Candidatus Kaistella beijingensis sp. nov., Isolated from a Municipal Wastewater Treatment Plant, Is Involved in Sludge Foaming.

Biological foaming (or biofoaming) is a frequently occurring problem in wastewater treatment plants (WWTPs) and is attributed to the overwhelming growth of filamentous bulking and foaming bacteria (BFB). Biological foaming has been intensively investigated, with BFB like Microthrix and Skermania having been identified from WWTPs and implicated in foaming. Nevertheless, studies are still needed to improve our understanding of the microbial diversity of WWTP biofoams and how microbial activities contribute to foaming. In this study, sludge foaming at the Qinghe WWTP of China was monitored, and sludge foams were investigated using culture-dependent and culture-independent microbiological methods. The foam microbiomes exhibited high abundances of Skermania, Mycobacterium, Flavobacteriales, and Kaistella. A previously unknown bacterium, Candidatus Kaistella beijingensis, was cultivated from foams, its genome was sequenced, and it was phenotypically characterized. Ca. K. beijingensis exhibits hydrophobic cell surfaces, produces extracellular polymeric substances (EPS), and metabolizes lipids. Ca. K. beijingensis abundances were proportional to EPS levels in foams. Several proteins encoded by the Ca. K. beijingensis genome were identified from EPS that was extracted from sludge foams. Ca. K. beijingensis populations accounted for 4 to 6% of the total bacterial populations in sludge foam samples within the Qinghe WWTP, although their abundances were higher in spring than in other seasons. Cooccurrence analysis indicated that Ca. K. beijingensis was not a core node among the WWTP community network, but its abundances were negatively correlated with those of the well-studied BFB Skermania piniformis among cross-season Qinghe WWTP communities. IMPORTANCE Biological foaming, also known as scumming, is a sludge separation problem that has become the subject of major concern for long-term stable activated sludge operation in decades. Biological foaming was considered induced by foaming bacteria. However, the occurrence and deterioration of foaming in many WWTPs are still not completely understood. Cultivation and characterization of the enriched bacteria in foaming are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the understanding of their relationships with foaming and performance of WWTPs.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

[Mechanism of hepatocyte transformation by HCV NS3 using two-dimensional electrophoresis and mass spectrometry].

OBJECTIVE: To investigate the proteome of hepatocyte transformation by hepatitis C virus (HCV) nonstructural protein 3 (NS3). METHODS: Human hepatocyte line QSG7701 stably expressing HCV NS3 C-terminal deleted protein was constructed, which was named pRcHCNS3/QSG. Two-dimensional electrophoresis (2-DE) was used to separate the total protein of pRcHCNS3/QSG and pRcCMV transfected cells (pRcCMV/QSG) respectively. Differentially expressed protein spots were identified by mass spectrometry. Western blot confirmed the differentially expressed proteins. RESULTS: 2-DE profiles with high resolution and reproducibility were obtained. The average spots of pRcHCNS3/QSG and pRcCMV/QSG were (1183+/-77) and (1095+/-82) respectively, and (920+/-60) spots were matched. Twenty-one differentially expressed protein spots were chosen randomly and 15 were identified by mass spectrometry. Some proteins such as Ras, P38 and HD53 which were involved in signal transduction were increased in pRcHCNS3/QSG cells. Western blot also showed strong expression of phosphorylated P44/42 and P38 in pRcHCNS3/QSG cells. Other differentially expressed proteins were related to cell cycle regulation, immunoreaction, tumor invasion and metastasis, and liver metabolizability. CONCLUSION: HCV NS3 might be involved in cell malignant transformation through affecting protein expression and signal transduction such as MAPK cascade. Further study on the signal transductions and their relationship would not only be helpful to explore the mechanism of HCV related HCC, but also provide a new idea for the molecular treatment of HCC.

[Cross-talk between ERK and NF-kappaB signal transduction pathways in the hepatocytes expressing hepatitis C virus nonstructural protein 3].

OBJECTIVE: To explore the cross-talk between extracellular signal-regulated kinase (ERK) and nuclear factor (NF-kappaB) signal transduction pathways in the hepatocytes expressing hepatitis C virus nonstructural protein 3 (HCV NS3). METHODS: A cell line QSG7701/NS3, which stably expressed HCV NS3 protein, was constructed by transfecting plasmid pcDNA3.1-NS3 into a human immortalized hepatocyte line QSG7701. Before and after QSG7701/NS3 cells were treated by MEK inhibitor PD98059, the phosphorylation level of ERK and the expression of cyclin D1 protein were detected by Western blot; the DNA binding activities of activator protein 1 (AP-1) and NF-kappaB were evaluated with electrophoretic mobility shift assay (EMSA). Cell cycles were measured by flow cytometry (FCM); the effects of PD98059 on the cell proliferation were determined by MTT assay. RESULTS: HCV NS3 protein could up-regulate the phosphorylation of ERK and the expression level of cyclin D1 in QSG7701 cells. PD98059 could inhibit the cell proliferation mediated by HCV NS3 protein, down-regulate the activities of AP-1 and NF-kappaB, and suppress the expression of cyclin D1. CONCLUSION: The inhibition of ERK can suppress the DNA binding activity of NF-kappaB and the cell proliferation mediated by HCV NS3 protein in a dose-dependent manner. There may be a cross-talk between ERK and NF-kappaB signal transduction pathways, which may exert synergistic action on the proliferation and malignant transformation of hepatocytes.

[The effects of hepatitis C virus core protein on biological behaviors of human hepatocytes].

OBJECTIVE: To investigate the effects of hepatitis C virus (HCV) core protein on the biological behaviors of human hepatocytes and their underlying mechanism. METHODS: A cell line expressing stably HCV core protein-QSG7701/core was constructed by transfecting the plasmid pcDNA3.1-core (expressing HCV core protein) into the human immortalized hepatocytes of the line QSG7701. The biological behaviors of these transfected cells were observed through plating-efficiency test, growth curve and flow cytometry (FCM). The association between HCV core protein and the expression of activated caspase-3 protein was evaluated by immunocytochemistry. The phosphorylation of mitogen-activate protein kinases (MAPKs) was detected with Western blotting. The activation of nuclear transcriptors AP-1, important effector molecule of MAPKs, and nuclear factor-kappa binding (NF-kappaB) were evaluated with luciferase assays and electrophoretic mobility shift assay (EMSA). RESULTS: HCV core protein was expressed in the QSG7701/core cells and not in the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. There were no significant differences in the expression levels of total P44/42(MAPK), p38(MAPK) and JNK among the QSG7701/core cells, QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. The expression levels of phophorylated P44/42(MAPK), p38(MAPK) and JNK in the QSG7701/core cells were significantly weaker than those in the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. Plating efficiency test showed that the clone formation rate of the QSG7701/core cells was 32.25%, significantly lower than those of QSG7701/pcDNA3.1 and untransfected QSG7701 cells (47.5% and 42.5% respectively, both P < 0.01). The growth curve showed that the multiplication time of the QSG7701/core cells was 36 hours, significantly longer than those of the QSG7701/pcDNA3.1 and untransfected QSG7701 cells (27 and 28 hours respectively). FCM showed that the apoptotic rate of the QSG770/1core was 1.04%, lower than those of the QSG7701/pcDNA3.1 and untransfected QSG7701 cells (1.68% and 3.7% respectively), and that the percentage of theQSG770/1core cells at the G(0)/G(1) stage increased and those in the S stage decreased. Immunocytochemistry showed that the expression intensity of caspase-3 in the QSG7701/core cells was significantly weaker than those of the QSG7701/pcDNA3.1 cells and untransfected QSG7701 cells. CONCLUSION: HCV core protein suppresses cell proliferation and apoptosis by downregmicrolating the phosphorylation of MAPKs and activating the transcriptors AP-1 and NF-kappaB, thus promoting the persistency of HCV infection which leads to chronic hepatitis C and hepatocellular cancer.

Effect of hepatitis C virus nonstructural protein NS3 on proliferation and MAPK phosphorylation of normal hepatocyte line.

AIM: To study the effect of hepatitis C virus nonstructural region 3 (HCV NS3) protein on proliferation and transformation of normal human liver cell line. METHODS: QSG7701 cells were transfected with pRcHCNS3-5', pRcHCNS3-3' and pRcCMV using lipofectamine transfecting technique and selected with G418 method. Expression of HCV NS3 protein was determined by immunohistochemistry. Biologic characteristics of transfected cells were evaluated by population doubling time and soft agar assays. Activation of MAPK was analyzed using Western blot with phosphospecific monoclonal antibody against dually phosphorylated MAPK. RESULTS: QSG7701 cells transfected with pRcHCNS3-5' showed strong intracellular expression of HCVNS3 protein, and the positive signal was localized in cytoplasm. The expressing strength of HCVNS3 protein in pRcHCNS3-3'-transfected cells was weaker than that in pRcHCNS3-5'-transfected cells. The population doubling time in the transfected cells with pRcHCNS3-5' (12 h) was much shorter than those with pRcHCNS3-3', pRcCMV and normal cells (24, 26, 28 h, respectively) (P<0.01). The transfected cells with pRcHCNS3-5' showed much more anchorage independent colonies than that in those with pRcHCNS3-3' and pRcCMV (P<0.01). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcHCNS3-3', pRcCMV and controls were 33%, 1.33%, 1.46%, 1.11% respectively. The level of phosphorylated MAPK in the cells with pRcHCNS3-5' was much higher than that in those with pRcHCNS3-3'and pRcCMV and normal cells (P<0.01). CONCLUSION: The results suggest that (1) QSG7701 cells are a better human liver cell line for investigating the pathogenesis of HCV NS3 protein. (2) 5' region of the HCV genome segment encoding HCV NS3 is involved in cell growth and cell phenotype. (3) HCV NS3 N-terminal peptide may up-regulate the activation of MAPK, but not affect the expression of MAPK.

[Regulation of hepatitis C virus core protein on the activity of signal transducers and activators of transcription 3].

OBJECTIVE: To investigate the regulation of hepatitis C virus (HCV) core protein on the activity of signal transducers and activators of transcription 3 (stat3). METHODS: A cell line expressing stable HCV core protein-QSG7701-core was constructed by transfecting the pcDNA3. 1-core (expressing HCV core protein) into the human immortalized hepatocyte line QSG7701. The phosphorylation and DNA binding activity of stat3 were detected by immunocytochemistry, Western blot, and electrophoretic mobility shift assay (EMSA). RESULTS: The expression level of phosphorylated stat3 in QSG7701-core cells was significantly lower than that in QSG7701-pcDNA3. 1 cells and untransfected QSG7701 cells, but there were no significant differences in the expression levels of total stat3 among the 3 groups. The positive signal of phosphorylated stat3 in nucleus of QSG7701-core cells was obviously weaker than that in QSG7701-pcDNA3. 1 cells and untransfected QSG7701 cells. EMSA showed that DNA binding activity of stat3 in QSG7701-core cells significantly decreased. CONCLUSION: The expressionof HCV core protein in human hepatocyte line may suppress the phosphorylation and DNA binding activity of stat3, which may be one of the causes for resistance against interferon.

[Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 N-terminal protein].

OBJECTIVE: To study the effect of hepatitis C virus nonstructural protein 3 N-terminal protein (HCV NS3-5') on hepatocyte transformation and tumor development. METHODS: QSG7701 cells were transfected with plasmid pRcHCNS3-5' (expressing HCV NS3 N-terminal protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological effect of transfected cells was observed through cell proliferation assay, anchor independent growth, and tumor development in nude mice. The expression of HCV NS3 and c-myc protein in the induced tumor was evaluated by immunohistochemistry. RESULTS: HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5' and the positive signal was located in cytoplasm. The HCV NS3 expression and c-myc protein in the induced cytoplasm. Cell proliferation assay showed that the population doubling time in the pRcHCNS3-5' transfected cells was much shorter than that in the pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcCMV and non-transfected cells were 33.0%, 1.5%, 1.1% respectively (P < 0.01). Tumor developed in nude mice inoculated with pRcHCNS3-5'transfected cells 15 days after the inoculation. HE staining showed hepatocarcinoma character and immunohistochemistry confirmed HCV NS3 and c-myc expression in the tumor tissue. The positive control group also showed tumor development, while no tumor mass obtained in the nude mice inoculated with pRcCMV and non-transfected cells even 40 days after the injection. CONCLUSION: HCV NS3 N-terminal protein showed cell transformation and tumorigenic features.

Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 c-terminal deleted protein.

AIM: To study the effect of hepatitis C virus nonstructural protein 3 c-terminal deleted protein (HCV NS3-5') on hepatocyte transformation and tumor development. METHODS: QSG7701 cells were transfected with plasmid pRcHCNS3-5' (expressing HCV NS3 c-terminal deleted protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological behavior of transfected cells was observed through cell proliferation assay, anchorage-independent growth and tumor development in nude mice. The expression of HCV NS3 and c-myc proteins in the induced tumor was evaluated by immunohistochemistry. RESULTS: HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5' and the positive signal was located in cytoplasm. Cell proliferation assay showed that the population doubling time in pRcHCNS3-5' transfected cells was much shorter than that in pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning ratio of cells transfected with pRcHCNS3-5', pRcCMV and non-transfected cells was 33 %, 1.46 %, 1.11 %, respectively, the former one was higher than that in the rest two groups (P<0.01). Tumor development was seen in nude mice inoculated with pRcHCNS3-5' transfected cells after 15 days. HE staining showed its feature of hepatocarcinoma, and immunohistochemistry confirmed the expressions of HCV NS3 and c-myc proteins in tumor tissue. The positive control group inoculated with HepG2 also showed tumor development, while no tumor developed in the nude mice injected with pRcCMV and non-transfected cells after 40 days. CONCLUSION: 1.HCV NS3 c-terminal deleted protein has transforming and oncogenic potential. 2. Human liver cell line QSG7701 may be used as a good model to study HCV NS3 pathogenesis.

[Expression of TGF beta 1 and TGF beta RI proteins in tissues of salivary adenoid cystic carcinoma].

OBJECTIVE: To determine the possible role of TGF beta 1 and TGF beta RI in facilitating neoplastic progression in human salivary adenoid cystic carcinoma (ACC). METHODS: The expression of TGF beta 1 and TGF beta RI protein in human salivary adenoid cystic carcinoma was detected by the streptavidin-peroxidase (S-P) immunohistochemical method, and their contents were compared with those of normal salivary gland tissues and salivary pleomorphic adenoma (PA). RESULTS: 1. TGF beta 1 and TGF beta RI proteins were expressed in most ductal epithelial cells and myo-epithelial cells, but they were not expressed in the acinar epithelial cells of any normal glands examined; 2. TGF beta 1 and TGF beta RI proteins were expressed in the duct-like arranging tumor cells and other solid proliferating tumor cells of salivary pleomorphic adenoma, and no difference of their contents existed between PA and normal salivary gland tissues (P > 0.05); 3. The contents of TGF beta 1 in ACC were significantly higher than those in normal salivary glands (P < 0.01), while the contents of TGF beta RI in ACC were significantly lower than those in normal salivary glands (P < 0.01). CONCLUSION: The abnormal expression of TGF beta 1 and TGF beta RI might play an important role in tumor genesis and the development of malignant salivary gland tumors such as ACC; the over-expression of TGF beta 1 and low-expression of TGF beta RI might involve the bad biological behavior of ACC.

[Relationship between the expression of PTEN protein and phosphorylation of MAPK in hepatocellular carcinomas and their surrounding liver tissues].

OBJECTIVE: To investigate the relationship between the expression of phosphatase and tensin homolog deleted on chromosome ten(PTEN) protein and phosphorylation of mitogen-activated protein kinase (MAPK) in hepatocellular carcinomas (HCC) and their surrounding liver tissues. METHODS: The expression of PTEN and the phosphorylation of MAPK were detected by the SP immunohistochemical technique in 75 cases of HCC and their pericarcinomatous tissues. RESULTS: The positive rate (62.7%) and the expression intensity of PTEN in HCC were lower and weaker than those in the pericarcinomatous liver tissues (89.3%)(P < 0.01), and the expression level of PTEN was significantly related to the differentiation degree of HCC. The poorer the differentiation of HCC, the weaker the expression of PTEN protein. The positive rate (85.3%) and the expression intensity of p42/44MAPK in HCC were higher and stronger than those in the pericarcinomatous liver tissues (34.7%)(P < 0.01), and the expression intensity of p42/44MAPK was not related to the differentiation degree of HCC. A significant negative relationship was observed between the expression intensity of PTEN protein and the phosphorylation level of MAPK in HCC and their surrounding liver tissues. CONCLUSION: The decrease or deletion of PTEN protein expression may not be able to effectively inhibit the over-activation of Ras/Raf/MAPK signaling pathway, and this will result in abnormal proliferation and malignant transformation of hepatocytes in hepatocellular carcinogenesis.