RESUMEN
Testicular nuclear receptor 4 (TR4) modulates the transcriptional activation of genes and plays important roles in many diseases. The regulation of TR4 on target genes involves direct interactions with DNA molecules via the DNA-binding domain (DBD) and recruitment of coregulators by the ligand-binding domain (LBD). However, their regulatory mechanisms are unclear. Here, we report high-resolution crystal structures of TR4DBD, TR4DBD-DNA complexes and the TR4LBD-JAZF1 complex. For DNA recognition, multiple factors come into play, and a specific mutual selectivity between TR4 and target genes is found. The coactivators SRC-1 and CREBBP can bind at the interface of TR4 originally occupied by the TR4 activation function region 2 (AF-2); however, JAZF1 suppresses the binding through a novel mechanism. JAZF1 binds to an unidentified surface of TR4 and stabilizes an α13 helix never reported in the nuclear receptor family. Moreover, the cancer-associated mutations affect the interactions and the transcriptional activation of TR4 in vitro and in vivo, respectively. Overall, our results highlight the crucial role of DNA recognition and a novel mechanism of how JAZF1 reinforces the autorepressed conformation and influences the transcriptional activation of TR4, laying out important structural bases for drug design for a variety of diseases, including diabetes and cancers.
Asunto(s)
Proteínas Co-Represoras , Regulación de la Expresión Génica , Receptores de Esteroides , Humanos , Proteínas Portadoras/genética , Proteínas Co-Represoras/metabolismo , ADN , Proteínas de Unión al ADN/genética , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: Due to the deep location of the prostate within the pelvic cavity, procedures of robot-assisted radical prostatectomy (RARP) might be challenged by the prostate size and the limited pelvic cavity space. This study aimed to investigate the roles of bony pelvic and prostate dimensions in RARP procedures by an original study coupled with a meta-analysis. METHODS: In the original study, patients undergoing multiport RARP between 2021 and 2022 were consecutively assessed. The associations of anatomic features with operative time (OT), estimated blood loss (EBL), and positive surgical margin (PSM) were evaluated using linear and logistic regression analyses as well as restricted cubic spline (RCS) analysis. Based on machine-learning algorithms, this study established predictive models for surgical difficulty and interpreted the model using SHapley Additive exPlanation (SHAP). In the meta-analysis, three databases were searched for eligible studies. Quantitative syntheses were subsequently performed. RESULTS: Overall, 219 patients were enrolled in the original study. Prostate volume (PV) and the prostate volume-to-pelvic cavity index (PCI) ratio (PV-to-PCI ratio) were significantly associated with longer OT (P < 0.05). In the RCS models, U-shaped associations were observed between the prostate anteroposterior diameter (PAD) and OT, and between the prostate height (PH) and EBL, and an L-shaped association was observed between the anteroposterior diameter of the pelvic inlet (API) and EBL. The XGBoost model was superior to the logistic regression model in predicting prolonged OT. The meta-analysis demonstrated that greater PV was significantly associated with longer OT (ß = 0.20; 95% confidence interval [CI] 0.12-0.27; odds ratio [OR] = 1.05; 95% CI 1.00-1.11), and a smaller PV could increase the risk of PSM (OR = 0.82; 95% CI 0.77-0.88). CONCLUSIONS: A large prostate within a narrow and deep pelvis might suggest increased surgical difficulty of RARP. The size of the pelvic inlet also had a great impact on RARP. For PAD and PH, there seemed to be an optimal range with the lowest surgical difficulty. Machine-learning models based on the XGBoost algorithm could be successfully applied to predict the surgical difficulty of RARP.
RESUMEN
Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate â × Channa argus â) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4ãCD8αãMHCIIαãTNFαãIL-1ß and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.
Asunto(s)
Enfermedades de los Peces , Nocardiosis , Nocardia , Vacunas de ADN , Animales , PecesRESUMEN
As a lymphocyte-specific surface receptor belonging to the cysteine-rich superfamily of scavenger receptors, CD6 acts as a pattern recognition receptor for microbial components and is involved in the regulation of inflammatory responses. However, the characteristics and functions of CD6 molecules in lower vertebrates represented by teleost fish are unknown. In this study, a CD6 homolog (designated OnCD6) was characterized from Nile tilapia (Oreochromis niloticus), and establishing its role as a PRRs that participates in immune recognition. OnCD6 contains an open reading frame of 1872 bp that encodes a peptide of 623 amino acids, and contains two conserved SR domain. Multiple sequence alignment revealed that OnCD6 shares a relatively high level of identity with those of other species. Transcriptional expression analysis revealed that OnCD6 was constitutively expressed in immunes tissues such as head kidney and thymus. The expression level of OnCD6 in mainly immune tissues were found significantly upregulated after the injection of Streptococcus agalactiae (S. agalactiae). Moreover, OnCD6 protein was located in the head kidney and brain, mainly over the plasma membrane of lymphocytes in these immune tissues. In vitro experiments showed that CD6 extracellular protein bound to and aggregated several Gram-positive and -negative bacterial strains through the recognition of bacterial surface conserved components LPS and LTA etc. In vivo experiments demonstrated that overexpression OnCD6 before S. agalactiae challenge significantly improved tilapia survival, and this was concomitant with reduced bacterial load and pro-inflammatory cytokines (IL-1ß and TNF-α). Taken together, our results illustrated the function of CD6 molecular pattern recognition receptors (PRRs) is conserved and plays an important role in antibacterial infection.
Asunto(s)
Cíclidos , Enfermedades de los Peces , Infecciones Estreptocócicas , Animales , Streptococcus agalactiae/fisiología , Secuencia de Aminoácidos , Citocinas/metabolismo , Inflamación , Proteínas de Peces/química , Infecciones Estreptocócicas/veterinaria , Regulación de la Expresión GénicaRESUMEN
Fish nocardiosis is a chronic disease mainly caused by Nocardia seriolae, which occurs in a variety of economically cultured freshwater and marine fish. Studies have shown that DNA vaccine is an effective treatment to protect fish from bacterial infection. In our previous experiment, an in vivo-induced gene of N. seriolae, encoding phosphoketolase (PK) family protein, was identified by in vivo-induced antigen technology. In the present study, the antigenic gene encoding PK family protein was analyzed by bioinformatics and further inserted into the eukaryotic expression vector pcDNA3.1-myc-his-A for DNA vaccine development. The immunological effects of pcDNA-PK DNA vaccine were assessed in hybrid snakehead (Channa maculata â × Channa argus â), showing induction in several serum enzyme activity parameters (including LZM, SOD, ACP and AKP), increasing in specific-antibody IgM levels, as well as up-regulation in six immune-related genes (CD4, CD8α, TNFα, IL-1ß, MHCIα and MHCIIα). Moreover, an immune-protection with a relative survival rate was provided at 53.82 % following artificial challenge with N. seriolae in vaccinated fish in comparison to the control group. In summary, these results indicate that pcDNA-PK DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, which may be applied in aquaculture to control fish nocardiosis.
Asunto(s)
Vacunas Bacterianas , Enfermedades de los Peces , Nocardiosis , Nocardia , Vacunas de ADN , Animales , Nocardia/inmunología , Nocardiosis/veterinaria , Nocardiosis/inmunología , Nocardiosis/prevención & control , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/prevención & control , Vacunas de ADN/inmunología , Vacunas Bacterianas/inmunología , Aldehído-Liasas/genética , Aldehído-Liasas/inmunología , Peces/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genéticaRESUMEN
Optimizing jamming strategies is crucial for enhancing the performance of cognitive jamming systems in dynamic electromagnetic environments. The emergence of frequency-agile radars, capable of changing the carrier frequency within or between pulses, poses significant challenges for the jammer to make intelligent decisions and adapt to the dynamic environment. This paper focuses on researching intelligent jamming decision-making algorithms for Intra-Pulse Frequency Agile Radar using deep reinforcement learning. Intra-Pulse Frequency Agile Radar achieves frequency agility at the sub-pulse level, creating a significant frequency agility space. This presents challenges for traditional jamming decision-making methods to rapidly learn its changing patterns through interactions. By employing Gated Recurrent Units (GRU) to capture long-term dependencies in sequence data, together with the attention mechanism, this paper proposes a GA-Dueling DQN (GRU-Attention-based Dueling Deep Q Network) method for jamming frequency selection. Simulation results indicate that the proposed method outperforms traditional Q-learning, DQN, and Dueling DQN methods in terms of jamming effectiveness. It exhibits the fastest convergence speed and reduced reliance on prior knowledge, highlighting its significant advantages in jamming the subpulse-level frequency-agile radar.
RESUMEN
CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules, and its mediated adhesion plays a crucial role in different physiological and pathological phenomena, especially related to leukocyte extravasation, immune synapse stability, T cell activation and proliferation. In this study, CD166 was identified from Nile tilapia (Oreochromis niloticus, OnCD166). OnCD166 contains an open reading frame of 1671 bp that encodes a peptide of 556 amino acids, and contains five consecutive extracellular immunoglobulin domains. It's tissue distribution and expression patterns after S. agalactiae challenge were also investigated. OnCD166 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnCD166 expressions were significantly up-regulated in all tested immune tissues. Meanwhile, the recombinant OnCD166 (rOnCD166E) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnCD166E could promote phagocytosis of macrophages. Taken together, our results illustrated that OnCD166 might as a receptor involved in the immune recognition and phagocytosis against invading pathogen, which play important roles in the immune responses of Nile tilapia against bacterial pathogens.
Asunto(s)
Cíclidos , Enfermedades de los Peces , Infecciones Estreptocócicas , Animales , Regulación de la Expresión Génica , Inmunidad , Macrófagos , Streptococcus agalactiae/fisiología , Proteínas de Peces/genéticaRESUMEN
Nocardia seriolae is the main pathogen of fish nocardiosis. In our previous study, alanine dehydrogenase was identified as a potential virulence factor of N. seriolae. On the basis of this fact, the alanine dehydrogenase gene of N. seriolae (NsAld) was knocked out to establish the strain ΔNsAld for vaccine development against fish nocardiosis in this study. The LD50 of strain ΔNsAld was 3.90 × 105 CFU/fish, higher than that of wild strain (5.28 × 104 CFU/fish) significantly (p < 0.05). When the strain ΔNsAld was used as a live vaccine to immunize hybrid snakehead (Channa maculata â × Channa argus â) at 2.47 × 105 CFU/fish by intraperitoneal injection, the non-specific immune indexes (LZM, CAT, AKP, ACP and SOD activities), specific antibody (IgM) titers and several immune-related genes (CD4, CD8α, IL-1ß, MHCIα, MHCIIα and TNFα) were up-regulated in different tissues, indicating that this vaccine could induce humoral and cell-mediated immune responses. Furthermore, the relative percentage survival (RPS) of ΔNsAld vaccine was calculated as 76.48% after wild N. seriolae challenge. All these results suggest that the strain ΔNsAld could be a potential candidate for live vaccine development to control fish nocardiosis in aquaculture.
Asunto(s)
Enfermedades de los Peces , Nocardiosis , Animales , Alanina-Deshidrogenasa/genética , Eliminación de Gen , Nocardiosis/prevención & control , Nocardiosis/veterinaria , Nocardiosis/genética , Peces/genética , Desarrollo de VacunasRESUMEN
The enteritis is a common disease in fish farming, but the pathogenesis is still not fully understood. The aim of the present study was to investigate the inducement of Dextran Sulfate Sodium Salt (DSS) intestinal inflammation on Orange-spotted grouper (Epinephelus coioides). The fish were challenged with 200 µl 3% DSS via oral irrigation and feeding, an appropriate dose based on the disease activity index of inflammation. The results indicated that the inflammatory responses induced by DSS were closely associated with the expression of pro-inflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL16, IL-10 and tumor necrosis factor α (TNF-α), as well as NF-κB and myeloperoxidase (MPO) activity. At day5 after DSS treatment, the highest levels of all parameters were observed. Also, the severe intestinal lesions (intestinal villus fusion and shedding), strong inflammatory cell infiltration and microvillus effacement were seen through histological examination and SEM (scanning electronic microscopy) analysis. During the subsequent 18 days of the experimental period, the injured intestinal villi were gradually recovery. These data is beneficial to further investigate the pathogenesis of enteritis in farmed fish, which is helpful for the control of enteritis in aquaculture.
Asunto(s)
Lubina , Enteritis , Animales , Lubina/metabolismo , Sulfato de Dextran/efectos adversos , Inflamación , Enteritis/inducido químicamente , Enteritis/veterinaria , Citocinas/metabolismoRESUMEN
BACKGROUND: Circular RNA spi-1 proto-oncogene (circ-SPI1) regulates cell proliferation, apoptosis, and bone marrow differentiation in acute myeloid leukemia (AML). This study aimed to assess the relationship of circ-SPI1 expression with the clinical features, induction therapy response, and survival of AML patients. METHODS: In total, 80 AML patients were included with bone marrow (BM) samples collected at baseline and after induction therapy. Additionally, 20 healthy donors (HDs) and 20 disease controls (DCs) were enrolled with BM samples collected after enrollment. BM circ-SPI1 expression was detected by reverse-transcription quantitative polymerase chain reaction assay. RESULTS: Circ-SPI1 expression was highest in AML patients, moderate in DCs, and lowest in HDs (median (interquartile range): 3.01 [2.02-4.14] versus 1.71 [1.01-2.85] versus 0.98 [0.74-1.71]) (p < 0.001). Moreover, lower circ-SPI1 expression was related to its decreased located gene SPI1 expression (p = 0.029), white blood cells (WBC) < 18.8 × 109 /L (p = 0.010), trisomy 8 (p = 0.025), and more favorable risk stratification (p = 0.014) in AML patients. Additionally, circ-SPI1 expression was reduced in AML patients after induction therapy (p < 0.001), and its low expression after induction therapy was correlated with the achievement of complete remission (p < 0.001). Furthermore, circ-SPI1 decline ≥30% during therapy (versus <30%) was independently related to longer event-free survival (EFS) (hazard ratio (HR): 0.445, p = 0.028) and overall survival (OS) (HR: 0.319, p = 0.025) in AML patients. CONCLUSION: Decreased circ-SPI1 expression is related to lower WBC, favorable risk stratification, and better therapy response; moreover, its decline during therapy is an independent factor to predict longer EFS and OS in AML patients.
Asunto(s)
Leucemia Mieloide Aguda , ARN Circular , Humanos , Quimioterapia de Inducción , Leucemia Mieloide Aguda/genética , Oncogenes , Médula Ósea , PronósticoRESUMEN
BACKGROUND: Sunitinib resistance can be classified into primary and secondary resistance. While accumulating research has indicated several underlying factors contributing to sunitinib resistance, the precise mechanisms in renal cell carcinoma are still unclear. METHODS: RNA sequencing and m6A sequencing were used to screen for functional genes involved in sunitinib resistance. In vitro and in vivo experiments were carried out and patient samples and clinical information were obtained for clinical analysis. RESULTS: We identified a tumor necrosis factor receptor-associated factor, TRAF1, that was significantly increased in sunitinib-resistant cells, resistant cell-derived xenograft (CDX-R) models and clinical patients with sunitinib resistance. Silencing TRAF1 increased sunitinib-induced apoptotic and antiangiogenic effects. Mechanistically, the upregulated level of TRAF1 in sunitinib-resistant cells was derived from increased TRAF1 RNA stability, which was caused by an increased level of N6-methyladenosine (m6A) in a METTL14-dependent manner. Moreover, in vivo adeno-associated virus 9 (AAV9) -mediated transduction of TRAF1 suppressed the sunitinib-induced apoptotic and antiangiogenic effects in the CDX models, whereas knockdown of TRAF1 effectively resensitized the sunitinib-resistant CDXs to sunitinib treatment. CONCLUSIONS: Overexpression of TRAF1 promotes sunitinib resistance by modulating apoptotic and angiogenic pathways in a METTL14-dependent manner. Targeting TRAF1 and its pathways may be a novel pharmaceutical intervention for sunitinib-treated patients.
Asunto(s)
Adenosina , Carcinoma de Células Renales , Neoplasias Renales , Metiltransferasas , Sunitinib , Factor 1 Asociado a Receptor de TNF , Adenosina/análogos & derivados , Inhibidores de la Angiogénesis/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Metiltransferasas/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Sunitinib/farmacología , Factor 1 Asociado a Receptor de TNF/genética , Factor 1 Asociado a Receptor de TNF/metabolismoRESUMEN
The glutamyl endopeptidase homolog of Nocardia seriolae (GluNS) has been proved to be a potential virulence factor in our previous study. Present investigation was carried out to construct an attenuated N. seriolae strain by deletion with GluNS gene and evaluate its protective immunity in head snakehead. A deletion strain (NS-ΔGluNS) was established by knockout of gene GluNS from wild strain N. seriolae ZJ0503 via homologous recombination. The LD50 of NS-ΔGluNS in 3.41 × 106 cfu/mL was significantly increased than that of wild strain in 4.75 × 105 cfu/mL, indicating that the virulence of N. seriolae has been attenuated with the knockout of GluNS. Meanwhile, applying NS-ΔGluNS as an attenuated live vaccine to immune hybrid snakehead, the non-specific immunity parameters (serum LYZ, POD, ACP, and AKP activities), specific antibody (IgM) titers production and immune-related genes (MHCIα, CD4, and IL-8) expression were up-regulated in different tissues, which indicated that they were able to trigger humoral and cell-mediated immune responses. Furthermore, the protective efficacy in hybrid snakehead after vaccination with NS-ΔGluNS shown 73.53% relative percentage survival (RPS). Taken together, the attenuated NS-ΔGluNS was obtained successfully and it could elicit strong immune response and supply protective efficacy to hybrid snakehead against N. seriolae wild strain.
Asunto(s)
Enfermedades de los Peces , Nocardiosis , Animales , Inmunoglobulina M , Interleucina-8 , Nocardia , Serina Endopeptidasas , Vacunas Atenuadas , Factores de Virulencia/genéticaRESUMEN
Nocardia seriolae, a Gram-positive facultative intercellular pathogen, has been identified as the causative agent of fish nocardiosis, causing substantial mortality and morbidity of a wide range of fish species. Looking into that fact, the effective vaccine against this pathogen is urgently needed to control the significant losses in aquaculture practices. In order to induct attenuated strains for developing the potential live vaccines, the mutagenic N. seriolae strain S-250 and U-20 were obtained from wild-type strain ZJ0503 through continuous passaging and ultraviolet (UV) irradiation, respectively. Additionally, the biological characteristic, virulence, stability, mediating immune response and supplying protective efficacy to hybrid snakehead of the S-250 and U-20 strains were determined in the present study. The results showed that U-20 strain displayed dramatic changes in morphological characteristic and significant decreased in the virulence to hybrid snakehead, while that of S-250 strain had no obvious different in comparison to ZJ0503 strain. When hybrid snakehead were intraperitoneally injected with ZJ0503, S-250 and U-20 strains at their respective sub-clinical dosage, the non-specific immunity parameters (serum LYZ, POD, ACP, AKP and SOD activities), specific antibody (IgM) titers production and immune-related genes (CC1, CC2, IL-1ß, IL-8, TNFα, IFNγ, MHCIα, MHCIIα, CD4, CD8α, TCRα and TCRß) expression were up-regulated, indicating that they were able to trigger humoral and cell-mediated immune responses. Furthermore, the protective efficacy in hybrid snakehead after vaccination with ZJ0503, S-250 and U-20 strains, in terms of relative percentage survival (RPS), were 28.85%, 56.89% and 89.65% respectively. Taken together, two attenuated N. seriolae strains S-250 and U-20 were obtained successfully and they could elicit strong immune response and supply protective efficacy to hybrid snakehead against N. seriolae, which suggested that these two attenuated strains were the potential candidates for live vaccine development to control fish nocardiosis in aquaculture.
Asunto(s)
Enfermedades de los Peces , Nocardiosis , Nocardia , Animales , Nocardiosis/prevención & control , Nocardiosis/veterinaria , Nocardiosis/genética , Peces , Vacunas AtenuadasRESUMEN
Mammals TRAF2 played a dual role in several immune signaling transduction processes. In this study, TRAF2 was cloned from Nile tilapia, Oreochromis niloticus, which named OnTRAF2. The open reading frame was 1797 bp, encoding 598 amino acids. Amino acid alignment and phylogenetic analysis indicated that OnTRAF2 showed relatively low identify with other teleost TRAF2 proteins, with the exception of TRAF2s from Epinephelus coioides. In healthy tilapia, OnTRAF2 was expressed widely in all the examined tissues, which had highest expression level in the brain. After Streptococcus agalactiae infection, the expression level of OnTRAF2 was increased significantly at different times in several organs, implying that OnTRAF2 may be involved in host defense against S. agalactiae infection. The result of subcellular localization showed that OnTRAF2 presented in cytoplasm and nucleus of HEK293T cells. Additionally, overexpression of OnTRAF2 significantly decreased the transcriptional activity of the NF-κB reporter in HEK293T cells, yeast two-hybrid results revealed that OnTRAF2 had no interaction with E3 ubiquitin ligase OnNEDD4. These results indicated that OnTRAF2 played important function during bacterial infection, and negatively mediated the immune signaling transduction in Nile tilapia, while the mechanism need further study.
Asunto(s)
Cíclidos , Enfermedades de los Peces , Infecciones Estreptocócicas , Animales , Proteínas de Peces , Regulación de la Expresión Génica , Células HEK293 , Humanos , Mamíferos/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Filogenia , Infecciones Estreptocócicas/genética , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiae , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Natural killer lysin (Nklysin) is a small molecule antimicrobial peptide produced by natural killer cells and T lymphocytes and widely expressed in vertebrates. Homologues of Nklysin have been found in several fish, but only several of biological activity was identified. In this study, we characterized a Nklysin from grouper (Epinephelus coioides), and explored its expression pattern and biological function in bacterial infection. We also investigated the role of Nklysin in viral replication and maturation. The nklysin gene of grouper encodes a 169 amino acid, sharing 92.90% identity to H. septemfasciatus NKlysin protein, containing a saposin B domain and six well-conserved cysteine residues that necessary for antimicrobial activity by forming three intrachain disulfide bonds. Analysis of qRT-PCR revealed that nklysin gene widely expressed in all tested tissues with the higher expressions in spleen. After bacterial challenge, the nklysin gene expression significantly varied in different tissues. In addition, a large-scale of the recombinant Nklysin protein was secreted in Pichia pastoris strain GS115. The MIC assay showed that the Nklysin protein directly inhibited growth of several pathogens, including Proteus mirabilis, Bacillus subtilis, Salmonella typhi, Escherichia coli, Shigella sonnei and Streptococcus agalactiae. Further analysis showed the Nklysin protein over-expression might prevent viral genes transcriptions and replication in FHM cells. Our findings suggested that the Nklysin of grouper might be a potential agent for antibacterial and antiviral infection in the future.
Asunto(s)
Lubina , Infecciones por Virus ADN , Enfermedades de los Peces , Animales , Lubina/genética , Lubina/metabolismo , Proteínas de Peces/química , Antivirales/farmacología , Secuencia de Aminoácidos , Antibacterianos/farmacología , Antibacterianos/metabolismo , Escherichia coli/genética , Proteínas Recombinantes/genética , Filogenia , Regulación de la Expresión GénicaRESUMEN
Neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) was a member of HECT E3 ubiquitin ligases, which participated in various biological processes. In this study, a NEDD4 was identified and analyzed in Nile tilapia, Oreochromis niloticus (OnNEDD4) and its open reading frame was 2781 bp, encoding 926 amino acids. Three conserved structure features were found in OnNEDD4, including C2 domain, WW domains and HECT domain. OnNEDD4 was constitutively expressed in all examined tissues and the highest expression level was observed in thymus. After Streptococcus agalactiae stimulation, OnNEDD4 was significantly induced in several tissues, including thymus, intestine, blood and gill. Moreover, yeast two-hybrid assay shown OnNEDD4 could interact with extracellular region of OnCD40, but this interaction didn't affect the phagocytosis of monocytes/macrophages (MO/MΦ) to S. agalactiae and A. hydrophila. Taken together, the present study suggested that OnNEDD4 participate in CD40-mediated immune response excluding phagocytosis.
Asunto(s)
Cíclidos , Enfermedades de los Peces , Infecciones Estreptocócicas , Animales , Proteínas de Peces/química , Regulación de la Expresión Génica , Secuencia de Aminoácidos , Streptococcus agalactiae/fisiología , Clonación Molecular , Inmunidad Innata/genéticaRESUMEN
Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein that interacts with Bcl-2 and mediate cell death. However, little is known about the roles of fish BAG3 during viral infection. In this study, we characterized a BAG3 homolog from orange-spotted grouper (Epinephelus coioides) (EcBAG3) and investigated its roles during viral infection. The EcBAG3 protein encoded 579 amino acids with typical WW, PXXP and BAG domains, which shared high identities with reported fish BAG3. Quantitative real-time PCR (qRT-PCR) analysis revealed that EcBAG3 was highly expressed in brain and heart. And the expression of EcBAG3 was significantly up-regulated after red-spotted grouper nervous necrosis virus (RGNNV) stimulation in vitro. EcBAG3 overexpression could promoted the expression of viral genes (coat protein (CP) and RNA-dependent RNA polymerase (RdRp)), which was enhanced by co-transfection with Hsp70 and Hsp22. Also, EcBAG3 overexpression up-regulated the expression of LC3-â ¡ and down-regulated the expression of Bax and BNIP3, the IFN- (IRF1, IRF3, IRF7, IFP35, Mx1) or inflammation-related (IL-1ß and TNFα) factors, as well as decreased the activities of NF-κB, ISRE and IFN-3. While knockdown of EcBAG3 decreased the transcripts of RGNNV CP gene and RdRp gene. Further studies showed that EcBAG3 knockdown impaired the expression level of autophagy factor LC3-â ¡, and promoted the expression level of Bax and BNIP3, inflammatory factors and interferon factors. These data indicate that EcBAG3 can affect viral infection through modulating virus-induced cell death, regulating the expression of IFN- and inflammation-related factors, which will be helpful to further explore the immune response of fish during viral infection.
Asunto(s)
Lubina , Enfermedades de los Peces , Nodaviridae , Infecciones por Virus ARN , Virosis , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Regulación de la Expresión Génica , Inmunidad Innata/genética , Nodaviridae/fisiología , Alineación de SecuenciaRESUMEN
According to the whole-genome bioinformatics analysis, a heme-binding protein from Nocardia seriolae (HBP) was found. HBP was predicted to be a bacterial secretory protein, located at mitochondrial membrane in eukaryotic cells and have a similar protein structure with the heme-binding protein of Mycobacterium tuberculosis, Rv0203. In this study, HBP was found to be a secretory protein and co-localized with mitochondria in FHM cells. Quantitative analysis of mitochondrial membrane potential value, caspase-3 activity, and transcription level of apoptosis-related genes suggested that overexpression of HBP protein can induce cell apoptosis. In conclusion, HBP was a secretory protein which may target to mitochondria and involve in cell apoptosis in host cells. This research will promote the function study of HBP and deepen the comprehension of the virulence factors and pathogenic mechanisms of N. seriolae.
Asunto(s)
Enfermedades de los Peces , Nocardiosis , Nocardia , Animales , Apoptosis , Proteínas Bacterianas/metabolismo , Enfermedades de los Peces/microbiología , Proteínas de Unión al Hemo , Nocardia/genética , Nocardia/metabolismo , Nocardiosis/microbiología , Nocardiosis/veterinariaRESUMEN
Drug options for the life-threatening Cushing's disease are limited, and surgical resection or radiation therapy is not invariably effective. Testicular receptor 4 (TR4) has been identified as a novel drug target to treat Cushing's disease. We built the structure model of TR4 and searched the TR4 antagonist candidate via in silico virtual screening. Bexarotene was identified as an antagonist of TR4 that can directly interact with TR4 ligand binding domain (TR4-LBD) and induces a conformational change in the secondary structure of TR4-LBD. Bexarotene suppressed AtT-20 cell growth, proopiomelanocortin (POMC) expression and adrenocorticotropin (ACTH) secretion. Mechanism dissection revealed that bexarotene could suppress TR4-increased POMC expression via promoting the TR4 translocation from the nucleus to the cytoplasm. This TR4 translocation might then result in reducing the TR4 binding to the TR4 response element (TR4RE) on the 5' promoter region of POMC. Results from in vivo mouse model also revealed that oral bexarotene administration markedly suppressed ACTH-secreting tumour growth, adrenal enlargement and the secretion of ACTH and corticosterone in mice with already established tumours. Together, these results suggest that bexarotene may be developed as a potential novel therapeutic drug to better suppress Cushing's disease.
Asunto(s)
Bexaroteno/farmacología , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/antagonistas & inhibidores , Proopiomelanocortina/metabolismo , Transducción de Señal/efectos de los fármacos , Adenoma Hipofisario Secretor de ACTH , Hormona Adrenocorticotrópica/biosíntesis , Animales , Bexaroteno/química , Sitios de Unión , Línea Celular Tumoral , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Conformación Molecular , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/química , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Hipersecreción de la Hormona Adrenocorticotrópica Pituitaria (HACT) , Proopiomelanocortina/genética , Unión Proteica , Transporte de Proteínas , Relación Estructura-Actividad , Transcripción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metabolism reprograming is a hallmark of cancer and plays an important role in tumor progression. The aberrant metabolism in renal cell carcinoma (RCC) leads to accumulation of the oncometabolite L-2-hydroxyglurate (L-2HG). L-2HG has been reported to inhibit the activity of some α-ketoglutarate-dependent dioxygenases such as TET enzymes, which mediate epigenetic alteration, including DNA and histone demethylation. However, the detailed functions of L-2HG in renal cell carcinoma have not been investigated thoroughly. In our study, we found that L-2HG was significantly elevated in tumor tissues compared to adjacent tissues. Furthermore, we demonstrated that L-2HG promoted vasculogenic mimicry (VM) in renal cancer cell lines through reducing the expression of PHLDB2. A mechanism study revealed that activation of the ERK1/2 pathway was involved in L-2HG-induced VM formation. In conclusion, these findings highlighted the pathogenic link between L-2HG and VM and suggested a novel therapeutic target for RCC.