RESUMO
Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.
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Hepatite B/patologia , Hepatite C/patologia , Cirrose Hepática/patologia , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Actinas/biossíntese , Adulto , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Coinfecção/patologia , Cadeia alfa 1 do Colágeno Tipo I/biossíntese , Feminino , Técnicas de Inativação de Genes , Hepacivirus/metabolismo , Células Estreladas do Fígado/patologia , Células Estreladas do Fígado/virologia , Vírus da Hepatite B/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Fígado/patologia , Cirrose Hepática/virologia , Masculino , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Inibidor Tecidual de Metaloproteinase-1/biossínteseRESUMO
Lactiplantibacillus plantarum T1 is an isolated probiotic lactic acid bacterium (LAB) from pickled vegetables in Chongqing, China. In this study, we evaluated the anti-inflammatory activity and the underlying mechanisms of L. plantarum T1 cell-free supernatant (CFS) on lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages in vitro. Reverse transcription quantitative PCR (RT-qPCR), immunofluorescence, Griess methods, and western blotting were utilized to assess the anti-inflammatory cytokines and antioxidative effect of L. plantarum T1 CFS. Our results showed that L. plantarum T1 CFS pretreatment significantly reduced pro-inflammatory cytokine levels, including nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor, interleukin (IL)-1ß, and IL-6, as well as reactive oxygen species. Interestingly, L. plantarum T1 CFS unregulated the antioxidant indicators, including superoxide dismutase, catalase, and glutathione in RAW264.7 cells. Furthermore, L. plantarum T1 CFS activated the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway. This study showed the excellent antioxidant and anti-inflammatory properties of L. plantarum T1 through multiple pathways, highlighting its potential for further research and application as a probiotic strain.IMPORTANCEL. plantarum T1 stood out in a series of acid and bile salt tolerance and bacterial inhibition tests as a probiotic isolated from paocai, which provides many health benefits to the host by inhibiting the growth of harmful pathogenic microorganisms and suppressing excessive levels of oxidative stress and inflammation. Not all LAB have good probiotic functions and are used in various applications. The anti-inflammatory antioxidant potential and mechanisms of L. plantarum T1 CFS have not been described and reported. By using RT-qPCR, Griess method, and western blotting, we showed that L. plantarum T1 CFS had anti-inflammatory and antioxidant effects. Griess assay, TBA assay, WST-8 assay, immunofluorescence assay, RT-qPCR, and western blotting data revealed that its anti-inflammatory and antioxidant mechanisms were associated with oxidative stress and NF-κB and MAPK signaling pathways. The anti-inflammatory and antioxidant effects of L. plantarum T1 CFS in paocai generates opportunities for probiotic product development.
Assuntos
Antioxidantes , NF-kappa B , Animais , Camundongos , NF-kappa B/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Sistema de Sinalização das MAP Quinases , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Células RAW 264.7 , Inflamação , Citocinas/metabolismo , Estresse Oxidativo , Lipopolissacarídeos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/farmacologiaRESUMO
BACKGROUND: Clostridioides difficile is a bacterium that causes antibiotic-associated infectious diarrhea and pseudomembranous enterocolitis. The impact of C. difficile infection (CDI) in China has gained significant attention in recent years. However, little epidemiological data are available from Chongqing, a city located in Southwest China. This study aimed to investigate the epidemiological pattern of CDI and explore the drug resistance of C. difficile isolates in Chongqing. METHODS: A case-control study was conducted to investigate the clinical infection characteristics and susceptibility factors of C. difficile. The features of the C. difficile isolates were evaluated by testing for toxin genes and using multi-locus sequence typing (MLST). The susceptibility of strains to nine antibiotics was determined using agar dilution technique. RESULTS: Out of 2084 diarrhea patients, 90 were tested positive for the isolation of toxigenic C. difficile strains, resulting in a CDI prevalence rate of 4.32%. Tetracycline, cephalosporins, hepatobiliary disease, and gastrointestinal disorders were identified as independent risk factors for CDI incidence. The 90 strains were classified into 21 sequence types (ST), with ST3 being the most frequent (n = 25, 27.78%), followed by ST2 (n = 10, 11.11%) and ST37 (n = 9, 10%). Three different toxin types were identified: 69 (76.67%) were A+B+CDT-, 12 (13.33%) were A-B+CDT-, and 9 (10%) were A+B+CDT+. Although substantial resistance to erythromycin (73.33%), moxifloxacin (62.22%), and clindamycin (82.22%), none of the isolates exhibited resistance to vancomycin, tigecycline, or metronidazole. Furthermore, different toxin types displayed varying anti-microbial characteristics. CONCLUSIONS: The strains identified in Chongqing, Southwest China, exhibited high genetic diversity. Enhance full awareness of high-risk patients with HA-CDI infection, particularly those with gastrointestinal and hepatocellular diseases, and emphasize caution in the use of tetracycline and capecitabine. These findings suggest that a potential epidemic of CDI may occur in the future, emphasizing the need for timely monitoring.
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Clostridioides difficile , Infecções por Clostridium , Humanos , Clostridioides difficile/genética , Tipagem de Sequências Multilocus , Clostridioides/genética , Centros de Atenção Terciária , Estudos de Casos e Controles , Infecções por Clostridium/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Tigeciclina , Tetraciclina , Diarreia/microbiologia , China/epidemiologia , Testes de Sensibilidade MicrobianaRESUMO
Pigments produced by micro-organisms could contribute to their pathogenesis and resistance. The investigation into the red pigment of R. mucilaginosa and its ability to survive and resist has not yet been explored. This study aimed to investigate the survival and resistance of the R. mucilaginosa CQMU1 strain following inhibition of pigment production by naftifine and its underlying mechanism. The red-pigmented Rhodotorula mucilaginosa CQMU1 yeast was isolated from an infected toenail of a patient with onychomycosis. Cultivation of R. mucilaginosa in liquid and solid medium showed the effect of naftifine after treatment. Then, analysis of phagocytosis and tolerance to heat or chemicals of R. mucilaginosa was used to evaluate the survival and resistance of yeast to different treatments. Naftifine reversibly inhibited the pigmentation of R. mucilaginosa CQMU1 in solid and liquid media. Depigmented R. mucilaginosa CQMU1 showed increased susceptibility toward murine macrophage cells RAW264.7 and reduced resistance toward different types of chemicals, such as 1.5-M NaCl and 0.5% Congo red. Inhibition of pigment production by naftifine affected the survival and growth of R. mucilaginosa and its resistance to heat and certain chemicals. The results obtained could further elucidate the target of new mycosis treatment.
Assuntos
Alilamina , Rhodotorula , Humanos , Animais , Camundongos , Alilamina/farmacologiaRESUMO
BACKGROUND: Severe respiratory and neurological diseases caused by human enterovirus D68 (EV-D68) pose a serious threat to public health, and there are currently no effective drugs and vaccines. Adenosine deaminase acting on RNA1 (ADAR1) has diverse biological functions in various viral infections, but its role in EV-D68 infections remains undetermined. METHODS: Rhabdomyosarcoma (RD) and human embryonic kidney 293 T (293 T) cells, and HeLa cells were used to evaluate the expression level of ADAR1 upon EV-D68 (Fermon strain) and human parainfluenza virus type 3 (HPIV3; NIH47885) infection, respectively. Knockdown through silencing RNA (siRNA) and overexpression of either ADAR1p110 or ADAR1p150 in cells were used to determine the function of the two proteins after viral infection. ADAR1p110 double-stranded RNA binding domains (dsRBDs) deletion mutation was generated using a seamless clone kit. The expression of ADAR1, EV-D68 VP1, and HPIV3 hemagglutinin-neuraminidase (HN) proteins was identified using western blotting. The median tissue culture infectious dose (TCID50) was applied to detect viral titers. The transcription level of EV-D68 mRNA was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and the viral 5'-untranslated region (5'-UTR)-mediated translation was analyzed using a dual luciferase reporter system. CONCLUSION: We found that the transcription and expression of ADAR1 was inhibited upon EV-D68 infection. RNA interference of endogenous ADAR1 decreased VP1 protein expression and viral titers, while overexpression of ADAR1p110, but not ADAR1p150, facilitated viral replication. Immunofluorescence assays showed that ADAR1p110 migrated from the nucleus to the cytoplasm after EV-D68 infection. Further, ADAR1p110 lost its pro-viral ability after mutations of the active sites in the deaminase domain, and 5'-UTR sequencing of the viral genome revealed that ADAR1p110 likely plays a role in EV-D68 RNA editing. In addition, after ADAR1 knockdown, the levels of both phosphorylated double-stranded RNA dependent protein kinase (p-PKR) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α) increased. Attenuated translation activity of the viral genome 5'-UTR was also observed in the dual-luciferase reporter assay. Lastly, the deletion of ADAR1p110 dsRBDs increased the level of p-PKR, which correlated with a decreased VP1 expression, indicating that the promotion of EV-D68 replication by ADAR1p110 is also related to the inhibition of PKR activation by its dsRBDs. Our study illustrates that ADAR1p110 is a novel pro-viral factor of EV-D68 replication and provides a theoretical basis for EV-D68 antiviral research.
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Enterovirus Humano D , Infecções por Enterovirus , Humanos , Células HeLa , Enterovirus Humano D/genética , Replicação Viral , RNA de Cadeia Dupla , Antivirais/farmacologiaRESUMO
Hyaluronidase (HAase) can depolymerize mucopolysaccharide hyaluronic acid (HA) to increase the efficacy of drug diffusion in the case of pathogenic bacteria. Due to its widespread medical applications, HAase originating from microorganisms has attracted significant attention. In this study, the HAase-producing bacterium Bacillus sp. CQMU-D was isolated from soil and identified based on its morphology and 16S rRNA gene sequencing. Enzyme activity was detected by measuring the content of reducing sugar in HA degradation products with 3,5-dinitrosalicylic acid (DNSA) or p-dimethylaminobenzaldehyde (DMAB) as the detection reagent. The results revealed that HAase reached maximum activity after 48 h of cultivation. Gene function annotation after full-length sequencing showed increased transport and metabolic activities associated with HAase. Additionally, the HAase gene of Bacillus sp. CQMU-D was different from the existing microbial HAase, and its protein was predicted to be a stable secretory protein with a conserved GAG_Lyase domain. These results characterized a new HAase-producing Bacillus from the soil via enzyme activity and bioinformatic analysis, expanding the knowledge on Bacillus HAase for potential industrial applications.
Assuntos
Bacillus , Hialuronoglucosaminidase , Bacillus/genética , Bacillus/metabolismo , Ácido Hialurônico , Hialuronoglucosaminidase/genética , Hialuronoglucosaminidase/metabolismo , Proteínas , RNA Ribossômico 16S/genética , Solo , AçúcaresRESUMO
Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Interferon-alfa/fisiologia , RNA Longo não Codificante/fisiologia , Células Cultivadas , HumanosRESUMO
Trichosporon asahii and Rhodotorula mucilaginosa are important fungal species causing disseminated disease in immunocompromised patients. Onychomycosis prevalence rate ranges from 2 to 30%, which were 50% of nail diseases and 30% of superficial mycosis, respectively. Although important, little is known about the co-habitation of T. asahii and R. mucilaginosa in the causation of onychomycosis. Here, we present the co-habitation of T. asahii and R. mucilaginosa as causative agents of onychomycosis in a healthy 41-year-old male in China. Direct microscopic examination, fungal culture and MALDI-TOF MS were employed in isolated pathogens; hence, antifungal susceptibility test was evaluated. T. asahii was sensitive to posaconazole, voriconazole and itraconazole, whereas R. mucilaginosa was sensitive to both 5-flucytosine and amphotericin B. This report highlights the co-habitation of T. asahii and R. mucilaginosa in the causation of onychomycosis and to raise the awareness of this infection among dermatologists.
Assuntos
Coinfecção , Unhas , Rhodotorula , Trichosporon , Adulto , Antifúngicos/farmacologia , Coinfecção/tratamento farmacológico , Coinfecção/microbiologia , Dermatomicoses/tratamento farmacológico , Farmacorresistência Fúngica , Humanos , Masculino , Testes de Sensibilidade Microbiana , Unhas/microbiologia , Unhas/patologia , Onicomicose/tratamento farmacológico , Onicomicose/microbiologia , Rhodotorula/efeitos dos fármacos , Rhodotorula/isolamento & purificação , Trichosporon/efeitos dos fármacos , Trichosporon/isolamento & purificação , Tricosporonose/tratamento farmacológico , Tricosporonose/microbiologiaRESUMO
Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication.IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.
Assuntos
Regulação da Expressão Gênica , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Hepatite C/metabolismo , MicroRNAs/metabolismo , Replicação Viral/fisiologia , Linhagem Celular Tumoral , Hepatite B/genética , Hepatite B/patologia , Hepatite C/genética , Hepatite C/patologia , Humanos , MicroRNAs/genética , Piruvato Quinase/genética , Piruvato Quinase/metabolismoRESUMO
Hepatitis B vaccination prevents 80-95% of transmission and reduces the incidence of HBV in children. The variations in the a determinant of HBV surface antigen (HBsAg) have been reported to be the most prevalent cause for vaccine or antibody escape. There is a conflicting evidence on as to whether escape mutants arise de novo in infected infants or whether the mutants, that have preexisted maternally, subsequently undergo selective replication in the infant under immune pressure. Here, we report that nearly 65% (55 of 85) vaccination failure in child patients has no amino acid substitution in a determinant as seen by Sanger sequencing. We further employed an Illumina sequencing platform-based method to detect HBV quasispecies in four immunoprophylaxis failure infants and their mothers. In our data, the substitution rate of amino acid located at a determinant is relatively low (< 10%), I/T126A, C124S, F134Y, K141Q, Q129H, D144A, G145V, and N146K, which showed no statistical difference to their mothers, proving that these vaccine escape mutants preexist maternally as minor variants. Besides that, bioinformatical analysis showed that the binding affinity of high variation epitopes (amino acid divergence in mother and their infants > 20%) to related HLA molecules was generally decreased, these traces of immune escape suggesting that immune pressure was present and was effective in all samples.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Imunização/efeitos adversos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Quase-Espécies/genética , Substituição de Aminoácidos/imunologia , Anticorpos Antivirais/biossíntese , Criança , Pré-Escolar , DNA Viral , Feminino , Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Evasão da Resposta Imune , Lactente , Masculino , Mães , Mutação , Gravidez , Complicações Infecciosas na Gravidez/epidemiologiaRESUMO
Adenosine deaminases acting on RNA 1(ADAR1), an RNA editing enzyme that converts adenosine to inosine by deamination in double-stranded RNAs, plays an important role in occurrence and progression of various types of cancer. Ferroptosis has emerged as a hot topic of cancer research in recent years. We have previously reported that ADAR1 promotes breast cancer progression by regulating miR-335-5p and METTL3. However, whether ADAR1 has effects on ferroptosis in breast cancer cells is largely unknown. In this study, we knocked down ADAR1 using CRISPR-Cas9 technology or over-expressed ADAR1 protein using plasmid expressing ADAR1 in MCF-7 and MDA-MB-231 breast cancer cell lines, then detected cell viability, and levels of ROS, MDA, GSH, Fe2+, GPX4 protein and miR-335-5p. We showed that the cell proliferation was inhibited, levels of ROS, MDA, Fe2+, and miR-335-5p were increased, while GSH and GPX4 levels were decreased after loss of ADAR1, compared to the control group. The opposite effects were observed after ADAR1 overexpression in the cells. Further, we demonstrated that ADAR1-controlled miR-335-5p targeted Sp1 transcription factor of GPX4, a known ferroptosis molecular marker, leading to inhibition of ferroptosis by ADAR1 in breast cancer cells. Moreover, RNA editing activity of ADAR1 is not essential for inducing ferroptosis. Collectively, loss of ADAR1 induces ferroptosis in breast cancer cells by regulating miR-335-5p/Sp1/GPX4 pathway. The findings may provide insights into the mechanism by which ADAR1 promotes breast cancer progression via inhibiting ferroptosis.
Assuntos
Adenosina Desaminase , Neoplasias da Mama , Ferroptose , Proteínas de Ligação a RNA , Ferroptose/genética , Humanos , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Linhagem Celular Tumoral , Proliferação de Células , Células MCF-7 , Espécies Reativas de Oxigênio/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Regulação Neoplásica da Expressão GênicaRESUMO
As an inflammatory disease with a disrupted immune system, cytokine disorders in atopic dermatitis (AD) are closely related to the abnormal activation of JAK-STAT signal pathway. The critical relevance of the JAK-STAT signaling pathway to the pathogenesis of AD provides a strong rationale for JAK inhibitor research. Baricitinib, a small-molecule oral JAK inhibitor, has been proven to inhibit JAK-STAT signaling in a variety of diseases, including AD. It is currently available in China for off-label use. However, its efficacy in China and its mechanism are rarely reported. In our study, we found that the immune status of patients with moderate and severe AD was hyperactive. Among the 49 known immunotherapy targets, JAK1 and JAK2 genes on lymphocytes of AD patients were significantly upregulated, which was closely related to the symptom severity in moderate and severe AD patients. Baricitinib can improve immune hyperresponsiveness and clinical symptoms in moderate and severe AD by inhibiting the activation of Th2 cell subsets and the secretion of Th2-type cytokines through MAPK, mTOR and PI3K-Akt signaling pathways, providing an important theoretical basis for clinical off-label use of Baricitinib to treat moderate and severe AD.
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Hyaluronidase (HAase) can enhance drug diffusion and dissipate edema by degrading hyaluronic acid (HA) in the extracellular matrix into unsaturated HA oligosaccharides in mammalian tissues. Microorganisms are recognized as valuable sources of HAase. In this study, a new hyaluronate lyase (HAaseD) from Bacillus sp. CQMU-D was expressed in Escherichia coli BL21, purified, and characterized. The results showed that HAaseD belonged to the polysaccharide lyase (PL) 8 family and had a molecular weight of 123 kDa. HAaseD could degrade chondroitin sulfate (CS) -A, CS-B, CS-C, and HA, with the highest activity toward HA. The optimum temperature and pH value of HAaseD were 40°C and 7.0, respectively. In addition, HAaseD retained stability in an alkaline environment and displayed higher activity with appropriate concentrations of metal ions. Moreover, HAaseD was an endolytic hyaluronate lyase that could degrade HA to produce unsaturated HA oligosaccharides. Together, our findings indicate that HAaseD from Bacillus sp. CQMU-D is a new hyaluronate lyase and with excellent potential for application in industrial production.
Assuntos
Bacillus , Animais , Bacillus/genética , Polissacarídeo-Liases/metabolismo , Hialuronoglucosaminidase/metabolismo , Ácido Hialurônico/metabolismo , Clonagem Molecular , Oligossacarídeos/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Mamíferos/metabolismoRESUMO
Introduction: Probiotic is adjuvant therapy for traditional drug treatment of ulcerative colitis (UC). In the present study, Lactobacillus acidophilus C4 with high acid and bile salt resistance has been isolated and screened, and the beneficial effect of L. acidophilus C4 on Dextran Sulfate Sodium (DSS)-induced colitis in mice has been evaluated. Our data showed that oral administration of L. acidophilus C4 remarkably alleviated colitis symptoms in mice and minimized colon tissue damage. Methods: To elucidate the underlying mechanism, we have investigated the levels of inflammatory cytokines and intestinal tight junction (TJ) related proteins (occludin and ZO-1) in colon tissue, as well as the intestinal microbiota and short-chain fatty acids (SCFAs) in feces. Results: Compared to the DSS group, the inflammatory cytokines IL-1ß, IL-6, and TNF-α in L. acidophilus C4 group were reduced, while the antioxidant enzymes superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) were found to be elevated. In addition, proteins linked to TJ were elevated after L. acidophilus C4 intervention. Further study revealed that L. acidophilus C4 reversed the decrease in intestinal microbiota diversity caused by colitis and promoted the levels of SCFAs. Discussion: This study demonstrate that L. acidophilus C4 effectively alleviated DSS-induced colitis in mice by repairing the mucosal barrier and maintaining the intestinal microecological balance. L. acidophilus C4 could be of great potential for colitis therapy.
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Protein sensors based on allosteric enzymes responding to target binding with rapid changes in enzymatic activity are potential tools for homogeneous assays. However, a high signal-to-noise ratio (S/N) is difficult to achieve in their construction. A high S/N is critical to discriminate signals from the background, a phenomenon that might largely vary among serum samples from different individuals. Herein, based on the modularized luciferase NanoLuc, we designed a novel biosensor called NanoSwitch. This sensor allows direct detection of antibodies in 1 µl serum in 45 min without washing steps. In the detection of Flag and HA antibodies, NanoSwitches respond to antibodies with S/N ratios of 33-fold and 42-fold, respectively. Further, we constructed a NanoSwitch for detecting SARS-CoV-2-specific antibodies, which showed over 200-fold S/N in serum samples. High S/N was achieved by a new working model, combining the turn-off of the sensor with human serum albumin and turn-on with a specific antibody. Also, we constructed NanoSwitches for detecting antibodies against the core protein of hepatitis C virus (HCV) and gp41 of the human immunodeficiency virus (HIV). Interestingly, these sensors demonstrated a high S/N and good performance in the assays of clinical samples; this was partly attributed to the combination of off-and-on models. In summary, we provide a novel type of protein sensor and a working model that potentially guides new sensor design with better performance.
Assuntos
Técnicas Biossensoriais , COVID-19 , Anticorpos Antivirais , COVID-19/diagnóstico , Humanos , Luciferases , SARS-CoV-2RESUMO
To investigate molecular epidemiology of dengue viruses (DENV) in southern China, a total of 14 dengue isolates were collected in southern China during each epidemic year between 1978 and 2006 and their full-length genome sequences were obtained by using RT-PCR method. The E gene sequences from additional 6 dengue fever patients in Guangzhou in 2006 were also obtained by using RT-PCR method. Combined with DENVs sequences published in GenBank, phylogenetic analysis and recombination analysis were performed. One hundred and twenty-five E gene sequences and 60 complete genome sequences published in the GenBank were also involved. Phylogenetic analysis showed that there was a wide genetic diversity of DENVs isolated in southern China. DENV-1 strains exist in almost all of the clades of genotype I and IV except the Asia 1 clade of genotype I; DENV-2 stains are grouped into four of the five genotypes except American genotype. DENV-4 strains are grouped into 2 genotypes (I and II). Phylogenetic analysis also showed that all DENV-4 isolates and two DENV-2 isolates were closely related to the prior isolates from neighboring Southeast Asia countries. The DENV-1 strain isolated during the 2006 epidemic is highly homologous to the strains isolated during the 2001 epidemic.Recombination analysis showed no inter-serotype recombination, but 22 intra-serotype recombination events were found across the 32 complete genomes of all Chinese isolates. The study suggested that dengue fever epidemic in Southern China over the past 30 years presented two important modes, 1) imported-cases-induced endemic prevalence; 2) endogenous epidemic outbreak with natural epidemic focus. Recombination may play an important role in dengue virus evolution and adaptation.
Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Dengue/epidemiologia , Dengue/virologia , Genoma Viral , RNA Viral/genética , Análise de Sequência de DNA , Animais , China/epidemiologia , Análise por Conglomerados , Vírus da Dengue/isolamento & purificação , Evolução Molecular , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Epidemiologia Molecular , Dados de Sequência Molecular , Polimorfismo Genético , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ABSTRACT: Hepatocellular carcinoma (HCC) is one of the tumors with a higher mortality rate globally, which significantly threatens people's health. Hepatitis C virus (HCV) infection is a major driving factor of HCC. This study aims to determine the key microRNA (miRNA), hub genes, and related pathways, construct potential miRNA-mRNA regulatory networks, and clarify the new molecular mechanism of HCV-related HCC. In this study, 16 differentially expressed miRNAs (DE miRNAs) were identified. The prediction of potential transcription factors and target genes not only found that SP1 and ERG1 may potentially regulate most of the screened DE miRNAs, but it also obtained 2923 and 1782 predicted target genes for the up-regulation and down-regulation of DE miRNAs, respectively. Subsequently, the introduction of differentially expressed genes dataset GSE62232 for target gene verification yielded 98 and 147 potential up-regulation and down-regulation target genes. The gene ontology (GO) and Kyoto encyclopedia of genes and genomes pathway enrichment analysis showed that they were mainly enriched in the cell cycle process, that is, subsequently, 20 hub genes were screened out through the protein-protein interaction network, and related genes were further evaluated using the GEPIA database. Based on the above analysis, the miRNA-hub gene regulatory network was constructed. In short, this research's hub genes and miRNAs closely related to HCV-related HCC were screened and identified through bioinformatics analysis and then built their connection. These results are expected to find potential therapeutic targets for HCV-related HCC.
Assuntos
Carcinoma Hepatocelular/etiologia , Hepatite C/complicações , Neoplasias Hepáticas/etiologia , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Hepatite C/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
8-Chloro-adenosine (8-Cl-Ado) has been shown to exhibit its antitumor activity by inducing apoptosis in human lung cancer A549 and H1299 cells or autophagy in chronic lymphocytic leukemia, and MDA-MB-231 and MCF-7 breast cancer cells. Adenosine deaminases acting on RNA 1 (ADAR1) is tightly associated with cancer development and progression. The aim of this study was to investigate the role of ADAR1 in the proliferation of MDA-MB-231 and SK-BR-3 breast cancer cell lines after 8-Cl-Ado exposure and its possible mechanisms. After 8-Cl-Ado exposure, CCK-8 assay was performed to determine the cell proliferation; flow cytometry was used to analyze the cell cycle profiles and apoptosis; and the protein levels of ADAR1, p53, p21, and cyclin D1 were measured by western blotting. The results showed that the cell proliferation was greatly inhibited, G1 cell cycle was arrested, and apoptosis was induced after 8-Cl-Ado exposure. ADAR1 and cyclin D1 protein levels were dramatically decreased, while p53 and p21 levels were increased after 8-Cl-Ado exposure. Moreover, the cell growth inhibition was rescued, apoptosis was reduced, and p53 and p21 protein levels were downregulated, while cyclin D1 was upregulated when cells were transfected with plasmids expressing ADAR1 proteins. More importantly, RNA-binding domain of ADAR1 is critical to the cell growth inhibition of breast cancer cells exposed to 8-Cl-Ado. Together, 8-Cl-Ado inhibits the cell proliferation, induces G1 phase arrest and apoptosis at least by targeting ADAR1/p53/p21 signaling pathway. The findings may provide us with insights into the role of ADAR1 in breast cancer progression and help us better understand the effects of 8-Cl-Ado in the treatment of breast cancer.
Assuntos
2-Cloroadenosina/análogos & derivados , Adenosina Desaminase/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , 2-Cloroadenosina/farmacologia , Adenosina Desaminase/química , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Domínios Proteicos , Proteínas de Ligação a RNA/química , Transdução de Sinais/efeitos dos fármacosRESUMO
Adenosine deaminase acting on RNA1 (ADAR1) is an RNA editing enzyme that modulates the replication of several viruses. Here, we provide evidence showing that ADAR1 stimulates hepatitis B virus (HBV) DNA replication in hepatocellular carcinoma cell lines that are transiently or stably-transfected with HBV. We show that overexpression of ADAR1 promotes the replication of all four HBV genotypes (A, B, C, and D). Furthermore, analysis by mutagenesis shows that RNA editing region, and to a lesser content, RNA binding region of ADAR is responsible for the promotion of replication. Together, these data show that ADAR1 stimulates HBV replication.
Assuntos
Adenosina Desaminase/genética , Vírus da Hepatite B/genética , Proteínas de Ligação a RNA/genética , Replicação Viral/genética , Adenosina Desaminase/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Interferência de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Genetic variation and genotype of Hepatitis B virus (HBV) are related to the efficiency of interferon alpha (IFN-α)-based antiviral therapy. However, the correlation of variation in interferon-stimulated response element (ISRE) and HBV genotype response to IFN-α therapy remains elusive.Differences of ISRE between genotype B and C HBV were explored using the HBV sequences retrieved from GenBank, and further investigated by ISRE region cloning and sequencing from 60 clinical samples post-IFN-α therapy. Additionally, ISRE mutants were constructed and their relation to responsiveness of IFN-α was evaluated by real-time PCR and Southern blot analysis.ISRE pattern between genotype B and C were found based on both clinical sample sequencing and full-length sequence alignment. The primary difference is the fourth base within the ISRE region, with T and C for genotype B and C, respectively. HBV with genotype C-type ISRE had a higher replicative capability as compared to HBV with genotype B-type ISRE after IFN-α treatment in huh7 cells. CONCLUSION:: Preference of ISRE between genotype B and C HBV are distinct. Single nucleotide difference (C to T) within the HBV ISRE region may link to the efficacy of IFN-α therapy to genotype B and C HBV. Therefore, this study provides a clue for the determination of IFN-α therapy response to HBV treatment.