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1.
Plants (Basel) ; 13(12)2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38931090

RESUMO

Leaf nutrient resorption is a critical process in plant nutrient conservation during leaf senescence. However, the ecological mechanisms underlying the large variability in nitrogen (NRE) and phosphorous (PRE) resorption efficiencies among trees remain poorly understood. We conducted a comprehensive study on NRE and PRE variability using 61 tree individuals of 10 temperate broad-leaved tree species. Three potentially interrelated intrinsic ecological mechanisms (i.e., leaf senescence phenology, leaf pigments, and energy residual) were verified. We found that a delayed leaf senescence date, increased degradation of chlorophylls and carotenoids, biosynthesis of anthocyanins, and reduced nonstructural carbohydrates were all positively correlated with NRE and PRE at the individual tree level. The intrinsic factors affecting resorption efficiency were ranked in decreasing order of importance: leaf pigments > energy residual > senescence phenology. These factors explained more variability in NRE than in PRE. Our findings highlight the significance of these three ecological mechanisms in leaf nutrient resorption and have important implications for understanding how nutrient resorption responds to climate change.

2.
Artigo em Inglês | MEDLINE | ID: mdl-38290458

RESUMO

Objective: To study the association of H. pylori infection with colorectal adenomas. Methods: Web searches of PubMed, Embase, and Scopus databases for randomized controlled trials, class-experimental studies, and cohort studies on the association between H. pylori and colorectal adenomas were performed from May 2000 to May 2023. Literature was screened based on inclusion and exclusion criteria, data were extracted and evaluated for quality, and statistical analyses were performed using RevMan 5.2 software. Results: A total of 15 studies were included, and meta-analysis showed a statistically significant difference between colorectal neoplastic polyp cases in the H. pylori-positive and H. pylori-negative groups [OR=1.80, 95%CI: (1.31, 2.47), P < .01, I2 = 95%]. Analysis based on subgroups of different H. pylori detection methods showed that the correlation between H. pylori infection and colorectal polyp incidence is not affected by their detection methods, with serological detection subgroup: [OR=0.13, 95%CI: (0.05, 0.21), P < .01, I2 = 88%], and non-serological detection subgroup: [OR=0.13, 95%CI: (0.04, 0.22), P < .01, I2 = 95%]. Subgroup analysis of pathological types showed that H. pylori infection is not significantly associated with the development of non-neoplastic polyps [OR=1.47, 95%CI: 0.98-2.22, P = .06], whereas it is correlated with the development of neoplastic polyps [95%CI: 1.69-3.22, P < .01]. In the subgroup analysis of geographic differences in the population, H. pylori infection was correlated with the development of colorectal polyps in different geographic populations (P < .01). Conclusion: H. pylori infection is a risk factor for colorectal polyp neoplasia, its infection is associated with colorectal neoplasia, and the correlation is not affected by the different methods of H. pylori detection and the different geographic regions of the population.

3.
Mol Oncol ; 18(3): 580-605, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38060346

RESUMO

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.


Assuntos
Neoplasias , Proteína Supressora de Tumor p14ARF , Animais , Camundongos , Proteína BRCA1/genética , Cromatina , DNA/metabolismo , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Neoplasias/genética , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo
4.
Trop Anim Health Prod ; 56(1): 19, 2023 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-38110604

RESUMO

In the current study, the role of the ovine IGF2 as a potential candidate gene was investigated as though marker-assisted selection in Chinese Tibetan sheep. The Sanger DNA sequencing method explored five single nucleotide polymorphisms (SNPs) in 5'UTR of the ovine IGF2 gene (C15640T, G15801A, G15870A, C15982G and G15991A) in Chinese Tibetan sheep. The frequencies of four SNPs were within the Hardy-Weinberg Equilibrium (chi-square test) except C15982G. The statistical analysis indicated that the C15640T and G15801A were significantly associated with body height, body length, chest circumference, and body weight (P < 0.05 or P < 0.01). Furthermore, C15982G variant exhibited significant correlation with the body weight (P < 0.01). These findings suggests that the promoter variants of IGF2 gene could be used as a candidate gene through marker-assisted selection for the body weight and body measurement traits in Tibetan sheep breeding program.


Assuntos
Peptídeos Semelhantes à Insulina , Polimorfismo de Nucleotídeo Único , Ovinos/genética , Animais , Tibet , Fenótipo , Peso Corporal/genética , Genótipo
5.
Biomimetics (Basel) ; 8(5)2023 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-37754169

RESUMO

A quick and effective way of segmenting images is the Otsu threshold method. However, the complexity of time grows exponentially as the number of thresolds rises. The aim of this study is to address the issues with the standard threshold image segmentation method's low segmentation effect and high time complexity. The two mutations differential evolution based on adaptive control parameters is presented, and the twofold mutation approach and adaptive control parameter search mechanism are used. Superior double-mutation differential evolution views Otsu threshold picture segmentation as an optimization issue, uses the maximum interclass variance technique as the objective function, determines the ideal threshold, and then implements multi-threshold image segmentation. The experimental findings demonstrate the robustness of the enhanced double-mutation differential evolution with adaptive control parameters. Compared to other benchmark algorithms, our algorithm excels in both image segmentation accuracy and time complexity, offering superior performance.

6.
Sensors (Basel) ; 23(16)2023 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-37631627

RESUMO

Traffic management is a critical task in software-defined IoT networks (SDN-IoTs) to efficiently manage network resources and ensure Quality of Service (QoS) for end-users. However, traditional traffic management approaches based on queuing theory or static policies may not be effective due to the dynamic and unpredictable nature of network traffic. In this paper, we propose a novel approach that leverages Graph Neural Networks (GNNs) and multi-arm bandit algorithms to dynamically optimize traffic management policies based on real-time network traffic patterns. Specifically, our approach uses a GNN model to learn and predict network traffic patterns and a multi-arm bandit algorithm to optimize traffic management policies based on these predictions. We evaluate the proposed approach on three different datasets, including a simulated corporate network (KDD Cup 1999), a collection of network traffic traces (CAIDA), and a simulated network environment with both normal and malicious traffic (NSL-KDD). The results demonstrate that our approach outperforms other state-of-the-art traffic management methods, achieving higher throughput, lower packet loss, and lower delay, while effectively detecting anomalous traffic patterns. The proposed approach offers a promising solution to traffic management in SDNs, enabling efficient resource management and QoS assurance.

7.
Reprod Domest Anim ; 58(5): 657-661, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36808762

RESUMO

In the current study, the difference between the sex-sorted and non-sex-sorted frozen semen of Holstein Friesian breed cattle was investigated. Significant variation (p < .05) was found in the semen quality parameters such as motility; vitality; acrosome integrity rate; the anti-oxidative enzyme activity including GSH (glutathione); SOD (superoxide dismutase); CAT (catalase); GSH-Px (glutathione peroxidase) and the rate of fertilization. The results showed that the sperm acrosome integrity and motility of the non-sorted sperm were higher compared to sex-sorted sperm (p < .05). The linearity index and mean coefficient analysis revealed that the percentage of 'grade a' in sex-sorted sperm were significantly (p < .05) lower than non-sorted sperm. Interestingly, low SOD level and high CAT level was found in the non-sexed semen than in the sexed semen (p < .05). Furthermore, the GSH and GSH-Px activity in the sexed semen was found lower than the non-sexed semen (p < .05). In conclusion, sperm motility characteristics were lower in sex-sorted semen than in non-sex-sorted semen. This might be related to the complex process of sexed semen production, which could reduce sperm motility and movement characteristics, acrosomal integrity, CAT, SOD, GSH and GSH-Px, and finally lead to the decline in the fertilization rate.


Assuntos
Preservação do Sêmen , Sêmen , Bovinos , Masculino , Animais , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides , Criopreservação/veterinária , Criopreservação/métodos , Espermatozoides , Glutationa , Superóxido Dismutase
8.
RSC Adv ; 13(7): 4553-4563, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36760271

RESUMO

The encapsulation of fullerenes by carbon nanorings has gained increasing attention because of the unique molecular structure and special properties of the formed complexes. The host-guest interactions between the fullerenes and the carbon nanorings can influence the metal ion orientation and the molecular electronic structure. In this study, we hooped a series of carbide cluster metallofullerenes, namely Sc2C2@C2v(5)-C80, Sc2C2@C3v(8)-C82, and Sc2C2@D2d(23)-C84, with molecular carbon nanorings of [12]cycloparaphenylene ([12]CPP) and perfluoro[12]cycloparaphenylene (PF[12]CPP). The formed complexes were computationally studied via dispersion-corrected density functional theory calculations. The results showed that the deformation rate of PF[12]CPP after the formation of the fullerene-containing complexes was significantly smaller than that of [12]CPP. The binding energy and thermodynamic information showed that PF[12]CPP was more suitable for fullerene encapsulation. Moreover, charge population analysis showed that PF[12]CPP transferred more electrons to Sc2C2@C2n (n = 40, 41, and 42) compared with [12]CPP. Energy decomposition and real-space function analyses of host-guest interactions revealed the characteristics and nature of the noncovalent interactions in the supramolecules. These results provide theoretical support for the study of host-guest systems based on metallofullerenes.

9.
Oncogene ; 41(44): 4877-4892, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36192478

RESUMO

PTEN is frequently mutated in human cancers, which leads to the excessive activation of PI3K/AKT signaling and thus promotes tumorigenesis and drug resistance. Met1-linked ubiquitination (M1-Ubi) is also involved in cancer progression, but the mechanism is poorly defined. Here we find that HOIP, one important component of linear ubiquitin chain assembly complex (LUBAC), promotes prostate cancer (PCa) progression by enhancing AKT signaling in a PTEN-dependent manner. Mechanistically, PTEN is modified by M1-Ubi at two sites K144 and K197, which significantly inhibits PTEN phosphatase activity and thus accelerates PCa progression. More importantly, we identify that the high-frequency mutants PTENR173H and PTENR173C in PCa patients showed the enhanced level of M1-Ubi, which impairs PTEN function in inhibition of AKT phosphorylation and cell growth. We also find that HOIP depletion sensitizes PCa cells to therapeutic agents BKM120 and Enzalutamide. Furthermore, the clinical data analyses confirm that HOIP is upregulated and positively correlated with AKT activation in PCa patient specimen, which may promote PCa progression and increase the risk of PCa biochemical relapse. Together, our study reveals a key role of PTEN M1-Ubi in regulation of AKT activation and PCa progression, which may propose a new strategy for PCa therapy.


Assuntos
PTEN Fosfo-Hidrolase , Neoplasias da Próstata , Masculino , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Ubiquitinação
10.
J Fish Dis ; 45(7): 1033-1043, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35475515

RESUMO

Largemouth bass ranavirus disease (LMBVD) caused by largemouth bass ranavirus (LMBV) has resulted in severe economic losses in the largemouth bass (Micropterus salmoides) farming industry in China. Early and accurate diagnosis is the key measure for the prevention and control of LMBVD. In this study, a quantitative polymerase chain reaction (qPCR) and a real-time recombinase-aided amplification (real-time RAA) assay were established for the detection of LMBV. The sensitivity and specificity of these two methods, and the efficacy for detection of LMBV from clinical samples were also evaluated. Results showed that the real-time RAA reaction was completed in <30 min at 39℃ with a detection limit of 58.3 copies, while qPCR reaction required 60 min with a detection limit of 5.8 copies. Both methods were specific for LMBV, where no cross-reactions observed with the other tested fish pathogens. Comparing the amplification results of both assays to the results obtained by virus isolation using 53 clinical tissue samples, results showed that the clinical sensitivity of real-time RAA and qPCR were 93.75% and 100% respectively, and the clinical specificity of both were 100%. Our results showed that qPCR is more suitable for quantitative analysis and accurate detection of LMBV in the laboratory, while real-time RAA is more suitable as a point-of-care diagnostic tool for on-site detection and screening of LMBV under farm conditions and in poorly equipped laboratories.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Ranavirus , Animais , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/diagnóstico , Ranavirus/genética , Recombinases , Sensibilidade e Especificidade
11.
Nat Commun ; 12(1): 5416, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518544

RESUMO

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. However, whether and how hypoxia regulates overall mRNA homeostasis is unclear. Here we show that hypoxia inhibits global-mRNA decay in cancer cells. Mechanistically, hypoxia induces the interaction of AGO2 with LUBAC, the linear ubiquitin chain assembly complex, which co-localizes with miRNA-induced silencing complex and in turn catalyzes AGO2 occurring Met1-linked linear ubiquitination (M1-Ubi). A series of biochemical experiments reveal that M1-Ubi of AGO2 restrains miRNA-mediated gene silencing. Moreover, combination analyses of the AGO2-associated mRNA transcriptome by RIP-Seq and the mRNA transcriptome by RNA-Seq confirm that AGO2 M1-Ubi interferes miRNA-targeted mRNA recruiting to AGO2, and thereby facilitates accumulation of global mRNAs. By this mechanism, short-term hypoxia may protect overall mRNAs and enhances stress tolerance, whereas long-term hypoxia in tumor cells results in seriously changing the entire gene expression profile to drive cell malignant evolution.


Assuntos
Proteínas Argonautas/genética , Regulação Neoplásica da Expressão Gênica , Homeostase/genética , Metionina/genética , RNA Mensageiro/genética , Ubiquitinação , Células A549 , Proteínas Argonautas/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Inativação Gênica , Células HEK293 , Células HeLa , Humanos , Hipóxia , Metionina/metabolismo , MicroRNAs/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células PC-3 , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo
12.
Mol Oncol ; 15(1): 279-298, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33155366

RESUMO

Deubiquitinase BAP1 is an important tumor suppressor in several malignancies, but its functions and critical substrates in prostate cancer (PCa) remain unclear. Here, we report that the mRNA and protein expression levels of BAP1 are downregulated in clinical PCa specimens. BAP1 can physically bind to and deubiquitinate PTEN, which inhibits the ubiquitination-mediated degradation of PTEN and thus stabilizes PTEN protein. Ectopically expressed BAP1 in PCa cells increases PTEN protein level and subsequently inhibits the AKT signaling pathway, thus suppressing PCa progression. Conversely, knockdown of BAP1 in PCa cells leads to the decrease in PTEN protein level and the activation of the Akt signaling pathway, therefore promoting malignant transformation and cancer metastasis. However, these can be reversed by the re-expression of PTEN. More importantly, we found that BAP1 protein level positively correlates with PTEN in a substantial fraction of human cancers. These findings demonstrate that BAP1 is an important deubiquitinase of PTEN for its stability and the BAP1-PTEN signaling axis plays a crucial role in tumor suppression.


Assuntos
Progressão da Doença , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/genética , Ligação Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
13.
Mol Carcinog ; 59(10): 1174-1187, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32805084

RESUMO

Long noncoding RNAs (LncRNAs) have emerged as important players in cancer biology. Increasing evidence suggests that LncRNAs are frequently dysregulated in cancer and may function as oncogenes or tumor suppressors. Urothelial carcinoma associated 1 (UCA1), a LncRNA, firstly identified in bladder transitional cell carcinoma, seems to act as an oncogene in many different types of human cancers by promoting cell proliferation and migration. In this study, we revealed a novel biological function of UCA1, which was different from that reported by previous studies, was responsible for maintaining the low-tumorigenic, nonmetastatic phenotypes in primary prostate epithelial cells. UCA1 could stabilize E-cadherin protein by preventing the interaction between E-cadherin and its E3 ligase MDM2, which suppressed MDM2-mediated ubiquitination and degradation of E-cadherin. In addition, we also found that UCA1 acted as a sponge of miR-296-3p, which targeted E-cadherin gene CDH1 messenger RNA at the posttranscription level. Taken together, these findings demonstrated that UCA1 had a new important role in effectively keeping E-cadherin at a high level through a dual mechanism, which maintained primary prostate cancer cells at the low-tumorigenic and nonmetastatic status.


Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias da Próstata/patologia , RNA Longo não Codificante/genética , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Caderinas/genética , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Neoplasia ; 21(7): 676-688, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31125786

RESUMO

Csk, a non-receptor tyrosine kinase, serves as an indispensable negative regulator of the Src family kinases (SFKs). However, little is known about regulation of Csk expression so far. SUMOylation, a reversible post-translational modification, has been shown to regulate many biological processes especially in tumor progression. Here we report that Csk is covalently modified by SUMO1 at lysine 53 (K53) both in vitro and in vivo. Treatment with hydrogen peroxide inhibited this modification to a certain extent, but PIAS3, identified as the main specific SUMO E3 ligase for Csk, could significantly enhance SUMO1-Csk level. In addition, phosphorylation at Ser364, the active site in Csk, had no effect on this modification. Ectopic expression of SUMO-defective mutant, Csk K53R, inhibited tumor cell growth more potentially than Csk wild-type. Consistent with the biological phenotype, the SUMO modification of Csk impaired its activity to interact with Cbp (Csk binding protein) leading to decreased c-Src phosphorylation at Y527. Our results suggest that SUMOylation of Csk mainly at lysine 53 negatively modulates its tumor suppressor function by reducing its binding with Cbp and consequently, inducing c-Src activation.


Assuntos
Proteína Tirosina Quinase CSK/genética , Neoplasias/genética , Proteína SUMO-1/genética , Sumoilação/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Lisina/genética , Chaperonas Moleculares/química , Neoplasias/patologia , Fosforilação , Ligação Proteica/genética , Proteínas Inibidoras de STAT Ativados/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Quinases da Família src/genética
15.
Oncogene ; 38(9): 1410-1431, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30305728

RESUMO

Argonaute2 (AGO2) is an effector of small RNA mediated gene silencing. Increasing evidence show that post-translational modifications of AGO2 can change miRNA activity at specific or global levels. Among the six mature miRNAs that are encoded by miR-17-92, miR-19b1 is the most powerful to exert the oncogenic properties of the entire cluster. Here we identify that AGO2 can be acetylated by P300/CBP and deacetylated by HDAC7, and that acetylation occurs at three sites K720, K493, and K355. Mutation of K493R/K720R, but not K355R at AGO2, inhibits miR-19b biogenesis. We demonstrate that acetylation of AGO2 specifically increases its recruiting pre-miR-19b1 to form the miPDC (miRNA precursor deposit complex), thereby to enhance miR-19b maturation. The motif UGUGUG in the terminal-loop of pre-miR-19b1, as a specific processing feature that is recognized and bound by acetylated AGO2, is essential for the assembly of miRISC (miRNA-induced silencing complex) loading complex. Analyses on public clinical data, xenograft mouse models, and IHC and ISH staining of lung cancer tissues, further confirm that the high levels of both AGO2 acetylation and miR-19b correlate with poor prognosis in lung cancer patients. Our finding reveals a novel function of AGO2 acetylation in increasing oncogenic miR-19b biogenesis and suggests that modulation of AGO2 acetylation has potential clinical implications.


Assuntos
Proteínas Argonautas/genética , Carcinogênese/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Histona Desacetilases/genética , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Fatores de Transcrição de p300-CBP/genética
16.
J Fish Dis ; 41(12): 1811-1819, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30255607

RESUMO

Currently, serological assays for grass carp reovirus genotype II (GCRV-II) diagnosis are not available. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against GCRV-II was developed. The structural protein VP38 of GCRV-II was used as the coating antigen. Monoclonal antibodies (mAb) against IgM of grass carp labelled with HRP were used as a secondary antibody. The antigen concentration and serum dilution were optimized using chess board titration. Furthermore, the specificity of indirect ELISA assay was confirmed by cross check with sera positive for other grass carp pathogens. In comparison with results obtained from indirect immunofluorescence assay (IFA) and Western blot by testing of 60 serum samples to evaluate the sensitivity and specificity of the ELISA, agreement between 90% and 96.7% was reached, respectively. A serological survey was performed using the assay with grass carp field serum samples. The seropositive rate of the 242 serum samples was 69.8%. In conclusion, the developed indirect ELISA is a very specific and sensitive test that will be useful for large-scale serological surveys to detect indirectly GCRV II infections as well as to monitor the changes of antibody level after immunization.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Western Blotting/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças dos Peixes/diagnóstico , Técnica Indireta de Fluorescência para Anticorpo/veterinária , Infecções por Reoviridae/veterinária , Reoviridae/isolamento & purificação , Animais , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Peixes/virologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Proteínas Recombinantes/metabolismo , Infecções por Reoviridae/diagnóstico , Infecções por Reoviridae/virologia , Sensibilidade e Especificidade , Proteínas Virais/metabolismo
17.
J Virol Methods ; 256: 111-115, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29559331

RESUMO

Grass carp reovirus (GCRV) is the causative agent of a hemorrhagic disease that causes severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Discrimination between wild-type field and vaccine strains of GCRV is crucial for meaningful disease diagnosis and epidemiological investigation, yet current detection methods do not discriminate between these different viruses. This study exploited sequence differences between vaccine viruses and the virulent and field strains in the S6 gene that is present in all GCRV strains to develop a high resolution melting curve assay to differentiate between virus strains. The high resolution melting curve analysis was as analytically sensitive as real-time quantitative-Polymerase Chain Reaction (qPCR) detection and at least 10 times sensitive than the conventional PCR. This one-step assay will facilitate grass carp hemorrhagic disease outbreak responses and control.


Assuntos
Carpas/virologia , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/virologia , Genótipo , Reação em Cadeia da Polimerase em Tempo Real , Reoviridae/genética , Vacinas Virais/genética , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/prevenção & controle , Reoviridae/classificação , Reoviridae/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vacinas Virais/imunologia
18.
J Autoimmun ; 89: 125-138, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29269245

RESUMO

Rho-associated kinase (ROCK) has been found to be involved in the pathogenesis of a variety of autoimmune diseases, but the role of ROCK in inflammatory bowel disease (IBD) is still elusive. In this study, we demonstrated that the levels of ROCK2, but not ROCK1, activity were significantly upregulated in peripheral blood mononuclear cells (PBMC) and inflamed mucosa from IBD patients using a ROCK activity assay, and that ROCK2 activity in intestinal mucosa was positively correlated with disease severity. Stimulation with TNF markedly upregulated ROCK2 activity in IBD CD4+ T cells through NF-κB signaling. Blockade of ROCK2 activity using Slx-2119 significantly suppressed proinflammatory cytokines in inflamed mucosa from IBD patients including IFX-unresponsive CD patients, and inhibited IBD CD4+ T cells to differentiate into Th1 and Th17 cells through downregulating phosphorylated Stat1 and Stat3, but promoted Treg cell differentiation through upregulating phosphorylated Stat5. Furthermore, oral administration of Slx-2119 markedly ameliorated intestinal mucosal inflammation in TNBS-induced colitis in mice and decreased proinflammatory cytokines productions in inflamed colon. Our data indicate that ROCK2 plays a critical role in inducing mucosal T cell activation and inflammatory responses in IBD and that inhibition of ROCK2 activity might serve as a novel therapeutic approach in the management of IBD.


Assuntos
Colite/imunologia , Inflamação/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Quinases Associadas a rho/metabolismo , Adulto , Animais , Diferenciação Celular , Células Cultivadas , Colite/induzido quimicamente , Modelos Animais de Doenças , Feminino , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Transdução de Sinais , Adulto Jovem
19.
Biochem Biophys Res Commun ; 485(1): 95-101, 2017 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-28189685

RESUMO

LRSAM1, a RING-type E3 ubiquitin ligase, is essential for regulating cargo sorting, signaling pathways, cell adhesion and anti-bacterial autophagy. It is important to elucidate the mechanism that underlies the regulation of LRSAM1 E3 ligase activity. Here, we reported that LRSAM1 exhibited self-association in vitro and in vivo. We found the self-association of LRSAM1 promotes intermolecular ubiquitination and proved a potential N-terminal ubiquitination. The E3 activity of LRSAM1 is amplified when the RING domain is present in tandem with its N-terminal domain(s). Furthermore, we found that the CC2-SAM domain had a strong inhibitory effect on the E3 activity of LRSAM1 in vitro and blocked ubiquitination of TSG101 in vivo; the tandem CC1 domain, but not the individual CC1 domain, could counteract this inhibition. Collectively, our data characterized the self-association of LRSAM1 and showed how its domains may contribute to its overall activity.


Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Ubiquitina-Proteína Ligases/química
20.
Protein Expr Purif ; 129: 158-161, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27154902

RESUMO

LRSAM1 is a typical RING-finger E3 ubiquitin ligase that plays an important role in many processes. The expression and purification of LRSAM1 from Escherichiacoli had not yet been reported. Here, strategies to clone, express and purify recombinant LRSAM1 in E. coli cells were developed. LRSAM1 was expressed with high yield as inclusion bodies and successfully recovered in soluble form by subsequent denaturation and renaturation steps. Refolded LRSAM1 was directly purified through two steps of ammonium sulfate precipitation, resulting in a purity of up to 95% and a yield of about 6 mg/L bacterial culture. Purified recombinant LRSAM1 exhibited a pH-dependent E3 ligase activity. Its ligase activity was RING-finger domain-dependent, and its ubiquitination favors K6-, K27-, K29- and K48-linkages in cooperation with UbcH5-type E2 enzymes.


Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Ubiquitina-Proteína Ligases , Escherichia coli/genética , Células HeLa , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificação , Ubiquitinação
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