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In a previous paper, we reported that triptolide (TP), a commonly used immunomodulator, could attenuate cardiac hypertrophy. This present study aimed to further explore the inhibition of cardiac fibrosis by TP and the possible mechanism from the perspective of the NOD-like receptor protein 3 (NLRP3) inflammasome. Hematoxylin-eosin and Masson's staining, immunohistochemistry, and immunofluorescence were performed to observe cardiac fibrotic changes in mice and mouse cardiac fibroblasts (CFs). The Western blot, colocalization, and immunoprecipitation were applied to detect protein expression and interactions. Results suggested that TP dose-dependently inhibited cardiac fibrosis induced by isoproterenol and collagen production of CFs induced by angiotensin II. TP exhibited an antifibrotic effect via inhibiting activation of the NLRP3 inflammasome, which sequentially decreased IL-1ß maturation, myeloid differentiation factor 88 (MyD88)-related phosphorylation of c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase 1/2 (ERK1/2), and TGF-ß1/Smad signaling, and ultimately resulted in less collagen production. Moreover, TP showed no antifibrotic effect in Nlrp3-knockout CFs. Notably, TP inhibited the expression of NLRP3 and apoptosis-associated speck-like proteins containing a caspase recruitment domain (ASC) as well as inflammasome assembly, by interrupting the NLRP3-ASC interaction to inhibit inflammasome activation. Finally, TP indeed inhibited the NLRP3-TGFß1-Smad pathway in vivo. Conclusively, TP was found to play a dual role in interrupting the activation of the NLRP3 inflammasome to attenuate cardiac fibrosis.
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Diterpenos/farmacologia , Inflamassomos/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fenantrenos/farmacologia , Angiotensina II , Animais , Colágeno/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Ventrículos do Coração/patologia , Isoproterenol , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND/AIMS: Adult cardiomyocytes can re-enter cell cycle as stimulated by prohypertrophic factors although they withdraw from cell cycle soon after birth. p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, has been implicated in cardiac hypertrophy, however, its precise contribution to this process remains largely unclear. METHODS: The gene expression profile in left ventricle (LV) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was determined using quantitative PCR array and verified by real-time PCR and Western blotting. Hypertrophic response of H9c2 cells and neonatal rat ventricular myocytes (NRVM) were induced by angiotensin II (1 µmol/L). Cardiac hypertrophy of mice was elicited by isoproterenol (ISO) infusion (40 mg/kg per day for 14 days). p21-adenovirus and p21-siRNA were employed to transfect NRVM, and sterigmatocystin (STE, 3 mg/kg, ip, qd) was used to inhibit p21 activity. mRNA and protein expression levels of α- and ß-myosin heavy chain (MHC), p21WAF1/CIP1, calcineurin (CaN) and atrial natriuretic peptide (ANP) were assayed by realtime PCR and WB, respectively. RESULTS: Sixteen genes showed two-fold or greater changes between SHR and WKY rats, in which the expression of p21WAF1/CIP1 was upregulated by 4.15-fold (P=0.002) and reversed by losartan. Surface area, protein content, mRNA and protein expressions of ß-MHC, ANP and p21WAF1/CIP1 in H9c2 cells treated with AngII elevated significantly compared with control group. p21-Ad transfection markedly increased the surface area and ß-MHC mRNA expression of normal NRVMs, and p21-siRNA transfection decreased them in AngII-treated NRVMs. STE treatment decreased HW/BW and cross-sectional area, expression levels of ß-MHC, ANP and p21 significantly in ISO-treated mice. CONCLUSION: Our findings suggest that p21 facilitates the development of cardiac hypertrophy, and regulating the expression of p21 may be an approach to attenuate hypertrophic growth of cardiomyocytes.
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Cardiomegalia/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Isoproterenol , Losartan/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Esterigmatocistina/farmacologia , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismoRESUMO
Tremendous development in the field of portable electronics and hybrid electric vehicles has led to urgent and increasing demand in the field of high-energy storage devices. In recent years, many research efforts have been made for the development of more efficient energy-storage devices such as supercapacitors, batteries, and fuel cells. In particular, supercapacitors have great potential to meet the demands of both high energy density and power density in many advanced technologies. For the last half decade, graphene has attracted intense research interest for electrical double-layer capacitor (EDLC) applications. The unique electronic, thermal, mechanical, and chemical characteristics of graphene, along with the intrinsic benefits of a carbon material, make it a promising candidate for supercapacitor applications. This Review focuses on recent research developments in graphene-based supercapacitors, including doped graphene, activated graphene, graphene/metal oxide composites, graphene/polymer composites, and graphene-based asymmetric supercapacitors. The challenges and prospects of graphene-based supercapacitors are also discussed.
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Irisin, a hormone-like adipo-myokine, has garnered considerable attention in recent years for its potential impact in metabolic diseases. Its physiological effects are similar to those of thyroid hormones, prompting numerous investigations into potential correlations and interactions between irisin and thyroid function through various in vitro and animal experiments. However, existing studies suggest that the relationship between irisin and thyroid diseases is highly complex and multifaceted. In this paper, we have summarized the research results on serum irisin and thyroid function, providing an overview of advancements and constraints in current research on irisin and thyroid hormones. The aim is to offer insights and directions for future clinical trials in this field.
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Dysmorphologists sometimes encounter challenges in recognizing disorders due to phenotypic variability influenced by factors such as age and ethnicity. Moreover, the performance of Next Generation Phenotyping Tools such as GestaltMatcher is dependent on the diversity of the training set. Therefore, we developed GestaltMatcher Database (GMDB) - a global reference for the phenotypic variability of rare diseases that complies with the FAIR-principles. We curated dysmorphic patient images and metadata from 2,224 publications, transforming GMDB into an online dynamic case report journal. To encourage clinicians worldwide to contribute, each case can receive a Digital Object Identifier (DOI), making it a citable micro-publication. This resulted in a collection of 2,312 unpublished images, partly with longitudinal data. We have compiled a collection of 10,189 frontal images from 7,695 patients representing 683 disorders. The web interface enables gene- and phenotype-centered queries for registered users (https://db.gestaltmatcher.org/). Despite the predominant European ancestry of most patients (59%), our global collaborations have facilitated the inclusion of data from frequently underrepresented ethnicities, with 17% Asian, 4% African, and 6% with other ethnic backgrounds. The analysis has revealed a significant enhancement in GestaltMatcher performance across all ethnic groups, incorporating non-European ethnicities, showcasing a remarkable increase in Top-1-Accuracy by 31.56% and Top-5-Accuracy by 12.64%. Importantly, this improvement was achieved without altering the performance metrics for European patients. GMDB addresses dysmorphology challenges by representing phenotypic variability and including underrepresented groups, enhancing global diagnostic rates and serving as a vital clinician reference database.
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The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.
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The present paper established the determination method of heavy metals such as As, Pb, Hg and Cd in cereals by inductively coupled plasma mass spectrometry (ICP-MS) with microwave digestion. The pretreatment conditions were improved and the instrument operating parameters were optimized. 72Ge, 115In and 209Bi were selected as the internal standard elements to overcome the matrix effects and instrument fluctuations effectively. Interference correction equations were used to eliminate the interference of polyatomic ions. Satisfactory linearity of standard curves was obtained with elemental correlation coefficients over 0.9999. The detection limits were in the range of 0.0006-0.016 mg x L(-1), the recoveries of samples were 90%-110%, and the RSD was within 5%. The accuracy of the method was evaluated with national standard reference materials and the interference test was experimented using standard solution. Studies have shown that the method is suitable for rapid determination of heavy metals As, Pb, Hg and Cd in cereals with wide linear range, good precision and high accuracy.
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Grão Comestível/química , Espectrometria de Massas , Metais Pesados/análise , Limite de Detecção , Mercúrio , Micro-Ondas , Espectrofotometria AtômicaRESUMO
Pathological cardiac hypertrophy is an independent risk factor of cardiovascular diseases. Although the function of p53 and p21 in pathological cardiac hypertrophy have been studied, the relationship between them in cardiomyocytes is still unclear. By using specific adenoviruses and siRNAs to modulate p53 or p21 expression in neonatal rat ventricular myocytes (NRVMs), we found that both upregulated p53 and p21 expression induced hypertrophic responses, and they promote each other's expression. Overexpression of p53 aggravated the hypertrophic response of cardiomyocytes in vitro and in vivo, while knockdown of p21 diminished the hypertrophic responses induced by angiotensin â ¡ and the increase of p53 expression. Additionally, Angiotensin â ¡ treatment promoted the nuclear translocation of p21 in NRVMs. Notably, increased p53 expression alone did not promote p21 translocation to the nucleus. Together, these data suggest a self-limiting bidirectional positive feedback interaction between p53 and p21 during cardiac hypertrophy.
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Angiotensina II , Proteína Supressora de Tumor p53 , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cardiomegalia/patologia , Retroalimentação Fisiológica , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Ratos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We investigated the expression of laminin ß1 and integrin α2 in the anterior temporal neocortex tissue of patients with intractable epilepsy and explored the role of these molecules in the pathogenesis of this disease. Immunohistochemistry and immunofluorescence were used to test the expression of laminin ß1 and integrin α2 in samples (from the brain bank of our department, n=32) of surgically removed anterior temporal neocortex tissues from intractable epilepsy patients, and the results were compared with those of controls (n=10). We found that laminin ß1 and integrin α2 protein expression was significantly increased in the anterior temporal neocortex as compared with controls (immunohistochemistry optical density: laminin ß1 = 0.36 ± 0.01 vs. 0.10 ± 0.03 for control; integrin α2=0.42 ± 0.02 vs. 0.04 ± 0.01 for control; p<.05). Immunofluorescence staining indicated that laminin ß1 and integrin α2 accumulated in the plasma membrane and cytoplasm, with strong fluorescence intensity in the anterior temporal neocortex tissue of patients with intractable epilepsy. Thus, our work demonstrates that laminin ß1 and integrin α2 expression is elevated in the anterior temporal neocortex tissue from patients with intractable epilepsy.
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Epilepsia/metabolismo , Integrina alfa2/metabolismo , Laminina/metabolismo , Lobo Temporal/metabolismo , Adolescente , Adulto , Lobectomia Temporal Anterior/métodos , Criança , Epilepsia/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Lobo Temporal/fisiopatologia , Regulação para Cima/fisiologia , Adulto JovemRESUMO
Adult mammalian cardiomyocytes may reenter the cell cycle and cause cardiac hypertrophy. Triptolide (TP) can regulate the expressions of various cell cycle regulators in cancer cells. However, its effects on cell cycle regulators during myocardial hypertrophy and mechanism are unclear. This study was designed to explore the profile of cell cycle of cardiomyocytes and the temporal expression of their regulators during cardiac hypertrophy, as well as the effects of TP. The hypertrophy models employed were neonatal rat ventricular myocytes (NRVMs) stimulated with angiotensin II (Ang II) for scheduled times (from 5 min to 48 h) in vitro and mice treated with isoprenaline (Iso) for from 1 to 21 days, respectively. TP was used in vitro at 1 µg/L and in vivo at 10 µg/kg. NRVMs were analyzed using flow cytometry to detect the cell cycle, and the expression levels of mRNA and protein of various cell cycle regulators were determined using real-time PCR and Western blot. It was found NRVM numbers in phases S and G2 increased, while that in the G1 phase decreased significantly after Ang II stimulation. The mRNA expression levels of p21 and p27 increased soon after stimulation, and thereafter, mRNA expression levels of all cell cycle factors showed a decreasing trend and reached their lowest levels in 1-3 h, except for cyclin-dependent kinase 1 (CDK1) and CDK4 mRNA. The mRNA expression levels of CDK1, p21, and p27 increased markedly after stimulation with Ang II for 24-48 h. In myocardium tissue, CDK and cyclin expression levels peaked in 3-7 days, followed by a decreasing trend, while those of p21 and p27 mRNA remained at a high level on day 21. Expression levels of all protein were consistent with the results of mRNA in NRVMs or mice. The influence of Ang II or Iso on protein expression was more obvious than that on mRNA. TP treatment effectively prevented the imbalance in the expression of cell cycle regulators in the hypertrophy model group. In Conclusion, an imbalance in the expression of cell cycle regulators occurs during cardiac hypertrophy, and triptolide corrects these abnormal expression levels and attenuates cardiac hypertrophy.
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This study reports a delicate electroless approach for the deposition of RuO2·nH2O nanoparticles on the VO x ·mH2O nanowires and this method can be extended to deposit RuO2·nH2O nanoparticles on various material surfaces. Electrochemical characterizations, including linear sweep voltammetry (LSV), electrochemical quartz crystal microbalance (QCM) analysis and rotating ring-disc electrode (RRDE) voltammetry, were carried out to investigate the growth mechanism. The deposition involves the catalytic reduction of dissolved oxygen by the V4+ species of VO x ·mH2O, which drives the oxidation of RuCl3 to proceed with the growth of RuO2·nH2O. This core/shell VO x ·mH2O/RuO2·nH2O shows a better catalytic activity of the oxygen reduction reaction (ORR) than RuO2·nH2O, which is ascribed to the pronounced dispersion of RuO2·nH2O. Such an electroless approach was applicable to the preparation of a RuO2-based nanoparticle suspension as well as the deposition of nanocrystalline RuO2·nH2O on other functional supports like TiO2 nanowires. The thus-obtained RuO2-decorated TiO2 nanorods exhibit significantly an enhanced photoactivity toward photoelectrochemical water oxidation. The versatility of the current electroless approach may facilitate the widespread deployment of nanocrystalline RuO2·nH2O in a variety of energy-related applications.
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For the first time a ZnO nanorod-based Z-scheme heterostructure system was proposed and realized for efficient photoelectrochemical water splitting. The samples were prepared by depositing a thin layer of SnO2 on the Au surface of Au particle-decorated ZnO nanorods. For ZnO-Au-SnO2 nanorods, the embedded Au can mediate interfacial charge transfer by promoting electron transfer from the conduction band of SnO2 to the valence band of ZnO. This vectorial charge transfer resulted in the situation that the photoexcited electrons accumulated at ZnO while the photogenerated holes concentrated at SnO2, giving ZnO-Au-SnO2 substantially high redox powers. Time-resolved photoluminescence spectra suggested that the interfacial charge transfer across the ZnO/Au/SnO2 interface was significantly improved as a result of the Z-scheme charge transfer mechanism. With the substantially high redox powers and significantly improved interfacial charge transfer, ZnO-Au-SnO2 nanorods performed much better as a photoanode in photoelectrochemical water splitting than pristine ZnO, plasmonic Au-decorated ZnO and type-II SnO2-coated ZnO nanorods did. The present study has provided a viable approach to exploit Z-scheme photoanodes in the design of efficient artificial photosynthesis systems for solar energy conversion.
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The forkhead/winged helix transcription factor (Fox) p3 can regulate the expression of various genes, and it has been reported that the transfer of Foxp3-positive T cells could ameliorate cardiac hypertrophy and fibrosis. Triptolide (TP) can elevate the expression of Foxp3, but its effects on cardiac hypertrophy remain unclear. In the present study, neonatal rat ventricular myocytes (NRVM) were isolated and stimulated with angiotensin II (1 µmol/L) to induce hypertrophic response. The expression of Foxp3 in NRVM was observed by using immunofluorescence assay. Fifty mice were randomly divided into five groups and received vehicle (control), isoproterenol (Iso, 5 mg/kg, s.c.), one of three doses of TP (10, 30, or 90 µg/kg, i.p.) for 14 days, respectively. The pathological morphology changes were observed after Hematoxylin and eosin, lectin and Masson's trichrome staining. The levels of serum brain natriuretic peptide (BNP) and troponin I were determined by enzyme-linked immunosorbent assay and chemiluminescence, respectively. The mRNA and protein expressions of α- myosin heavy chain (MHC), ß-MHC and Foxp3 were determined using real-time PCR and immunohistochemistry, respectively. It was shown that TP (1, 3, 10 µg/L) treatment significantly decreased cell size, mRNA and protein expression of ß-MHC, and upregulated Foxp3 expression in NRVM. TP also decreased heart weight index, left ventricular weight index and, improved myocardial injury and fibrosis; and decreased the cross-scetional area of the myocardium, serum cardiac troponin and BNP. Additionally, TP markedly reduced the mRNA and protein expression of myocardial ß-MHC and elevated the mRNA and protein expression of α-MHC and Foxp3 in a dose-dependent manner. In conclusion, TP can effectively ameliorate myocardial damage and inhibit cardiac hypertrophy, which is at least partly related to the elevation of Foxp3 expression in cardiomyocytes.
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Fe-MCM-41 was first used for the treatment of m-cresol in catalytic ozonation. The effect of the percentage of Fe dopping mass, catalyst dosage and the natural concentration of substrate on m-cresol conversion and TOC removal were studied. The structural property of Fe-MCM-41 was characterized by X-ray diffraction, temperature-programmed reduction, Mössbauer spectra and BET of catalysts. The results showed that Fe dopping mass had a great effect on the catalytic activity of Fe-MCM-41 in catalytic ozonation and the optimal percentage of dopping mass was 4.4% (wt). The results showed that with Fe dopping mass increase, the degree of crystallinity became weaker, the crystal surface distance reduced, as well as the specific surface area, pore volume and aperture. γ-Fe2O3 was the only form staying on the surface of MCM-41, and the catalyst had good ferromagnetism and stability. Ozonation played a role of both direct oxidation and indirect oxidation in the reaction, approximately the same ratio. Under the experimental condition of the natural pH of model wastewater,using 4.4% (wt) Fe-MCM-41 as catalyst, natural concentration of m-cresol 500 mg x L(-1), catalyst dosage 0.1 g x L(-1) and reaction time 30 min, m-cresol conversion and TOC removal were 100% and 26.8%, respectively.
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Cresóis/química , Compostos Férricos/química , Dióxido de Silício/química , Águas Residuárias/química , Catálise , Oxirredução , Poluentes Químicos da Água/química , Difração de Raios XRESUMO
During 2009-2012, the Nam Dinh virus (NDiv) was detected from the samples of Culex pipiens quinquefasciatus in Shenzhen China. In this study, cell culture,SYBR Green I based real time RT-PCR and RT-PCR were performed to analyze the cell susceptibility and other biological characteristics of the NDiV isolates. The results showed that C6/36 cell line was susceptible to four isolates of Culex pipiens quinquefasciatus. The "S" type amplification curve and specific melting curve were obtained in the realtime fluorescence quantitative RT-PCR based on SYBR Green I for the detection of the NDiV from the mosquito. The target bands from the RdRp gene and partial fragment of ZmHel1 gene were observed using agarose gel electrophoresis. Both the nucleotide and amino acid sequences of four Shenzhen isolates showed more than 99.00% homology with the Vietnam representative NDiV strain (02VN178). Phylogenetic analysis showed that four Shenzhen isolates shared the same evolution branch as the Vietnam representative NDiV strain. This is the first report of NDiV in China.
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Culex/virologia , Nidovirales/isolamento & purificação , Animais , China , Nidovirales/classificação , Nidovirales/genética , FilogeniaRESUMO
We selected useful antibody fragments against rabies virus from a human single chain variable fragment (scFv) gene library using ribosome display technique. The recombinant rabies virus glycoprotein (RVGp) was used as an antigen to isolate specific scFvs. After five rounds of selection, the analysis demonstrated that scFv-ribosome-mRNA complexes were specifically selected against RVGp. Sequence analysis showed that mutations were introduced at random by PCR among the rounds of selection and variants with high affinity were isolated. The obtained scFvs with high affinity could recognize RVGp specifically and showed binding activity to rabies virus. These scFvs were potential for inclusion in a combination of several human monoclonal antibodies (MAbs) aimed for application in rabies post-exposure prophylaxis. Ribosome display technology is a robust tool for rapid isolation of human antibody fragments, and has exceptional strength in affinity maturation and molecular evolution in vitro.
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Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Biblioteca Gênica , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Vírus da Raiva/imunologia , Ribossomos/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Biotecnologia/métodos , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Seleção Genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Recently, much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among many genes in the MDV genome, several genes such as Meq, RLORF4, RLORF12, and 132bpr have been considered recently associated with virulence of MDV. In this paper, primers of Meq, RLORF4, RLORF12 and 132bpr genes were designed and synthesized, based on the published whole genome sequence of MDV strain GA. The genes of Meq, RLORF4 and RLORF12 from four Chinese epidemic MDV strains highly passaged on chicken embryo fibroblast (CEF), i. e. L-SYp85C, L-MSp75C, L-CZp75C, and L-ZYp75C, as well as their corresponding parent strains, i. e. L-SY, L-MS, L-CZ, and L-ZY, the reference virulent strain J-1 and the vaccine strain 814 were amplified by PCR respectively. Then the PCR products of interest were cloned and sequenced respectively. The results of sequence comparison and analysis of Meq genes in the study indicated that Meq genes from the two strains L-ZYp75C and L-CZp75C contained single nucleotide insertion and deletion. The Meq gene from strain L-ZYp75C contained an extra cytidine (C) insertion at nucleotide position 529 and a single thymidine (T) deletion at nucleotide position 602, resulting in a frameshift mutation. And this frameshift mutation could lead to changes in deduced amino acid sequence from position 177 to 200 of Meq gene. The extra C insertion at nucleotide position 625 in Meq gene of strain L-CZp75C was also predicted to cause frameshift mutation in three overlapping genes (Meq, RLORF6 and 23KD genes). The comparison of nucleotide sequences of RLORF4 genes in the study revealed that the RLORF4 gene of strain L-SYp85C contained a fragment deletion in Open Reading Frame (ORF) from nucleotide position 215 to 265, resulting in 17 amino acids deletions, which were not found in other sequenced strains. Comparison of nucleotide sequences of RLORF12 genes in the study revealed several mutations. The RLORF12 gene of strain L-MSp75C contained a single T deletion at nucleotide position 67 and of 814 vaccine strain a large fragment deletion from nucleotide position 18 to 86, both of the deletions located in Origin of replication site (Ori) of MDV genome. But strain L-ZYp75C possessed an unique "TGTTGGG" deletion in its RLORF12 gene. When the four Chinese epidemic MDV strains were serially passaged on CEF, the number of copies of the 132bp repeats increased from 2 to more than 10 copies. All of above results indicated that deletion and/or insertion mutation occurred in Meq, RLORF4, RLORF12 and 132bpr after serial passage of these four Chinese epidemic MDV strains on CEF.
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Doença de Marek/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Análise Mutacional de DNA , Fibroblastos , Doença de Marek/genética , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/químicaRESUMO
AIM: To prepare distinct human McAbs to TNF-alpha with high neutralizing potency using ribosome display technology. METHODS: The immunoglobulin heavy and light chain variable (VH, VL) genes were prepared from the peripheral blood lymphocytes in three arthritis patients by PCR. The genes encoding VH/K fragments were prepared by randomly combining VH and VL genes by SOE PCR. TNF-alpha binding fragments were selected over three cycles of ribosome display and the selected VH/Ks genes were cloned into pET22b(+)/BL21(DE3), from which soluble VH/K fragments were prepared. The expressed products of selected clones were analyzed by ELISA. Then the positive clones were characterized. RESULTS: A human VH/K gene library with 6.7x10(12) numbers used for ribosome display was constructed. Among the 180 selected clones, two clones TRB21 and TRB409 exhibiting the highest ELISA signals were isolated. The analysis of the sequence of TRB21 and TRB409 showed that they were new human immunoglobulin V genes to TNF-alpha and they recognize TNF-alpha specifically and antagonize the cytolytic effect of TNF-alpha on 1929 cell. CONCLUSION: The selected VH/Ks to TNF-alpha will be useful for constructing engineering antibodies with high affinity against arthritis. Ribosome display is a rapid means of generating fully human antibody fragments in vitro.
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Anticorpos Monoclonais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Artrite Reumatoide/imunologia , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Camundongos , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/genéticaRESUMO
Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV (Marek's disease virus) encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq has been suggested as the MDV-associated oncogene. In this paper, based on the published sequence of Meq gene of GA strain of MDV, a pair of primers were designed and synthesized. Meq gene ORF (Open reading frame) of the four Chinese local MDV isolates, the reference strain J-1 and the vaccine strain 814 were amplified by using polymerase chain reaction(PCR). Then the PCR products were cloned and sequenced respectively. The results of sequence comparison indicated that the sequences of Meq gene in different strains are relatively conserved and homology of the amino acid sequences is 96.5%-99.7%. The proline-rich repeats of Meq gene of four MDV isolates have site mutations, and it is related to MDV's virulence. Two unique site mutations appear in Meq gene of Chinese local MDV isolates, but they aren't present in Meq gene of the published MDV strains from abroad and the early domestic strains. It seems that some regularities exist between such mutations in four Chinese local MDV isolates and the virulence of MDV, but the regularities need further research.
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Herpesvirus Galináceo 2/genética , Proteínas Oncogênicas Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Galinhas , Clonagem Molecular , Dados de Sequência Molecular , MutaçãoRESUMO
Effect of Ce(III) on photochemical reaction activity in soybean seedlings exposed to ultraviolet-B radiation (UV-B, 280 - 320 nm) was studied with hydroponics under laboratory conditions. The results showed that CeCl3 (20 mg x L(-1)) could improve photosynthesis action and net photosynthesis rate, the Mg(2+) -ATPase activeness, the photosynthetic phosphorylation active and stimulate the activity of Hill reaction (23.2% - 14.3%). And Ce(III) could alleviate the inhibition of UV-B (T1 = 0.15 W x m(-2) and T2 = 0.45 W x m(2-)) on the four indexes above in plants to a certain extent, and the decreasing degrees of the indexes under Ce(III) and UV-B stress were lower than those only under UV-B stress, which were 36.6% - 15.9% [Ce(III) + T1 ], 65.6% - 33.2% [Ce(III) + T2], 50.9% - 23.3% (T1), 77.3% - 51.4% (T2) respectively. The dynamic data indicated that Ce(III) could alleviate the inhibition of UV-B on the four indexes above while promoting without UV-B stress. The results showed that Ce(III) could alleviate the decrease in the net photosynthesis rate by lightening the damage of the photochemical reaction activity on the soybean seedings under UV-B stress.