAs at some cell clusters located near the melano-macrophage centers. CD8α and CD8ß mRNAs were detected at the same cell clusters in the spleen and head kidney. These results suggest that CD4-1 and CD4-2 are expressed in different cells and that CD4-2-positive cells, rather than CD4-1-positive cells, have a main role in Th1-related immune responses collaborating with CD8α- and CD8ß-positive cells in Japanese flounder.

Relationship between peak VO2 and subcutaneous fat thickness of the thigh measured by ultrasonography.

BACKGROUND: Measured physical activity and percent body fat obtained by bioelectrical impedance analysis have been used as indicators of cardiorespiratory fitness in middle-aged women and children. However, the relationship between subcutaneous fat thickness in various regions of the thigh determined by ultrasonography and cardiorespiratory fitness has not been previously reported. METHODS: We measured subcutaneous fat thickness in the frontal, lateral, and medial regions of both thighs using ultrasonography, and also determined peak VO2 and percent body fat in 13 healthy and generally well female college students. RESULTS: There were significant negative correlations found between peak VO2 and the 3 frontal portions of the thigh, as well as the vastus lateralis (both sides), while correlations were not seen with the right and left medial regions 15 cm above the medial epicondyle. CONCLUSIONS: Our results suggest that increases in peak VO2 are reflected by decreases in subcutaneous fat thickness in the frontal and lateral regions of the thigh, but not in decreases in that in both sides of the medial region of the thigh. In addition, subcutaneous fat thickness may indicate partial or segmental activation of the frontal and lateral regions of the legs, such as that obtained by cycling. We concluded that measurement of subcutaneous fat thickness in the region above the muscle mass of the thigh such as the rectus femoris or lateral vastus using ultrasonography may reasonably represent physical activity. Further study is needed to be done with larger groups of subjects and to evaluate the effects of training on activation of regions of the thigh.