RESUMO
LincRNA-EPS is an important regulator in inflammation. However, the role of lincRNA-EPS in the host response against viral infection is unexplored. Here, we show that lincRNA-EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV-1. Overexpression of lincRNA-EPS facilitates viral infection, while deficiency of lincRNA-EPS protects the host against viral infection in vitro and in vivo. LincRNA-EPS-/- macrophages show elevated expression of antiviral interferon-stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN-ß, the key upstream inducer of these ISGs, is downregulated in lincRNA-EPS-/- macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA-EPS binds to PKR and antagonizes the viral RNA-PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN-I induction. LincRNA-EPS inhibits PKR-STAT1-ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA-EPS promoting the induction of PKR-STAT1-dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.
Assuntos
RNA Longo não Codificante , Antivirais , Imunidade Inata , Interferon beta/genética , Interferons , RNA Longo não Codificante/genética , RNA Viral/metabolismoRESUMO
XIAP-associated factor 1 (XAF1) is an interferon (IFN)-stimulated gene (ISG) that enhances IFN-induced apoptosis. However, it is unexplored whether XAF1 is essential for the host fighting against invaded viruses. Here, we find that XAF1 is significantly upregulated in the host cells infected with emerging RNA viruses, including influenza, Zika virus (ZIKV), and SARS-CoV-2. IFN regulatory factor 1 (IRF1), a key transcription factor in immune cells, determines the induction of XAF1 during antiviral immunity. Ectopic expression of XAF1 protects host cells against various RNA viruses independent of apoptosis. Knockout of XAF1 attenuates host antiviral innate immunity in vitro and in vivo, which leads to more severe lung injuries and higher mortality in the influenza infection mouse model. XAF1 stabilizes IRF1 protein by antagonizing the CHIP-mediated degradation of IRF1, thus inducing more antiviral IRF1 target genes, including DDX58, DDX60, MX1, and OAS2. Our study has described a protective role of XAF1 in the host antiviral innate immunity against RNA viruses. We have also elucidated the molecular mechanism that IRF1 and XAF1 form a positive feedback loop to induce rapid and robust antiviral immunity. IMPORTANCE Rapid and robust induction of antiviral genes is essential for the host to clear the invaded viruses. In addition to the IRF3/7-IFN-I-STAT1 signaling axis, the XAF1-IRF1 positive feedback loop synergistically or independently drives the transcription of antiviral genes. Moreover, XAF1 is a sensitive and reliable gene that positively correlates with the viral infection, suggesting that XAF1 is a potential diagnostic marker for viral infectious diseases. In addition to the antitumor role, our study has shown that XAF1 is essential for antiviral immunity. XAF1 is not only a proapoptotic ISG, but it also stabilizes the master transcription factor IRF1 to induce antiviral genes. IRF1 directly binds to the IRF-Es of its target gene promoters and drives their transcriptions, which suggests a unique role of the XAF1-IRF1 loop in antiviral innate immunity, particularly in the host defect of IFN-I signaling such as invertebrates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Fator Regulador 1 de Interferon , Infecções por Vírus de RNA , Vírus de RNA , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Humanos , Imunidade Inata , Fator Regulador 1 de Interferon/imunologia , Camundongos , Camundongos Knockout , Infecções por Vírus de RNA/imunologia , Replicação ViralRESUMO
Adhesion and degranulation-promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS. Furthermore, overexpression of ADAP enhances, whereas loss of ADAP reduces, the LPS-mediated phosphorylation and activity of STAT3, suggesting ADAP acts as a coactivator of STAT3 activity and function. Furthermore, the coactivator function of ADAP mostly depends on the tyrosine phosphorylation at Y571 in the motif YDSL induced by LPS. Mutation of Y571 to F severely impairs the stimulating effect of ADAP on STAT3 activity and the ability of ADAP to inhibit M1-like polarization in TLR4-activated mouse macrophages. Moreover, ADAP interacts with STAT3, and loss of ADAP renders mouse macrophages less sensitive to IL-6 stimulation for STAT3 phosphorylation. Collectively, our findings revealed an additional layer of regulation of TLR4-mediated mouse macrophage plasticity whereby ADAP phosphorylation on Y571 is required to prime STAT3 for activation in TLR4-stimulated mouse macrophages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Fator de Transcrição STAT3/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Feminino , Camundongos , Camundongos Knockout , Fosforilação/genética , Fosforilação/imunologia , Fator de Transcrição STAT3/genética , Receptor 4 Toll-Like/genéticaRESUMO
IFN-γ-inducible protein 16 (IFI16) recognizes viral DNAs from both nucleus-replicating viruses and cytoplasm-replicating viruses. Isoform 2 of IFI16 (IFI16-iso2) with nuclear localization sequence (NLS) has been studied extensively as a well-known DNA sensor. However, the characteristics and functions of other IFI16 isoforms are almost unknown. Here, we find that IFI16-iso1, with exactly the same length as IFI16-iso2, lacks the NLS and locates in the cytoplasm. To distinguish the functions of IFI16-iso1 and IFI16-iso2, we have developed novel nuclear viral DNA mimics that can be recognized by the nuclear DNA sensors, including IFI16-iso2 and hnRNPA2B1. The hexanucleotide motif 5'-AGTGTT-3' DNA form of the nuclear localization sequence (DNLS) effectively drives cytoplasmic viral DNA nuclear translocation. These nuclear viral DNA mimics potently induce IFN-ß and antiviral IFN-stimulated genes in human A549 cells, HEK293T cells, and mouse macrophages. The subcellular location difference of IFI16 isoforms determines their differential functions in recognizing viral DNA and activating type I IFN-dependent antiviral immunity. IFI16-iso1 preferentially colocalizes with cytoplasmic HSV60mer and cytoplasm-replicating vaccinia virus (VACV), whereas IFI16-iso2 mainly colocalizes with nuclear HSV60-DNLS and nucleus-replicating HSV-1. Compared with IFI16-iso2, IFI16-iso1 induces more transcription of IFN-ß and IFN-stimulated genes, as well as stronger antiviral immunity upon HSV60mer transfection or VACV infection. IFI16-iso2, with the ability of nuclear-cytoplasmic shuttling, clears both invaded HSV type 1 and VACV significantly. However, IFI16-iso2 induces more type I IFN-dependent antiviral immunity than IFI16-iso1 upon HSV60-DNLS transfection or HSV type 1 infection. Our study has developed potent agonists for nuclear DNA sensors and also has demonstrated that IFI16 isoforms with cytoplasmic and nuclear locations play differential roles in innate immunity against DNA viruses.
Assuntos
Núcleo Celular/imunologia , Vírus de DNA/imunologia , Proteínas Nucleares/imunologia , Fosfoproteínas/imunologia , Células Cultivadas , Humanos , Isoformas de Proteínas/imunologiaRESUMO
Interferon-γ-inducible factor 16 (IFI16) triggers stimulator of interferon (IFN) genes (STING)-dependent type I IFN production during host antiviral immunity and facilitates p53-dependent apoptosis during suppressing tumorigenesis. We have previously reported that STING-mediated IFI16 degradation negatively regulates type I IFN production. However, it is unknown whether STING also suppresses IFI16/p53-dependent apoptosis via degradation of IFI16. Here, our results from flow cytometry apoptosis detection and immunoblot assays show that IFI16 and nutlin-3, a p53 pathway activator, synergistically induce apoptosis in U2OS and A549 cells. Protein kinase R-triggered phosphorylation of p53 at serine 392 is critical for the IFI16-p53-dependent apoptosis. However, overexpression of STING suppresses p53 serine 392 phosphorylation, p53 transcriptional activity, expression of p53 target genes, and p53-dependent mitochondrial depolarization and apoptosis. In summary, our current study demonstrates that STING-mediated IFI16 degradation negatively regulates IFI16-mediated p53-dependent apoptosis in osteosarcoma and non-small cell lung cancer cells, which suggests a protumorigenic role for STING in certain cancer types because of its potent ability to degrade upstream IFI16.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína Supressora de Tumor p53 , Apoptose , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Proteínas de Drosophila , Humanos , Imunidade Inata , Neoplasias Pulmonares , Fosforilação , Transdução de SinaisRESUMO
Acute pancreatitis (AP), an inflammatory disorder of the pancreas with a high hospitalization rate, frequently leads to systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). However, therapeutic targets for effective treatment and early intervention of AP are still urgently required to be identified. Here, we have observed that the expression of pancreatic lincRNA-EPS, a long intergenic non-coding RNA, is dynamically changed during both caerulein-induced AP (Cer-AP) and sodium taurocholate-induced severe AP (NaTc-SAP). The expression pattern of lincRNA-EPS is negatively correlated with the typical inflammatory genes such as IL-6, IL-1ß, CXCL1, and CXCL2. Further studies indicate that knockout of lincRNA-EPS aggravates the pathological symptoms of AP including more induction of serum amylase and lipase, severe edema, inflammatory cells infiltration and acinar necrosis in both experimental AP mouse models. Besides these intrapancreatic effects, lincRNA-EPS also protects against tissue damages in the extra-pancreatic organs such as lung, liver, and gut in the NaTc-SAP mouse model. In addition, we have observed more serum pro-inflammatory cytokines TNF-α and IL-6 in the lincRNA-EPS-/- NaTc-SAP mice and more extracellular HMGB1 around injured acinar cells in the pancreas from lincRNA-EPS-/- NaTc-SAP mice, compared with their respective controls. Pharmacological inhibition of NF- κ B activity by BAY11-7082 significantly abolishes the suppressive effect of lincRNA-EPS on TLR4 ligand-induced inflammatory genes in macrophages. Our study has described a protective role of lincRNA-EPS in alleviating AP and SAP, outlined a novel pathway that lincRNA-EPS suppresses HMGB1-NF- κ B-dependent inflammatory response in pancreatic macrophages and provided a potential therapeutic target for SAP.
Assuntos
Inflamação/genética , Macrófagos/fisiologia , Pâncreas/patologia , Pancreatite/genética , RNA Longo não Codificante/genética , Animais , Ceruletídeo , Modelos Animais de Doenças , Células HEK293 , Proteína HMGB1/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , NF-kappa B/metabolismo , Necrose , Índice de Gravidade de Doença , Ácido TaurocólicoRESUMO
A new strain producing high yield of D-arabitol was isolated from hyperosmotic environments and the ITS rDNA sequencing analysis revealed it as Zygosaccharomyces rouxii. In addition, using a pH control and repeated-batch fermentation strategy in a 5-L reactor, the maximum yield and the highest volumetric productivity of D-arabitol were 93.48 ± 2.79 g/L and 1.143 g/L h, respectively. Volumetric productivity was successfully improved from 0.86 to 1.143 g/L h, which was increased by 32.9 % after 72 h of fermentation. Z. rouxii JM-C46 has potential to be used for D-arabitol and xylitol production from glucose via D-arabitol route.
Assuntos
Fermentação , Álcoois Açúcares/metabolismo , Zygosaccharomyces/isolamento & purificação , Zygosaccharomyces/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Xilitol/biossíntese , Zygosaccharomyces/classificação , Zygosaccharomyces/genéticaRESUMO
BACKGROUND: Hepatocellular carcinoma closely related to metabolic disorders is a common and aggressive liver malignancy. The dysregulation of bile acid homeostasis has emerged as a key factor for the development and progression of HCC. We aimed to investigate the relationship between bile acids and HCC diagnosis and progression. METHODS: A total of 744 HBV-related patients (including 396 HCC patients and 348 patients with chronic liver diseases) were enrolled in the current study. The baseline characteristics of patients were collected from electronic medical records, and the levels of bile acid profiles were determined by LC-MS/MS. Propensity score matching analysis was conducted to reduce the effect of selection bias, and receiver operating characteristic analysis was performed to evaluate the clinical application values of bile acid. RESULTS: Significant differences were observed for most characteristics between the HCC group and the CLD group before PSM analysis. Patients with HCC were older and fatter (p < 0.05). After adjusting with a 1:1 ratio for age, gender and BMI, 42 HCC patients and 42 non-HCC patients were matched in 2 groups, respectively. The total bile acid level in HCC patients was lower than that in patients with chronic liver diseases before and after PSM analysis (p < 0.05). However, patients with HCC had significantly higher levels of DCA, LCA, and GLCA and lower levels of TCDCA, GUDCA, and TUDCA (p < 0.05, respectively). Besides, the TCDCA, TUDCA, GLCA, and GUDCA were significantly correlated with tumor procession. Moreover, the BAs profiles had a superior predictive ability for predicting the development of HCC even in patients with low serum AFP levels. CONCLUSION: Patients with HCC had significantly lower levels of total bile acid, but higher levels of secondary bile acids (DCA, LCA, and GLCA). The levels of primary bile acid (TCDCA) were closely related to tumor size and stage, which indicated that the bile acids were involved in the HCC procession and had important clinical application values.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ácido Tauroquenodesoxicólico , Humanos , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/patologia , Vírus da Hepatite B , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida , Ácidos e Sais Biliares , Espectrometria de Massas em TandemRESUMO
U1 small nuclear RNA (snRNA) is an abundant and evolutionarily conserved 164-nucleotide RNA species that functions in pre-mRNA splicing, and it is considered to be a housekeeping non-coding RNA. However, the role of U1 snRNA in regulating host antiviral immunity remains largely unexplored. Here, we find that RNVU1-18, a U1 pseudogene, is significantly upregulated in the host infected with RNA viruses, including influenza and respiratory syncytial virus. Overexpression of U1 snRNA protects cells against RNA viruses, while knockdown of U1 snRNA leads to more viral burden in vitro and in vivo. Knockout of RNVU1-18 is sufficient to impair the type I interferon-dependent antiviral innate immunity. U1 snRNA is required to fully activate the retinoic acid-inducible gene I (RIG-I)-dependent antiviral signaling, since it interacts with tripartite motif 25 (TRIM25) and enhances the RIG-I-TRIM25 interaction to trigger K63-linked ubiquitination of RIG-I. Our study reveals the important role of housekeeping U1 snRNA in regulating host antiviral innate immunity and restricting RNA virus infection.
Assuntos
Fatores de Transcrição , Ubiquitina-Proteína Ligases , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína DEAD-box 58/metabolismo , Imunidade Inata , RNA Nuclear Pequeno , Ubiquitinação , Proteínas com Motivo Tripartido/metabolismoRESUMO
We aimed to build a deep learning-based pathomics model to predict the early recurrence of non-muscle-infiltrating bladder cancer (NMIBC) in this work. A total of 147 patients from Xuzhou Central Hospital were enrolled as the training cohort, and 63 patients from Suqian Affiliated Hospital of Xuzhou Medical University were enrolled as the test cohort. Based on two consecutive phases of patch level prediction and WSI-level predictione, we built a pathomics model, with the initial model developed in the training cohort and subjected to transfer learning, and then the test cohort was validated for generalization. The features extracted from the visualization model were used for model interpretation. After migration learning, the area under the receiver operating characteristic curve for the deep learning-based pathomics model in the test cohort was 0.860 (95% CI 0.752-0.969), with good agreement between the migration training cohort and the test cohort in predicting recurrence, and the predicted values matched well with the observed values, with p values of 0.667766 and 0.140233 for the Hosmer-Lemeshow test, respectively. The good clinical application was observed using a decision curve analysis method. We developed a deep learning-based pathomics model showed promising performance in predicting recurrence within one year in NMIBC patients. Including 10 state prediction NMIBC recurrence group pathology features be visualized, which may be used to facilitate personalized management of NMIBC patients to avoid ineffective or unnecessary treatment for the benefit of patients.
Assuntos
Aprendizado Profundo , Recidiva Local de Neoplasia , Neoplasias não Músculo Invasivas da Bexiga , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/patologia , Neoplasias não Músculo Invasivas da Bexiga/patologia , Curva ROC , Medição de Risco/métodosRESUMO
In adult hippocampal neurogenesis, chromatin modification plays an important role in neural stem cell self-renewal and differentiation by regulating the expression of multiple genes. Histone deacetylases (HDACs), which remove acetyl groups from histones, create a non-permissive chromatin that prevents transcription of genes involved in adult neurogenesis. HDAC inhibitors have been shown to promote adult neurogenesis and have also been used to treat nervous system disorders, such as epilepsy. However, most HDAC inhibitors are not specific and may have other targets. Therefore, it is important to decipher the role of individual HDACs in adult hippocampal neurogenesis. HDACs 1, 2, and 3 have been found expressed at different cellular stages during neurogenesis. Conditional deletion of HDAC2 in neural stem cells impairs neuronal differentiation in adult hippocampus. HDAC3 supports proliferation of adult hippocampal neural stem/progenitor cells. The role of HDAC1 in adult neurogenesis remains still open. Here, we used a conditional knock-out mouse to block HDAC1 expression in neural stem cells (Nestin+ cells) during hippocampal neurogenesis. Our results showed that both HDAC1 and HDAC2 are expressed in all cellular stages during hippocampal neurogenesis. Moreover, we found that deletion of HDAC1 by viral infection of neural stem cells is sufficient to compromise neuronal differentiation in vitro. However, we were unable to reduce the expression of HDAC1 in vivo using Nestin-CreERT2 mice. Understanding the role of HDAC1 may lead to ways to control stem cell proliferation and neuronal regeneration in the adult hippocampus, and to more specific HDAC therapeutics for neurological disorders.
RESUMO
In the mammalian hippocampus, adult-born granule cells (abGCs) contribute to the function of the dentate gyrus (DG). Disruption of the DG circuitry causes spontaneous recurrent seizures (SRS), which can lead to epilepsy. Although abGCs contribute to local inhibitory feedback circuitry, whether they are involved in epileptogenesis remains elusive. Here, we identify a critical window of activity associated with the aberrant maturation of abGCs characterized by abnormal dendrite morphology, ectopic migration, and SRS. Importantly, in a mouse model of temporal lobe epilepsy, silencing aberrant abGCs during this critical period reduces abnormal dendrite morphology, cell migration, and SRS. Using mono-synaptic tracers, we show silencing aberrant abGCs decreases recurrent CA3 back-projections and restores proper cortical connections to the hippocampus. Furthermore, we show that GABA-mediated amplification of intracellular calcium regulates the early critical period of activity. Our results demonstrate that aberrant neurogenesis rewires hippocampal circuitry aggravating epilepsy in mice.
Assuntos
Epilepsia/fisiopatologia , Hipocampo/fisiopatologia , Neurogênese/fisiologia , Animais , Cálcio/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacologia , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia do Lobo Temporal/fisiopatologia , Feminino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Pilocarpina/farmacologia , Retroviridae/genética , Convulsões/fisiopatologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.
Assuntos
Antígeno B7-H1 , Fatores de Iniciação em Eucariotos , Neoplasias Pulmonares , Antígeno B7-H1/genética , Fatores de Iniciação em Eucariotos/genética , Heme/biossíntese , Humanos , Neoplasias Pulmonares/genéticaRESUMO
γ-interferon-inducible protein-16 (IFI16), a key DNA sensor, triggers downstream STING-dependent type I interferon (IFN-I) production and antiviral immunity. However, it is still unclear how to negatively regulate IFI16 to avoid excessive IFN-I production and autoimmunity. Here, we find that STING directly interacts with IFI16 and facilitates IFI16 degradation via the ubiquitin-proteasome pathway by recruiting the E3 ligase TRIM21. The 1-pyrin region of IFI16 is responsible for the IFI16-STING interaction, and the first three lysines in the N-terminal region of IFI16 are the key sites that lead to STING-mediated IFI16 ubiquitination and degradation. Compared to wild-type IFI16, a higher level of viral DNA triggered IFN-ß and antiviral IFN-stimulated gene expression, and thus less HSV-1 infection, was observed in the cells transfected with IFI16-K3/4/6R, an IFI16 mutant that is resistant to degradation. STING-mediated negative feedback regulation of IFI16 restricts IFN-I overproduction during antiviral immunity to avoid autoimmune diseases.
Assuntos
Interferon beta/biossíntese , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteólise , Linhagem Celular , Humanos , Lisina/metabolismo , Modelos Biológicos , Proteínas Nucleares/química , Fosfoproteínas/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Estabilidade Proteica , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. Although PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types. Given that PCDH7 is a potentially targetable molecule on the surface of cancer cells, further investigation of its role in tumorigenesis in vivo is needed to evaluate the therapeutic potential of its inhibition. Here, we report the analysis of novel PCDH7 gain- and loss-of-function mouse models and provide compelling evidence that this cell-surface protein acts as a potent lung cancer driver. Employing a Cre-inducible transgenic allele, we demonstrated that enforced PCDH7 expression significantly accelerates KrasG12D -driven lung tumorigenesis and potentiates MAPK pathway activation. Furthermore, we performed in vivo somatic genome editing with CRISPR/Cas9 in KrasLSL-G12D ; Tp53fl/fl (KP) mice to assess the consequences of PCDH7 loss of function. Inactivation of PCDH7 in KP mice significantly reduced lung tumor development, prolonged survival, and diminished phospho-activation of ERK1/2. Together, these findings establish a critical oncogenic function for PCDH7 in vivo and highlight the therapeutic potential of PCDH7 inhibition for lung cancer. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the new inducible transgenic model described here provides a robust experimental system for broadly elucidating the effects of PCDH7 overexpression in vivo. IMPLICATIONS: In this study, we establish a critical oncogenic function for PCDH7 in vivo using novel mouse models and CRISPR/Cas9 genome editing, and we validate the therapeutic potential of PCDH7 inhibition for lung cancer.
Assuntos
Adenocarcinoma de Pulmão/genética , Caderinas/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Caderinas/deficiência , Caderinas/metabolismo , Carcinogênese , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Protocaderinas , Transdução de SinaisRESUMO
In the present study, a new strain of Lactobacillus brevis producing d-tagatose was isolated and identified. Then, the l-arabinose isomerase (L-AI) of this strain was displayed on the spore surface of Bacillus subtilis DB403 by using an anchoring protein CotG and a peptide linker (Gly-Gly-Gly-Gly-Ser). This displayed L-AI with high specific activity and stability was used as a novel immobilized biocatalyst for producing d-tagatose through batch and semi-continuous biotransformation. The conversion rate of d-tagatose from 125â¯g/L d-galactose was achieved 79.7% at 28â¯h, and the volumetric productivity reached 4.3â¯g/L/h at 20â¯h. Furthermore, the displayed L-AI showed a good performance on the reusability and remained 87% of the specific activity and 40.7% of the conversion rate after five recycles. A high efficient immobilized method for producing food-grade d-tagatose was established using spore surface-displayed L-AI.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Hexoses/metabolismo , Levilactobacillus brevis/enzimologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Biotransformação , Galactose , Lisina/análogos & derivados , Esporos BacterianosRESUMO
Adult neurogenesis is significantly increased in the hippocampus of rodent models of temporal lobe epilepsy (TLE). These adult-generated neurons have recently been shown to play a contributing role in the development of spontaneous recurrent seizures (SRS). In order to eventually target pro-epileptic adult neurogenesis in the clinical setting, it will be important to identify molecular players involved in the control of aberrant neurogenesis after seizures. Here, we focused on NeuroD1 (ND1), a member of the bHLH family of transcription factors previously shown to play an essential role in the differentiation and maturation of adult-generated neurons in the hippocampus. Wild-type mice treated with pilocarpine to induce status epilepticus (SE) showed a significant up-regulation of NeuroD1+ immature neuroblasts located in both the granule cell layer (GCL), and ectopically localized to the hilar region of the hippocampus. As expected, conditional knockout (cKO) of NeuroD1 in Nestin-expressing stem/progenitors and their progeny led to a reduction in the number of NeuroD1+ adult-generated neurons after pilocarpine treatment compared to WT littermates. Surprisingly, there was no change in SRS in NeuroD1 cKO mice, suggesting that NeuroD1 cKO fails to reduce aberrant neurogenesis below the threshold needed to impact SRS. Consistent with this conclusion, the total number of adult-generated neurons in the pilocarpine model, especially the total number of Prox1+ hilar ectopic granule cells were unchanged after NeuroD1 cKO, suggesting strategies to reduce SRS will need to achieve a greater removal of aberrant adult-generated neurons.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Epilepsia/genética , Epilepsia/patologia , Hipocampo/fisiopatologia , Neurogênese/fisiologia , Regulação para Cima/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Epilepsia/induzido quimicamente , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Agonistas Muscarínicos/toxicidade , N-Metilescopolamina/toxicidade , Nestina/genética , Nestina/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/metabolismo , Neurônios/patologia , Neuropeptídeos/metabolismo , Pilocarpina/toxicidade , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Xylitol has numerous applications in food and pharmaceutical industry, and it can be biosynthesized by microorganisms. In the present study, xdh gene, encoding xylitol dehydrogenase (XDH), was cloned from the genome of Gluconobacter oxydans CGMCC 1.49 and overexpressed in Escherichia coli BL21. Sequence analysis revealed that XDH has a TGXXGXXG NAD(H)-binding motif and a YXXXK active site motif, and belongs to the short-chain dehydrogenase/reductase family. And then, the enzymatic properties and kinetic parameter of purified recombinant XDH were investigated. Subsequently, transformations of xylitol from d-xylulose and d-arabitol, respectively, were studied through mixed culture of resting cells of G. oxydans wild-type strain and recombinant strain BL21-xdh. We obtained 28.80 g/L xylitol by mixed culture from 30 g/L d-xylulose in 28 h. The production was increased by more than three times as compared with that of wild-type strain. Furthermore, 25.10 g/L xylitol was produced by the mixed culture from 30 g/L d-arabitol in 30 h with a yield of 0.837 g/g, and the max volumetric productivity of 0.990 g/L h was obtained at 22 h. These contrast to the fact that wild-type strain G. oxydans only produced 8.10 g/L xylitol in 30 h with a yield of 0.270 g/g. To our knowledge, these values are the highest among the reported yields and productivity efficiencies of xylitol from d-arabitol with engineering strains.