The exoprotein Gbp of Fusobacterium nucleatum promotes THP-1 cell lipid deposition by binding to CypA and activating PI3K-AKT/MAPK/NF-κB pathways.

INTRODUCTION: Growing evidence has shown the correlation between periodontitis and atherosclerosis, while our knowledge on the pathogenesis of periodontitis-promoting atherosclerosis is far from sufficient. OBJECTIVES: Illuminate the pathogenic effects of Fusobacterium nucleatum (F. nucleatum) on intracellular lipid deposition in THP-1-derived macrophages and elucidate the underlying pathogenic mechanism of how F. nucleatum promoting atherosclerosis. METHODS AND RESULTS: F. nucleatum was frequently detected in different kinds of atherosclerotic plaques and its abundance was positively correlated with the proportion of macrophages. In vitro assays showed F. nucleatum could adhere to and invade THP-1 cells, and survive continuously in macrophages for 24 h. F. nucleatum stimulation alone could significantly promote cellular inflammation, lipid uptake and inhibit lipid outflow. The dynamic gene expression of THP-1 cells demonstrated that F. nucleatum could time-serially induce the over-expression of multiple inflammatory related genes and activate NF-κB, MAPK and PI3K-AKT signaling pathways. The exoprotein of F. nucleatum, D-galactose-binding protein (Gbp), acted as one of the main pathogenic proteins to interact with the Cyclophilin A (CypA) of THP-1 cells and induced the activation of the NF- κB, MAPK and PI3K-AKT signaling pathways. Furthermore, use of six candidate drugs targeting to the key proteins in NF- κB, MAPK and PI3K-AKT pathways could dramatically decrease F. nucleatum induced inflammation and lipid deposition in THP-1 cells. CONCLUSIONS: This study suggests that the periodontal pathogen F. nucleatum can activate macrophage PI3K-AKT/MAPK/NF-κB signal pathways, promotes inflammation, enhances cholesterol uptake, reduces lipid excretion, and promotes lipid deposition, which may be one of its main strategies promoting the development of atherosclerosis.

Pangenomic Study of Fusobacterium nucleatum Reveals the Distribution of Pathogenic Genes and Functional Clusters at the Subspecies and Strain Levels.

Fusobacterium nucleatum is a prevalent periodontal pathogen and is associated with many systemic diseases. Our knowledge of the genomic characteristics and pathogenic effectors of different F. nucleatum strains is limited. In this study, we completed the whole genome assembly of the 4 F. nucleatum strains and carried out a comprehensive pangenomic study of 30 strains with their complete genome sequences. Phylogenetic analysis revealed that the F. nucleatum strains are mainly divided into 4 subspecies, while 1 of the sequenced strains was classified into a new subspecies. Gene composition analysis revealed that a total of 517 "core/soft-core genes" with housekeeping functions widely distributed in almost all the strains. Each subspecies had a unique gene cluster shared by strains within the subspecies. Analysis of the virulence factors revealed that many virulence factors were widely distributed across all the strains, with some present in multiple copies. Some virulence genes showed no consistent occurrence rule at the subspecies level and were specifically distributed in certain strains. The genomic islands mainly revealed strain-specific characteristics instead of subspecies level consistency, while CRISPR types and secondary metabolite biosynthetic gene clusters were identically distributed in F. nucleatum strains from the same subspecies. The variation in amino acid sites in the adhesion protein FadA did not affect the monomer and dimer 3D structures, but it may affect the binding surface and the stability of binding to host receptors. This study provides a basis for the pathogenic study of F. nucleatum at the subspecies and strain levels. IMPORTANCE We used F. nucleatum as an example to analyze the genomic characteristics of oral pathogens at the species, subspecies, and strain levels and elucidate the similarities and differences in functional genes and virulence factors among different subspecies/strains of the same oral pathogen. We believe that the unique biological characteristics of each subspecies/strain can be attributed to the differences in functional gene clusters or the presence/absence of certain virulence genes. This study showed that F. nucleatum strains from the same subspecies had similar functional gene compositions, CRISPR types, and secondary metabolite biosynthetic gene clusters, while pathogenic genes, such as virulence genes, antibiotic resistance genes, and GIs, had more strain level specificity. The findings of this study suggest that, for microbial pathogenicity studies, we should carefully consider the subspecies/strains being used, as different strains may vary greatly.

Study of the inflammatory activating process in the early stage of Fusobacterium nucleatum infected PDLSCs.

Fusobacterium nucleatum (F. nucleatum) is an early pathogenic colonizer in periodontitis, but the host response to infection with this pathogen remains unclear. In this study, we built an F. nucleatum infectious model with human periodontal ligament stem cells (PDLSCs) and showed that F. nucleatum could inhibit proliferation, and facilitate apoptosis, ferroptosis, and inflammatory cytokine production in a dose-dependent manner. The F. nucleatum adhesin FadA acted as a proinflammatory virulence factor and increased the expression of interleukin(IL)-1ß, IL-6 and IL-8. Further study showed that FadA could bind with PEBP1 to activate the Raf1-MAPK and IKK-NF-κB signaling pathways. Time-course RNA-sequencing analyses showed the cascade of gene activation process in PDLSCs with increasing durations of F. nucleatum infection. NFκB1 and NFκB2 upregulated after 3 h of F. nucleatum-infection, and the inflammatory-related genes in the NF-κB signaling pathway were serially elevated with time. Using computational drug repositioning analysis, we predicted and validated that two potential drugs (piperlongumine and fisetin) could attenuate the negative effects of F. nucleatum-infection. Collectively, this study unveils the potential pathogenic mechanisms of F. nucleatum and the host inflammatory response at the early stage of F. nucleatum infection.

[Biology behavior of head and neck squamous cell cancer cells changes after knocking down heat shock protein 27].

OBJECTIVE: This study aimed to observe the metastatic behavior of head and neck squamous cell carcinoma cells after knocking down heat shock protein (Hsp) 27. METHODS: The experiment was divided into three groups: the lentivirus vector plasmid of pLenti-shRNA-Hsp27 was transfected into UM-SCC-22B cells as experimental group (shHsp27 group), routine culture of UM-SCC-22B cells as blank control (ctrl group), UM-SCC-22B cells transfection of pLenti-shRNA-ctrl lentivirus vector as negative control (shctrl group). Through real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay to detect the mRNA expression of Hsp27 in three groups. MTS assay was performed to detect cell-proliferation changes, wounding healing assay was performed to detect cell-migration changes, and Matrigel Transwell invasion assay was performed to detect cell-invasion changes. RESULTS: The expression of Hsp27 in shHsp27 group decreased signifi-cantly; MTS assay showed that UM-SCC-22B before and after Hsp27 knockdown had similar proliferation rates after being cultured for 24 or 48 h. Compared with the ctrl group, the shHsp27 group decreased the metastatic behavior by 4.38-fold in migration and 2.03-fold in cell invasion. CONCLUSIONS: Stably transfected lentivirus vector plasmid of pLenti-shRNA-Hsp27 can efficiently decrease Hsp27 expression and reduce the metastasis ability of UM-SCC-22B.

Rapamycin and hydroxychloroquine combination alters macrophage polarization and sensitizes glioblastoma to immune checkpoint inhibitors.

INTRODUCTION: The failure of immune checkpoint inhibitor (ICPi) on glioblastoma (GBM) treatment underscores the need for improving therapeutic strategy. We aimed to change tumor associated macrophage (TAM) from M2 type (anti-inflammatory) to M1 (pro-inflammatory) type to increase the therapeutic response of ICPi. We proposed that combined rapamycin (R) and hydroxychloroquine (Q) preferentially induce M2 cells death, as fatty acid oxidation was their major source of energy. METHODS: Macrophage polarization was characterized on mice and human macrophage cell lines by specific cytokines stimulation with or without RQ treatment under single culture or co-culture with GBM cell lines. Tumor sizes were evaluated on subcutaneous and intracranial GL261 mice models with or without RQ, anti-PD1 mAb treatment. Tumor volumes assessed by MRI scan and proportions of tumor infiltrating immune cells analyzed by flow cytometry were compared. RESULTS: In vitro RQ treatment decreased the macrophages polarization of M2, increased the phagocytic ability, and increased the lipid droplets accumulation. RQ treatment decreased the expression levels of CD47 and SIRPα on tumor cells and macrophage cells in co-culture experiments. The combination of RQ and anti-PD1 treatment was synergistic in action. Enhanced the intra-tumoral M1/M2 ratio, the CD8/CD4 ratio in the intracranial GL261 tumor model after RQ treatment were evident. CONCLUSION: We provide a rationale for manipulating the macrophage phenotype and increased the therapeutic effect of ICPi. To re-educate and re-empower the TAM/microglia opens an interesting avenue for GBM treatment.

Application of 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolyte in capillary zone electrophoresis for the simultaneous determination of five anthraquinones in Rhubarb.

A capillary zone electrophoresis method using only 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolyte for the simultaneous determination of five anthraquinone derivatives including aloe-emodin, emodin, chrysophanol, physcion and rhein in Rhubarb species was described. Ion association constants, K(ass), between anthraquinone anions and imidazolium cations were determined by analyzing the electrophoretic mobility change of anthraquinone anions using a non-linear least-squares method and factors contributing to ion associability were systematically clarified. For method optimization, several parameters such as ionic liquids concentration, background electrolyte pH and applied voltage, on the separation were evaluated and the optimum conditions were obtained as follows: 90mM 1-butyl-3-methylimidazolium tetrafluoroborate (pH 11.0) with an applied voltage of 20kV. Under these conditions, the method has been successfully applied to the determination of anthraquinones in extracts of two kinds of Rhubarb plants (R. palmatum and R. hotaoense) within 12min. The method proposed herein was shown to be much simpler than the previously reported methods.