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BACKGROUND: The diagnosis, severity assessment, and development of therapeutic strategies for asthma are crucial aspects of disease management. Since biomarkers are reliable tools in disease management, we aimed to identify and explore asthma-associated biomarkers and investigate their mechanisms. METHODS: Lipidomics was used to profile serum glycerophospholipids in asthmatic patients and controls. The absolute concentration of lysophosphatidylglycerol (LPG) 18:0 was quantified in various asthma subtypes. Mouse asthma models were used to confirm its potential as a biomarker and investigate its mechanisms in vivo. The effects of LPG 18:0 on CD4+ T cell differentiation, proliferation, and apoptosis were assessed in vitro by flow cytometry, while mitochondrial dysfunction was evaluated through mitochondrial membrane potential, reactive oxygen species, and ATP production measurements. The intracellular mechanism of LPG 18:0 in Tregs was investigated using small molecule inhibitors. RESULTS: The serum glycerophospholipid profile varied between asthmatic patients and control group, with LPG 18:0 levels being notably higher in asthmatic patients, correlating with asthma severity and control level. In vivo and in vitro studies revealed that LPG18:0 impaired naïve CD4+ T cell differentiation into Tregs and compromised their suppressive function. Further investigation demonstrated that LPG18:0 treatment reduced the FOXP3 protein level via SIRT1-mediated deacetylation during Treg differentiation. CONCLUSIONS: This study identifies that serum levels of LPG 18:0 are generally elevated in asthmatics and serve as a biomarker for asthma. LPG 18:0 impairs Treg function via the NAD+/SIRT1/FOXP3 pathway. Our research reveals the potential of LPG18:0 as a biomarker for asthma, elucidating its role in asthma diagnosis and treatment.
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BACKGROUND: Interleukin (IL)-2 is a key cytokine capable of modulating the immune response by activating natural killer (NK) cells. This study was recruited to explore the therapeutic potential of IL-2-activated NK-92 cells in endometriosis in vitro. METHODS: Ectopic endometrial stromal cells (EESCs) were isolated and co-cultured with IL-2-activated NK-92 cells at varying effector-to-target (E:T) ratios (1:0 [Control], 1:1, 1:3, and 1:9). The viability, cytotoxicity, and cell surface antigen expression of IL-2-activated NK-92 cells were assessed. The viability, apoptosis, invasion, and migration ability of EESCs co-cultured with NK-92 cells at different ratios were evaluated. The apoptosis-related proteins, invasion and migration-related proteins as well as MEK/ERK pathway were examined via western blot. Each experiment was repeated three times. RESULTS: IL-2 activation enhanced NK-92 cytotoxicity in a concentration-dependent manner. Co-culturing EESCs with IL-2-activated NK-92 cells at E:T ratios of 1:1, 1:3, and 1:9 reduced EESC viability by 20%, 45%, and 70%, respectively, compared to the control group. Apoptosis rates in EESCs increased in correlation with the NK-92 cell proportion, with the highest rate observed at a 1:9 ratio. Moreover, EESC invasion and migration were significantly inhibited by IL-2-activated NK-92 cells, with a 60% reduction in invasion and a 50% decrease in migration at the 1:9 ratio. Besides, the MEK/ERK signalling pathway was down-regulated in EESCs by IL-2-activated NK-92 cells. CONCLUSION: IL-2-activated NK-92 cells exhibit potent cytotoxic effects against EESCs. They promote EESC apoptosis and inhibit viability, invasion, and migration through modulating the MEK/ERK signalling pathway.
Endometriosis is a common chronic systemic disease affecting approximately 190 million women worldwide. However, clinical treatments for endometriosis remain challenging due to the scarcity of high-quality scientific evidence and conflicting available guidelines. This research was designed to explore whether interleukin (IL)-2 affected the progression of endometriosis by modulating endometrial stromal cell apoptosis and natural killer (NK) cell-mediated cytotoxicity, thereby providing new therapeutic methods for endometriosis.
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Apoptosis , Técnicas de Cocultivo , Endometriosis , Interleucina-2 , Células Asesinas Naturales , Humanos , Endometriosis/patología , Endometriosis/inmunología , Femenino , Interleucina-2/farmacología , Interleucina-2/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Apoptosis/efectos de los fármacos , Adulto , Endometrio/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Progresión de la Enfermedad , Supervivencia Celular/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células CultivadasRESUMEN
BACKGROUND: The airway microbiota plays an important role in asthma pathophysiology. However, the relationship between the airway microbiota and asthma phenotypes is still poorly understood. OBJECTIVE: We aimed to characterize the airway microbiota in asthma patients and determine its correlation with airway inflammatory phenotypes and other phenotypic characteristics. METHODS: The microbial composition of induced sputum specimens collected from asthma patients was determined using 16S rDNA gene sequencing. RESULTS: Patients with asthma had a higher abundance of bacterial taxa associated with Bacteroidetes, Fusobacteria and Proteobacteria and a reduced abundance of Firmicutes and Actinobacteria compared to healthy controls. This study classified the asthma-associated lung microbiota into three community types based on DMM models, which were defined as three pulmotypes (P1, P2 and P3). The lungs of patients with pulmotype 3 (P3) were dominated by Faecalibacterium and Bacteroides, while patients with pulmotype 1 (P1) had a greater abundance of Pasteurellaceae, Streptococcus and Rothia. P1 patients were older (p = .045) and had lower blood TGF levels (p = .028). P3 patients had fewer eosinophils (p = .016) and more neutrophils (p = .039) in induced sputa than P1 patients. CONCLUSIONS: Differences in asthma-associated airway microbiota pulmotypes are associated with and might influence asthma, particularly inflammatory phenotypes.
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Asma , Microbiota , Bacterias/genética , Eosinófilos , Humanos , Pulmón , ARN Ribosómico 16S/genética , Esputo/microbiologíaRESUMEN
Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by in vitro passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the in vitro and in vivo infection of TMUV.IMPORTANCE Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.
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Sustitución de Aminoácidos , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/virología , Flavivirus/genética , Proteínas del Envoltorio Viral/genética , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Neutralizantes , Línea Celular , China/epidemiología , Patos/virología , Femenino , Infecciones por Flavivirus/epidemiología , Infecciones por Flavivirus/patología , Mutagénesis Sitio-Dirigida , Mutación , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/mortalidad , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/virología , Carga Viral , Virulencia , Replicación ViralRESUMEN
BACKGROUND: In China, during the cultivation process of Pleurotus ostreatus, the yield and quality of fruiting bodies are easily affected by high temperatures in summer. Nitric oxide (NO) plays an important regulatory role in the response to abiotic stress, and previous studies have found that NO can induce alternative oxidase (aox) experssion in response to heat stress (HS) by regulating aconitase. However, the regulatory pathway of NO is complex, and the function and regulation of the aox gene in the response to HS remain unclear. RESULTS: In this study, we found that NO affected nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) levels, reduced hydrogen peroxide (H2O2) and superoxide anion (O2-) contents, and slowed O2- production. Further RNA-Seq results showed that NO regulated the oxidation-reduction process and oxidoreductase activity, affected the cellular respiration pathway and activated aox gene expression. The function of aox was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the OE-aox strains exhibited obviously improved growth recovery after exposure to HS. During exposure to HS, the OE-aox strains exhibited reduced levels of NADH, the product of the tricarboxylic acid (TCA) cycle, and decreased synthesis of ATP, which reduced the production and accumulation of reactive oxygen species (ROS), whereas the RNAi-aox strains exhibited the opposite result. In addition, aox mediated the expression of antioxidant enzyme genes in the mycelia of P. ostreatus under HS through the retrograde signaling pathway. CONCLUSIONS: This study shows that the expression of the aox gene in P. ostreatus mycelia can be induced by NO under HS, that it regulates the TCA cycle and cell respiration to reduce the production of ROS, and that it can mediate the retrograde signaling pathway involved in the mycelial response to HS.
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Regulación Fúngica de la Expresión Génica/genética , Respuesta al Choque Térmico/genética , Proteínas Mitocondriales/genética , Óxido Nítrico/metabolismo , Oxidorreductasas/genética , Proteínas de Plantas/genética , Pleurotus/enzimología , Pleurotus/genética , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , China , Proteínas Mitocondriales/metabolismo , Micelio/crecimiento & desarrollo , NAD/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Pleurotus/crecimiento & desarrolloRESUMEN
BACKGROUND: Trehalose, an intracellular protective agent reported to mediate defense against many stresses, can alleviate high-temperature-induced damage in Pleurotus ostreatus. In this study, the mechanism by which trehalose relieves heat stress was explored by the addition of exogenous trehalose and the use of trehalose-6-phosphate synthase 1 (tps1) overexpression transformants. RESULTS: The results suggested that treatment with exogenous trehalose or overexpression of tps1 alleviated the accumulation of lactic acid under heat stress and downregulated the expression of the phosphofructokinase (pfk) and pyruvate kinase (pk) genes, suggesting an ameliorative effect of trehalose on the enhanced glycolysis in P. ostreatus under heat stress. However, the upregulation of hexokinase (hk) gene expression by trehalose indicated the involvement of the pentose phosphate pathway (PPP) in heat stress resistance. Moreover, treatment with exogenous trehalose or overexpression of tps1 increased the gene expression level and enzymatic activity of glucose-6-phosphate dehydrogenase (g6pdh) and increased the production of both the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH), confirming the effect of trehalose on alleviating oxidative damage by enhancing PPP in P. ostreatus under heat stress. Furthermore, treatment with exogenous trehalose or overexpression of tps1 ameliorated the decrease in the oxygen consumption rate (OCR) caused by heat stress, suggesting a relationship between trehalose and mitochondrial function under heat stress. CONCLUSIONS: Trehalose alleviates high-temperature stress in P. ostreatus by inhibiting glycolysis and stimulating PPP activity. This study may provide further insights into the heat stress defense mechanism of trehalose in edible fungi from the perspective of intracellular metabolism.
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Glucosiltransferasas/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Pleurotus/metabolismo , Trehalosa/farmacología , Proteínas Fúngicas/metabolismo , Glucólisis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacosRESUMEN
A novel two-fold interpenetrated metal-organic framework, namely Co-EDDA, was synthesized by hydrothermal reaction of 5,5'-[ethane-1,2-diylbis(oxy)]diisophthalic acid (H4EDDA), Co(NO3)2·6H2O, and 1,4-di(1H-imidazol-1-yl)benzene in water in an alkaline environment and structurally characterized. Co-EDDA could display clear dual-emission signals at 350 and 430 nm, representing the charge transfer emission between metal ions and the ligand and the ligand-based emission, respectively, which represents the ratiometric luminescence response to chromium(III) with high selectivity and sensitivity (limit of detection of 0.54 µM). Comprehensive studies indicate that the detection can be attributed to the interaction between the Cr3+ ions and the O atoms on the ether bond in Co-EDDA.
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The endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis, which can lead to oxidative stress and inflammation. The role of autophagy in the process of atherosclerosis has drawn increasing attention. The human umbilical vein endothelial cells (HUVECs), whose Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac3 was knockdown, were used to detect whether the possible molecular mechanisms of Rac1 and Rac3 for anti-inflammatory in endothelial cells was effected by downregulation of autophagy. The HUVECs were incubated with ox-LDL. The inflammatory factors and autophagy proteins were evaluated to ascertain and compare the effect of Rac1 and Rac3 on autophagy. Then, 3-methyladenine (3-MA) as an inhibiter of autophagy was used to detect whether the effect of Rac1 and Rac3 was related to autophagy. ox-LDL-induced cell dysfunction in HUVECs was determined by testing the formation of foam cells, the expression of nuclear factor (NF)-κB and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 and NF-κB p65 and other inflammatory factors, the release of reactive oxygen species by oxidative stress and the dysfunction of the cytomembrane. And ApoE-/- mice on a high-fat diet were used as an animal model to detect the effect of Rac1 and Rac3 in vivo. The results showed that when Rac1 and Rac3 were decreased in HUVECs, the cell dysfunction caused by ox-LDL was inhibited. If 3-MA was used to inhibit autophagy in Rac1 and Rac3 knockdown cells, the injury induced by ox-LDL on the cells was recovered. These results indicated that the effect of Rac1 and Rac3 was combined with ox-LDL, which was related to inhibition of autophagy. The effect of Rac3 was more significant than that of Rac1.
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Aterosclerosis/metabolismo , Autofagia/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Lipoproteínas LDL/toxicidad , Proteínas de Unión al GTP rac/metabolismo , Animales , Aterosclerosis/patología , Dieta Alta en Grasa , Regulación hacia Abajo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Tembusu virus (TMUV) is a newly emerging flavivirus and has caused significant economic loss to the poultry industry in China. To date, the entry of TMUV into host cells remains poorly understood. Here, the mechanism of TMUV entry into BHK-21 cells was investigated. The depletion of cellular cholesterol by methyl-ß-cyclodextrin led to a significant decline in the titers and RNA levels of the infectious TMUV. This reduction was restored by supplementation of exogenous cholesterol. Membrane cholesterol depletion mainly blocked viral internalization but not attachment. However, viral infection was unaffected by genistein treatment or caveolin-1 silencing by small interfering RNA. In addition, clathrin-mediated endocytosis might be utilized in TMUV entry given that the viral infection was inhibited by knockdown of clathrin heavy chain and treatment of chlorpromazine (CPZ). Moreover, the number of internalized virus particles decreased under CPZ treatment. Dynasore inhibited TMUV entry suggesting a role for dynamin. Our results reveal that TMUV entry into BHK-21 cells is dependent on cholesterol, clathrin and dynamin but not caveolae.
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Colesterol , Clatrina , Endocitosis , Flavivirus , Internalización del Virus , Animales , Línea Celular , Cricetinae , Flavivirus/fisiologíaRESUMEN
MicroRNA (miRNA) plays a key role in virus-host interactions. Here, we employed deep sequencing technology to determine cellular miRNA expression profiles in chicken dendritic cells infected with H9N2 avian influenza virus (AIV). A total of 66 known and 36 novel miRNAs were differently expressed upon H9N2 infection, including 72 up-regulated and 30 down-regulated miRNAs. Functional analysis showed that the predicted targets of these miRNAs were significantly enriched in several pathways including endocytosis, notch, lysosome, p53, RIG-I-like and NOD-like receptor signaling pathways. These data provide valuable information for further investigating the roles of miRNA in AIV pathogenesis and host defense response.
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Pollos/genética , Regulación de la Expresión Génica , Subtipo H9N2 del Virus de la Influenza A/fisiología , Gripe Aviar/virología , MicroARNs/genética , Enfermedades de las Aves de Corral/virología , Animales , Pollos/metabolismo , Regulación hacia Abajo , Interacciones Huésped-Patógeno , MicroARNs/metabolismo , Regulación hacia Arriba , Replicación ViralRESUMEN
OBJECTIVE: To reveal the distribution signature of cancer susceptibility genes in patients with gastric adenocarcinoma, offering a diagnostic and prognostic surrogate for disease risk management and therapeutic decisions. METHODS: A total of 282 patients with gastric adenocarcinoma (182 males and 100 females) were enrolled in this study, with peripheral blood genomic DNA extracted. Mutations of 69 canonical cancer susceptibility genes or presumably tumor-related genes were analyzed by targeted capture-based high-throughput sequencing. Candidate mutations were particularly selected for discussion on tumor pathogenesis according to the American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: In this study, 7.1% (20/282) of patients with gastric adenocarcinoma were found to harbor mutations of canonical or presumable cancer susceptibility genes. The detection rate in male patients (3.8%, 7/182) was significantly lower than that in female patients (13%, 13/100) (P=0.004). The most recurrent mutations were in A-T mutated (ATM) (1.1%, 3/282), followed by BRCA1, BRIP1 and RAD51D, all showed a detection rate of 0.7% (2/282). Mutations in three genes associated with hereditary gastric cancer syndromes were detected, namely, PMS2 and EPCAM associated with Lynch syndrome and CDH1 associated with hereditary diffuse gastric cancer. The detection frequencies were all 0.4% (1/282). Notwithstanding no significant difference observed, the age of patients with pathogenic mutations or likely pathogenic mutations is slightly younger than that of non-carriers (median age: 58.5 vs. 60.5 years old), while the age of patients with ATM mutations was the youngest overall (median age: 49.3 years old). CONCLUSIONS: Our study shed more light on the distribution signature and pathogenesis of mutations in gastric cancer susceptibility genes, and found the detection rate of pathogenic and likely pathogenic mutations in male patients was significantly lower than that in female patients. Some known and unidentified mutations were found in gastric cancer, which allowed us to gain more insight into the hereditary gastric cancer syndromes from the molecular perspective.
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Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. The cellular factors required for DTMUV replication have been poorly studied. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates diverse cellular processes, including endocytosis and signal transduction, which may be involved in the entry of virus. In the present study, we explored the interplay between DTMUV replication and the UPS in BHK-21â¯cells and found that treatment with proteasome inhibitor (MG132 and lactacystin) significantly decreased the DTMUV progency at the early infection stage. We further revealed that inhibition of the UPS mainly occurs on the level of viral protein expression and RNA transcription. In addition, using specific siRNAs targeting ubiquitin reduces the production of viral progeny. In the presence of MG132 the staining for the envelope protein of DTMUV was dramatically reduced in comparison with the untreated control cells. Overall, our observations reveal an important role of the UPS in multiple steps of the DTMUV infection cycle and identify the UPS as a potential drug target to modulate the impact of DTMUV infection.
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Flavivirus/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Replicación Viral/fisiología , Acetilcisteína/análogos & derivados , Acetilcisteína/antagonistas & inhibidores , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Patos , Flavivirus/efectos de los fármacos , Flavivirus/patogenicidad , Técnicas de Silenciamiento del Gen , Leupeptinas/antagonistas & inhibidores , Enfermedades de las Aves de Corral/virología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , ARN Interferente Pequeño , Transfección , Ubiquitina/efectos de los fármacos , Ubiquitina/genética , Proteínas del Envoltorio Viral , Internalización del VirusRESUMEN
BACKGROUND: Tembusu virus (TMUV), classified in the genus Flavivirus, causes reduced egg production and neurological problems in poultry. Flavivirus replication depends on the host endoplasmic reticulum (ER) and induces ER stress that leads to activation of the cellular unfolded protein response (UPR), an important signalling pathway that regulates many biological functions involved in viral pathogenesis and innate immunity. However, the mechanism of TMUV-induced UPR activation remains unclear. RESULTS: In this study, we systematically investigated the three UPR pathways in TMUV-infected BHK-21 cells. Our results showed that expression of glucose-related protein 78 (GRP78) and GRP94 was upregulated during the course of TMUV infection. We then demonstrated that TMUV activated the PERK pathway in the early stage of infection, resulting in upregulation of ATF4, GADD34 and CHOP, with CHOP induction leading to caspase-3 activation. We also found the IRE1 pathway to be activated, leading to splicing of X box binding protein 1 (XBP1) mRNA and enhanced expression of p58IPK. Finally, we observed increased expression of ATF6 and activity of ER stress-response elements, suggesting stimulation of the ATF6 pathway. In addition, ATF6 pathway activation correlated with the induction of downstream chaperones calnexin, calreticulin, ERp57 and PDI. UPR activity was also observed by the marked elevation in GRP78 and sXBP1 levels in TMUV-infected DF-1 cells. CONCLUSIONS: This is the first report that TMUV infection-induced ER stress activates three branches of the UPR, and these results lay the foundation for elucidating the pathogenesis of TMUV and understanding the inherent mechanism of TMUV infection as well as the host response.
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Infecciones por Flavivirus/metabolismo , Flavivirus/metabolismo , Respuesta de Proteína Desplegada , Animales , Western Blotting , Caspasa 1/metabolismo , Línea Celular , Pollos/virología , Cricetinae/virología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , eIF-2 Quinasa/metabolismoRESUMEN
BACKGROUND: Tembusu virus is a newly emerging flavivirus that caused egg-drop syndrome in ducks in China. TMUV envelope protein is a major structural protein locates at the surface of tembusu virus particle. During tembusu virus infection, envelope protein plays a pivotal role in induction of neutralizing antibody. However, B cell epitopes within envelope protein have not been well studied. METHOD: A series of 13 peptides derived from E protein of tembusu virus were synthesized and screened by Dot blot with tembusu virus-positive duck serum. Potential B-cell epitopes were respectively fused with GST tag and expressed in E. coli. The immunogenicity and protective efficiency of epitopes were assessed in ducks. RESULTS: Dot blot assay identified the peptides P21 (amino acids 301-329), P23 (amino acids 369-387), P27 (amino acids 464-471) and P28 (amino acids 482-496) as potential B-cell epitopes within the envelope protein of tembusu virus. Immunization of prokaryotically expressed epitopes elicited specific antibodies in ducks and the specific antibody elicited by P21, P27 and P28 could neutralized tembusu virus. In addition, protective test suggested that P21 and P27 could completely protect immunized ducks from TMUV challenge. CONCLUSION: Four potential B cell epiotpes within tembusu virus envelope protein were identified and analyzed in vitro and in vivo. It was demonstrated that two of them (P21 and P27) could elicit neutralizing antibodies in ducks and offer complete protection against tembusu virus challenge. This findings will contribute to the development of epitope vaccine for tembusu virus prevention.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Epítopos de Linfocito B/inmunología , Flavivirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , China , Patos , Infecciones por Flavivirus/inmunología , Infecciones por Flavivirus/veterinaria , Infecciones por Flavivirus/virología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virologíaRESUMEN
The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P < 0.05), fractional esterification rate of HDL cholesterol (FERHDL) (P < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) (P < 0.05) and HDL cholesterol (HDL-C) (P < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.
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Cromatografía Líquida de Alta Presión/métodos , Pruebas de Enzimas/métodos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Esteroles/metabolismo , Adulto , Anciano , Cromatografía Líquida de Alta Presión/economía , Deshidrocolesteroles/sangre , Deshidrocolesteroles/aislamiento & purificación , Deshidrocolesteroles/metabolismo , Pruebas de Enzimas/economía , Esterificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/aislamiento & purificación , Reproducibilidad de los Resultados , Esteroles/sangre , Esteroles/aislamiento & purificación , Especificidad por Sustrato , Adulto JovenRESUMEN
BACKGROUND: Tembusu virus (TMUV) is a member of the genus Flavivirus. Outbreak of this virus infection in duck flocks was first observed in China in April 2010, causing severe egg drop and neurological signs in laying ducks. Recently reported duck infections in southeastern Asia highlighted the need for well-validated diagnostic methods of TMUV surveillance to understand its epidemiological characteristics and maintenance in nature. Several enzyme-linked immunosorbent assays (ELISAs) for the detection of TMUV infection have been reported, but none have been applied to high-throughput diagnostics. RESULTS: In this study, a monoclonal antibody (MAb) against TMUV was generated and characterized. MAb 9E4 was shown to bind specifically to a disulfide bond-dependent epitope on the domain I/II of TMUV E protein, and a blocking ELISA was established based on this MAb. The cut-off percentage inhibition value for negative sera was set at 30%. By comparison with the virus neutralization test, the specificity and sensitivity of the blocking ELISA were 96.37% and 100%, respectively, and the kappa value was 0.966, based on 416 serum samples collected from both experimentally and clinically infected ducks, geese and chickens. A good correlation (r2 = 07998, P < 0.001) was observed between the blocking ELISA and plaque reduction neutralization test (PRNT) titers. Using archived duck serum samples collected between 2009 and 2015, the seroprevalence in duck flocks raised in Northern China was estimated by blocking ELISA. CONCLUSIONS: Our MAb-based blocking ELISA provides a reliable and rapid diagnostic tool for serological monitoring of TMUV infection and evaluation of immune status following TMUV vaccination in multiple poultry species.
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Anticuerpos Antivirales/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Infecciones por Flavivirus/veterinaria , Flavivirus , Enfermedades de las Aves de Corral/diagnóstico , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting/veterinaria , Pollos/inmunología , Pollos/virología , Patos/inmunología , Patos/virología , Flavivirus/inmunología , Infecciones por Flavivirus/diagnóstico , Infecciones por Flavivirus/virología , Técnica del Anticuerpo Fluorescente/veterinaria , Gansos/inmunología , Gansos/virología , Pruebas de Neutralización/veterinaria , Enfermedades de las Aves de Corral/virología , Reproducibilidad de los ResultadosRESUMEN
This study aims to investigate the interaction between 3 flavonoids (quercetin, apigenin, and naringenin) and fat mass and obesity-associated protein by fluorescence, ultraviolet-visible absorption spectroscopy, and molecular modeling. Results indicate that the intrinsic fluorescence of fat mass and obesity-associated protein can be quenched by the 3 flavonoids through a static quenching procedure. Thermodynamic analysis and molecular modeling results suggest that hydrophobic interaction and hydrogen bond forces play the major roles in the binding process. Moreover, results also show that the rank order of quenching constant and binding constant is quercetin > apigenin > naringenin.
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Tejido Adiposo/química , Flavonoides/farmacología , Leptina/química , Simulación del Acoplamiento Molecular/métodos , Tejido Adiposo/metabolismo , Apigenina/química , Apigenina/farmacología , Sitios de Unión , Flavanonas/química , Flavanonas/farmacología , Flavonoides/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Leptina/metabolismo , Modelos Moleculares , Unión Proteica , Quercetina/química , Quercetina/farmacología , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Batai virus (BATV) belongs to the genus Orthobunyavirus of the family Bunyaviridae. It has been isolated from mosquitos, pigs, cattle, and humans throughout Africa, Asia, and Europe, and causes clinical signs in domestic animals and humans. Here, we report the isolation of BATV from a domestic duck flock. Genome sequence analysis revealed clustering of this isolate in the Africa-Asia lineage. The virus replicated in mosquitos and vertebrate host cells, showing different phenotypic characteristics, and showed the potential to infect mice. This is the first report of BATV in domestic birds and indicates the wide circulation of BATV in China.
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Animales Domésticos , Virus Bunyamwera/clasificación , Patos/virología , Animales , Virus Bunyamwera/genética , Virus Bunyamwera/aislamiento & purificación , Virus Bunyamwera/ultraestructura , Infecciones por Bunyaviridae/virología , Técnicas de Cultivo de Célula , Línea Celular , Efecto Citopatogénico Viral , Genoma Viral , Ratones , Filogenia , ARN Viral , Análisis de Secuencia de ADN , Replicación ViralRESUMEN
In this paper, The binding of twelve 1,3-diazaheterocyclic compounds (1a-1 l) to the fat mass and obesity-associated (FTO) protein was investigated by fluorescence, UV-vis absorption spectroscopy and molecular modeling. Results indicated that the intrinsic fluorescence of FTO is quenched by the nine compounds (1a-1i) with a static quenching procedure. No interaction was observed between FTO protein and compounds (1j-1 l). The thermodynamic parameters obtained from the fluorescence data showed that the hydrophobic force played a major role in stabilizing the complex. The results of synchronous and three-dimensional fluorescence spectra showed that the conformation of FTO was changed. In addition, the influence of molecular structure on the quenching effect has been investigated.
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Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Compuestos Aza/química , Compuestos Aza/metabolismo , Modelos Moleculares , Espectrometría de Fluorescencia/métodos , Sitios de Unión , Fluorescencia , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
In this work, the interaction between camptothecin (CPT) analogs and fat mass and obesity associated (FTO) was investigated using spectroscopy and molecular docking. From the experimental results, it was found that the CPT analogs caused the fluorescence quenching of FTO through a static quenching procedure. The binding constants and thermodynamic parameters at three different temperatures, the number of binding sites were obtained, which suggested that the hydrophobic interaction and electrostatic force played major role in the reaction between CPT analogs and FTO. Results revealed that 10-hydroxycamptothecin was the strongest quencher.