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1.
Rheumatology (Oxford) ; 60(3): 1252-1259, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32911538

RESUMO

OBJECTIVE: The relationship between serum lipid variations in SS and healthy controls was investigated to identify potential predictive lipid biomarkers. METHODS: Serum samples from 230 SS patients and 240 healthy controls were collected. The samples were analysed by ultrahigh-performance liquid chromatography coupled with Q Exactive™ spectrometry. Potential lipid biomarkers were screened through orthogonal projection to latent structures discriminant analysis and further evaluated by receiver operating characteristic analysis. RESULTS: A panel of three metabolites [phosphatidylcholine (18:0/22:5), triglyceride (16:0/18:0/18:1) and acylcarnitine (12:0)] was identified as a specific biomarker of SS. The receiver operating characteristic analysis showed that the panel had a sensitivity of 84.3% with a specificity of 74.8% in discriminating patients with SS from healthy controls. CONCLUSION: Our approach successfully identified serum biomarkers associated with SS patients. The potential lipid biomarkers indicated that SS metabolic disturbance might be associated with oxidized lipids, fatty acid oxidation and energy metabolism.


Assuntos
Lipidômica , Síndrome de Sjogren/sangue , Biomarcadores/sangue , Carnitina/análogos & derivados , Carnitina/sangue , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Análise Discriminante , Feminino , Humanos , Masculino , Espectrometria de Massas , Fosfatidilcolinas/sangue , Sensibilidade e Especificidade , Triglicerídeos/sangue
2.
Clin Exp Rheumatol ; 39 Suppl 133(6): 23-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251320

RESUMO

OBJECTIVES: At present, the pathogenesis of Sjögren's syndrome (SS) remains unclear. This research aimed to identify differential metabolites that contribute to SS diagnosis and discover the disturbed metabolic pathways. METHODS: Recent advances in mass spectrometry have allowed the identification of hundreds of unique metabolic signatures and the exploration of altered metabolite profiles in disease. In this study, 505 candidates including healthy controls (HCs) and SS patients were recruited and the serum samples were collected. A non-targeted gas chromatography-mass spectrometry (GC-MS) serum metabolomics method was used to explore the changes in serum metabolites. RESULTS: We found SS patients and HCs can be distinguished by 21 significant metabolites. The levels of alanine, tryptophan, glycolic acid, pelargonic acid, cis-1-2-dihydro-1-2-naphthalenediol, diglycerol, capric acid, turanose, behenic acid, dehydroabietic acid, stearic acid, linoleic acid, heptadecanoic acid, valine, and lactic acid were increased in serum samples from SS patients, whereas levels of catechol, anabasine, 3-6-anhydro-D-galactose, beta-gentiobiose, 2-ketoisocaproic acid and ethanolamine were decreased. The significantly changed pathways included the following: Linoleic acid metabolism; unsaturated fatty acid biosynthesis; aminoacyl-tRNA biosynthesis; valine, leucine, and isoleucine biosynthesis; glycerolipid metabolism; selenocompound metabolism; galactose metabolism; alanine, aspartate and glutamate metabolism; glyoxylate and dicarboxylate metabolism; glycerophospholipid metabolism; and valine, leucine and isoleucine degradation. CONCLUSIONS: These findings enhance the informative capacity of biochemical analyses through the identification of serum biomarkers and the analysis of metabolic pathways and contribute to an improved understanding of the pathogenesis of SS.


Assuntos
Síndrome de Sjogren , Biomarcadores , Humanos , Metabolômica , Síndrome de Sjogren/diagnóstico
3.
Analyst ; 143(6): 1444-1453, 2018 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-29469149

RESUMO

Nicking enzyme assisted amplification (NEAA) is an extremely rapid method for molecular diagnosis. However, this technology is not widely applied for real sample analysis because the overproduced non-specific products limit its sensitivity and raise the threshold of detection methods. Here, we have found that the non-specific amplification is mainly caused by the coexistence of Bst polymerase, nicking primers and dNTP. The highly active nicking enzyme directs and accelerates the non-specific amplification in a way which favors nicking. To suppress the non-specific amplification, the nicking enzyme concentration, reaction temperature, and magnesium ion concentration are optimized. The compatibility of Bst polymerase with the concentration of the monovalent cation is also crucial. Besides, the sensitivity could be enhanced by shortening the target sequences and priming the 3' end of the target.


Assuntos
Primers do DNA , DNA Polimerase Dirigida por DNA , Técnicas de Amplificação de Ácido Nucleico
4.
Molecules ; 23(1)2018 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-29361795

RESUMO

A sensitive and rapid ultra high-performance liquid-chromatography tandem mass spectrometry (UHPLC-MS/MS) method has been applied to investigate the influence of rheumatoid arthritis (RA) on the pharmacokinetics of nine analytes (daphnetin, daphnoretin, 7-hydroxycoumarin, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizin, and glycyrrhetinic acid), which are major active components in Zushima-Gancao extract. The analytes and internal standard (IS) were separated in a Hypersil Gold C18 column and detected on a triple-stage quadrupole mass spectrometer using the validated method. All analytes exhibited good linearities (R² > 0.98), and the lower limit of quantification (LLOQs) were sufficient for quantitative analysis. Intra- and inter-batch precision were all within 14.96% while the accuracy of nine analytes ranged from -17.99 to 14.48%, and these results were all within acceptance criteria. The extraction recoveries, matrix effects, and stabilities were all satisfactory. Main pharmacokinetic parameters of each compound were compared, and significant differences were found in parameters of daphnetin, daphnoretin, liquiritin, isoliquiritin, isoliquiritigenin, glycyrrhizin, and glycyrrhetinic acid, especially the last one, between the two groups. Therefore, adjuvant-induced arthritis has different effects on the pharmacokinetics of ingredients in Zushima-Gancao extract. The comparative pharmacokinetic study between normal and adjuvant-induced arthritis rats might provide more comprehensive information to guide the clinical usage of Zushima-Gancao extract for treating RA.


Assuntos
Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Compostos Fitoquímicos/química , Espectrometria de Massas em Tandem , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Estabilidade de Medicamentos , Medicamentos de Ervas Chinesas/farmacocinética , Masculino , Estrutura Molecular , Ratos , Reprodutibilidade dos Testes
5.
Anal Chem ; 89(8): 4413-4418, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28345860

RESUMO

On-site monitoring the plantation of genetically modified (GM) crops is of critical importance in agriculture industry throughout the world. In this paper, a simple, visual and instrument-free method for instant on-site detection of GTS 40-3-2 soybean has been developed. It is based on body-heat recombinase polymerase amplification (RPA) and followed with naked-eye detection via fluorescent DNA dye. Combining with extremely simplified sample preparation, the whole detection process can be accomplished within 10 min and the fluorescent results can be photographed by an accompanied smart phone. Results demonstrated a 100% detection rate for screening of practical GTS 40-3-2 soybean samples by 20 volunteers under different ambient temperatures. This method is not only suitable for on-site detection of GM crops but also demonstrates great potential to be applied in other fields.


Assuntos
DNA de Plantas/análise , Glycine max/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismo , DNA de Plantas/química , DNA de Plantas/metabolismo , Corantes Fluorescentes/química , Limite de Detecção , Fotografação , Plantas Geneticamente Modificadas/genética , Proteínas de Soja/genética , Temperatura
6.
Front Plant Sci ; 14: 1132881, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37063208

RESUMO

Temperature affects seed germination and seedling growth, which is a critical and complex stage in plant life cycle. However, comprehensive metabolic basis on temperature implicating seed germination and seedling growth remains less known. Here, we applied the high-throughput untargeted metabolomic and advanced shotgun lipidomic approaches to profile the Arabidopsis 182 metabolites and 149 lipids under moderate (22°C, 28°C) and extreme high (34°C, 40°C) temperatures. Our results showed that a typical feature of the metabolism related to organic acids/derivates and amines was obviously enriched at the moderate temperature, which was implicated in many cellular responses towards tricarboxylic acid cycle (TCA), carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis and indole 3-acetate (IAA) biosynthetic pathway. Whereas, under extreme high temperatures, there was no seed germination, but 148 out of total 182 metabolites were highly enriched, involving in the galactose metabolism, fatty acid degradation, tryptophan/phenylalanine metabolism, and shikimic acid-mediated pathways especially including alkaloids metabolism and glucosinolate/flavone/flavonol biosynthesis. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) also exhibited the gradually increased tendency from moderate temperatures to extreme high temperatures; whereas phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) were contrary to decrease. Another typical feature of the distinguished metabolites between 22°C and 28°C, the TCA, disaccharides, nucleotides, polypeptides, SQDG and the biosynthesis of fatty acids and glucobrassicin-mediated IAA were obviously decreased at 28°C, while amino acids, trisaccharides, PE, PC, PA, PS, MGDG, DGDG and diacylglycerol (DAG) preferred to enrich at 28°C, which characterized the alteration of metabolites and lipids during fast seedling growth. Taking together, our results provided the comprehensive metabolites phenotyping, revealed the characteristics of metabolites necessary for seed germination and/or seedling growth under different temperatures, and provided insights into the different metabolic regulation of metabolites and lipid homeostasis for seed germination and seedling growth.

7.
J Pharm Biomed Anal ; 236: 115677, 2023 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-37651923

RESUMO

Bronchoalveolar lavage (BAL) has been widely applied for the diagnosis of pulmonary diseases in clinical as it was recognized as a minimally invasive, well-tolerated and easily performed procedure. Lipid analysis of BAL fluid is a comprehensive strategy to observe lipid phenotypes, explore potential biomarkers, and elucidate the biological mechanisms of respiratory diseases. However, the highly diverse concentration of lipids in BAL fluid due to the deviation between the retrieved and injected aliquot volumes during lavage raised a challenge in obtaining high-quality lipidomic data. Here, this study aims to investigate what volume of BAL fluid is suitable for lipidomic analysis. Specifically, the BAL fluid harvested from H1N1 infected mice and controls was concentrated to varying degrees by freeze-drying technique before preparation for lipidomic analysis. The optimal concentration multiple of BAL fluid was approved by comparing the coverage and quality of identified lipids, as well as the number of differentially expressed lipids in the H1N1 infection model. Sixty-two differential lipids were identified respectively in the positive and negative modes when the BAL fluid was condensed five times, and they were classified into glycerolipids, phospholipids and fatty acids. This study focuses on the alterations of phospholipids, since they are the main constituents of pulmonary surfactants. Several phospholipids significantly accumulated in the BAL fluid of H1N1-infected mice, while most of them contained omega-3 polyunsaturated fatty acids, indicating disrupted inflammatory homeostasis in lungs. This study recommends freeze-drying/reconstitution prior to lipid extraction from BAL fluid for lipidomic analysis, as this procedure increased the richness and abundance of lipids.

8.
Drug Metab Dispos ; 39(4): 617-26, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21191083

RESUMO

Gambogic acid (GA) is a promising natural anticancer candidate. Although the anticancer activity of GA has been well demonstrated, information regarding the metabolic fate of GA is limited. Previous studies suggested that GA is mainly excreted into intestinal tract in rats through bile after intravenous administration, whereas only traces appeared in the feces, suggesting that GA is metabolized extensively in the intestine. However, there has been no report about the intestinal metabolism of GA either in animals or humans. In this study, large amounts of two sulfonic acid metabolites of GA were found in the feces samples of rats after intravenous administration, and their structures were identified as 10-α sulfonic acid GA and 10-ß sulfonic acid GA by comparison of the retention times and spectral data with those of synthesized reference substances using liquid chromatography-diode array detector-tandem mass spectrometry. This rare intestinal metabolic pathway mainly involves Michael addition of the sulfite ion to the 9,10 carbon-carbon double bond of α,ß-unsaturated ketone. In addition, a more detailed metabolic profile in rats is proposed, according to the results of in vitro and in vivo studies. It was found that GA can be metabolized by a variety of routes, including monooxidation, hydration, glutathionylation, glucuronidation, and glucosidation in the liver of rats. These findings provide information on the major metabolic soft spot of GA in the intestine and liver of rats, which is not only useful in the future human metabolic study of this compound but also of value in the metabolic studies of GA analogs.


Assuntos
Antineoplásicos/metabolismo , Bile/metabolismo , Produtos Biológicos/metabolismo , Fezes/química , Mucosa Intestinal/metabolismo , Redes e Vias Metabólicas , Xantonas/metabolismo , Animais , Antineoplásicos/análise , Bile/química , Produtos Biológicos/análise , Intestinos/química , Fígado/metabolismo , Microssomos Hepáticos , Ratos , Xantonas/análise
9.
Front Endocrinol (Lausanne) ; 12: 781417, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34956093

RESUMO

In diabetes mellitus (DM), disorders of glucose and lipid metabolism are significant causes of the onset and progression of diabetic nephropathy (DN). However, the exact roles of specific lipid molecules in the pathogenesis of DN remain unclear. This study recruited 577 participants, including healthy controls (HCs), type-2 DM (2-DM) patients, and DN patients, from the clinic. Serum samples were collected under fasting conditions. Liquid chromatography-mass spectrometry-based lipidomics methods were used to explore the lipid changes in the serum and identify potential lipid biomarkers for the diagnosis of DN. Lipidomics revealed that the combination of lysophosphatidylethanolamine (LPE) (16:0) and triacylglycerol (TAG) 54:2-FA18:1 was a biomarker panel for predicting DN. The receiver operating characteristic analysis showed that the panel had a sensitivity of 89.1% and 73.4% with a specificity of 88.1% and 76.7% for discriminating patients with DN from HCs and 2-DM patients. Then, we divided the DN patients in the validation cohort into microalbuminuria (diabetic nephropathy at an early stage, DNE) and macroalbuminuria (diabetic nephropathy at an advanced stage, DNA) groups and found that LPE(16:0), phosphatidylethanolamine (PE) (16:0/20:2), and TAG54:2-FA18:1 were tightly associated with the stages of DN. The sensitivity of the biomarker panel to distinguish between patients with DNE and 2-DM, DNA, and DNE patients was 65.6% and 85.9%, and the specificity was 76.7% and 75.0%, respectively. Our experiment showed that the combination of LPE(16:0), PE(16:0/20:2), and TAG54:2-FA18:1 exhibits excellent performance in the diagnosis of DN.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/diagnóstico , Lipidômica/métodos , Lipídeos/sangue , Adulto , Idoso , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
Artigo em Inglês | MEDLINE | ID: mdl-19661561

RESUMO

In the present study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI- MS/MS) method was developed for the screening and the structural elucidation of the metabolites of ecabet bismuth in rat bile. Solid-phase extraction cartridges were used for sample pre-treatment and a gradient liquid chromatographic system composed of 10 mM ammonium acetate buffer and methanol was used for chromatographic separation on a Phenomenex Kromasil C(18) column. The triple quadrupole mass spectrometer was employed to thoroughly detect and acquire the detailed MS/MS spectra of ecabet and its metabolites. By comparing the chromatographic retention behaviors, as well as the changes in molecular weight and full-scan MS/MS spectra of the potential metabolites with those of the parent compound, two main metabolites were identified as glucuronide conjugate of carbonylated ecabet (7-oxo-ecabet) and glucuronide conjugate of ecabet. Both two metabolites have not been reported in the literatures. The metabolic pathways of ecabet in rat were also proposed in this paper.


Assuntos
Abietanos/análise , Abietanos/metabolismo , Antiulcerosos/análise , Antiulcerosos/metabolismo , Bile/química , Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos
11.
Yao Xue Xue Bao ; 44(7): 771-7, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19806919

RESUMO

A sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method was established for the determination of eplerenone (EP) in human plasma. The plasma samples of EP were extracted with ethyl acetate and separated by HPLC on a reversed phase C18 column with a mobile phase of 10 mmol x L(-1) ammonium acetate water solution-methanol (30 : 70, v/v). EP was determined with electrospray ionization-mass spectrometry (ESI-MS) in the selected ion monitoring (SIM) mode. The calibration curves were linear over the range of 2-4 000 ng x mL(-1) for EP. The lower limit of quantification was 2 ng x mL(-1). The method has been successfully applied in the pharmacokinetic study of the EP tablets. The main pharmacokinetic parameters of EP after oral administration of 25 mg, 50 mg, 100 mg were as follows, t1/2: (4.9 +/- 2.1), (4.7 +/- 1.5), (5.9 +/- 1.2) h; AUC(0-infinity): (4 402 +/- 1 735), (8 150 +/- 2 509), (13 783 +/- 4 102) microg x h x L(-1); and MRT: (6.2 +/- 2.1), (6.6 +/- 1.3), and (7.2 +/- 1.6) h. Parameters of EP after oral administration of multiple doses of 50 mg were as follows, t1/2: (6.1 +/- 1.7) h; AUC(ss): (10 071 +/- 4220) microg x h x L(-1); MRT: (8.1 +/- 2.3) h; and DF: (3.2 +/- 1.0).


Assuntos
Espironolactona/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Eplerenona , Humanos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espironolactona/sangue , Espironolactona/farmacocinética
12.
Metabolites ; 9(1)2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30609645

RESUMO

Pyrexia is considered as a part of host's defense response to the invasion of microorganisms or inanimate matter recognized as pathogenic or alien, which frequently occurs in children. Jinxin oral liquid (JXOL) is a traditional Chinese medicine formula that has been widely used to treat febrile children in China. Experimental fever was induced by injecting yeast into young male Sprague-Dawley rats (80 ± 20 g) and the rectal temperature subsequently changed. Four hours later, the excessive production of interleukin (IL)-1ß and prostaglandin (PG) E2 induced by yeast was regulated to normal by JXOL administration. A rat brain metabolomics investigation of pyrexia of yeast and antipyretic effect of JXOL was performed using gas chromatography-mass spectrometry (GC-MS). Clear separation was achieved between the model and normal group. Twenty-two significantly altered metabolites were found in pyretic rats as potential biomarkers of fever. Twelve metabolites, significantly adjusted by JXOL to help relieve pyrexia, were selected out as biomarkers of antipyretic mechanism of JXOL, which were involved in glycolysis, purine metabolism, tryptophan mechanism, etc. In conclusion, the brain metabolomics revealed potential biomarkers in the JXOL antipyretic process and the associated pathways, which may aid in advanced understanding of fever and therapeutic mechanism of JXOL.

13.
Metabolites ; 9(4)2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31027159

RESUMO

Surfactant lipid metabolism is closely related to pulmonary diseases. Lipid metabolism disorder can cause lung diseases, vice versa. With this rationale, a useful method was established in this study to determine the lipidome in bronchoalveolar lavage fluid (BALF) of mice. The lipid components in BALF were extracted by liquid-liquid extraction (methanol and methyl tert-butyl ether, and water). Ultra-high-performance liquid chromatography coupled to hybrid Quadrupole-Exactive Orbitrap mass spectrometry was used to analyze the extracted samples, which showed a broad scanning range of 215-1800 m/z. With MS-DIAL software and built-in LipidBlast database, we identified 38 lipids in positive, and 31 lipids in negative, ion mode, including lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), etc. Then, the changes of lipids in BALF of mice with acute lung injury (ALI) induced by lipopolysaccharide (LPS) was investigated, which may contribute to further exploration of the pathogenesis of ALI.

14.
J Pharm Biomed Anal ; 162: 242-248, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30268025

RESUMO

An UHPLC-ESI-Q Exactive HF MS-based lipidomics method was successfully applied to profile various lipids from the plasma and lungs of mice intranasally challenged with lipopolysaccaride (LPS). Response trends of lipids to LPS were graphically represented by variable importance in projection (VIP) plot, heat map, and bar plot. As a result, 77 differential lipids in the lung and 13 differential lipids in the plasma were identified by comparison between healthy and LPS- induced mice. These results revealed the correlation between inflammation and lipids metabolism. The differentially regulated lipids could also be potentially used as biomarkers for inflammation.


Assuntos
Lesão Pulmonar Aguda/sangue , Cromatografia Líquida de Alta Pressão , Mediadores da Inflamação/sangue , Lipídeos/sangue , Lipopolissacarídeos , Pulmão/metabolismo , Metabolômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C
15.
Front Pharmacol ; 9: 891, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30154719

RESUMO

The Zushima tablet (ZT) has been used for decades in the clinical treatment of rheumatoid arthritis (RA) in China. However, its therapeutic mechanism is unclear. In this study, we aimed to explore the distinctive metabolic patterns in collagen-induced arthritis (CIA) rats and evaluate the therapeutic effects of ZT on RA using untargeted serum and fecal metabolomics approaches based on gas chromatography coupled with mass spectrometry. Body weight, hind paw swelling, TNF-α and IL-1ß levels, arthritis scores, and histopathological parameters were assessed. In the metabolomics study, 31 altered metabolites in the serum and 30 in the feces were identified by comparing the model with the control group using statistical processing. These altered metabolites revealed that the tricarboxylic acid cycle, glycolysis metabolism, fatty acid metabolism, and purine metabolism were disturbed in CIA rats, and most of these altered metabolites including l-isoleucine, l-aspartic acid, pyruvic acid, cholic acid, and hypoxanthine, were rectified by ZT. Furthermore, short-chain fatty acids in feces were quantitatively determined, and the results showed that ZT could regulate the levels of propionate, butyrate, and valerate in CIA rats. Then, gut microbiota were analyzed by 16S rRNA analysis. Our results showed that Firmicutes and Bacteroidetes were the most abundant bacteria in rats. The levels of 19 types of bacteria at the family level were altered in RA rats, and most of them could be regulated by ZT. This study demonstrated that metabolomics analysis is a powerful tool for providing novel insight into RA and for elucidating the potential mechanism of ZT.

16.
J Agric Food Chem ; 66(22): 5473-5480, 2018 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-29781618

RESUMO

' Candidatus Liberibacter asiaticus' (Las) is the most prevalent bacterium associated with huanglongbing, which is one of the most destructive diseases of citrus. In this paper, an extremely rapid and simple method for field detection of Las from leaf samples, based on recombinase polymerase amplification (RPA), is described. Three RPA primer pairs were designed and evaluated. RPA amplification was optimized so that it could be accomplished within 10 min. In combination with DNA crude extraction by a 50-fold dilution after 1 min of grinding in 0.5 M sodium hydroxide and visual detection via fluorescent DNA dye (positive samples display obvious green fluorescence while negative samples remain colorless), the whole detection process can be accomplished within 15 min. The sensitivity and specificity of this RPA-based method were evaluated and were proven to be equal to those of real-time PCR. The reliability of this method was also verified by analyzing field samples.


Assuntos
Citrus/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças das Plantas/microbiologia , Rhizobiaceae/isolamento & purificação , Primers do DNA/genética , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA/química , Rhizobiaceae/classificação , Rhizobiaceae/genética , Rhizobiaceae/fisiologia , Sensibilidade e Especificidade
17.
Artigo em Inglês | MEDLINE | ID: mdl-29890405

RESUMO

Flos Lonicerae Japonica-Fructus Forsythiae herb pair (Yin-Qiao in Chinese, YQ), is used clinically for the treatment of viral pneumonia due to its heat-clearing and detoxifying functions. In the present work, the effect of YQ in H1N1-induced inflammation in mice was investigated by metabolomics based on GC-MS. Body weight and histological results were used to assess the lung injury, while the levels of IL-6 and TNF-α in plasma were used to evaluate the extent of inflammation. The acquired GC-MS data were further subjected to multivariate data analysis, and the significantly altered metabolites identified. After statistical and pathway analysis, 17 significantly altered metabolites and 3 possible metabolic pathways were found in plasma between normal and H1N1-induced pneumonia mice, while 17 significant differential metabolites were identified when YQ treatment group was compared with model group. This work indicates that oral administration of YQ could protect mice from H1N1-induced inflammation partially by ameliorating the associated metabolic disturbances.


Assuntos
Medicamentos de Ervas Chinesas/administração & dosagem , Forsythia/química , Inflamação , Metaboloma/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Animais , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Frutas/química , Cromatografia Gasosa-Espectrometria de Massas , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/virologia , Vírus da Influenza A Subtipo H1N1 , Lonicera , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/metabolismo , Extratos Vegetais/farmacologia
18.
RSC Adv ; 8(51): 29368-29377, 2018 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-35548018

RESUMO

Respiratory syncytial virus (RSV) is a leading viral pathogen responsible for lower respiratory tract infections, particularly in children under five years worldwide, often resulting in hospitalization. At present, the molecular-level interactions between RSV and its host and the underlying mechanisms of RSV-induced inflammation are poorly understood. Herein, we describe an untargeted high-resolution lipidomics platform based on UHPLC-Q-Exactive-MS to assess the lipid alterations of lung tissues and plasma from a mouse model of RSV pneumonia. Untargeted lipidomics using LC-MS with multivariate analysis was applied to describe the lipidomic profiling of the lung tissues and plasma in RSV pneumonia mice. Lipid identification was conducted via an in silico MS/MS LipidBlast library using the MS-DIAL software. We observed distinct compartmental lipid signatures in the mice lung tissues and plasma and significant lipid profile changes between the systematic and localized host responses to RSV. A total of 87 and 68 differential lipids were captured in the mice lung tissue and plasma, respectively, including phospholipids, sphingolipids, acylcarnitine, and fatty acids. Some of these lipids belong to pulmonary surfactants, illustrating that RSV pneumonia-induced aberrations of the pulmonary surfactant system may play a vital role in the etiology of respiratory inflammation. Our findings reveal that the host responses to RSV and various lipid metabolic pathways were linked to disease pathology. Furthermore, our findings could provide mechanistic insights into RSV pneumonia.

19.
Chem Commun (Camb) ; 54(9): 1105-1108, 2018 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-29333552

RESUMO

An ultrafast and extremely simple approach was proposed to count the number of DNA molecules without any microfluidic-based device. By directly counting the number of amplicon clusters in a capillary, the absolute amount of DNA molecules could be easily determined.


Assuntos
DNA/análise , DNA/efeitos da radiação , Corantes Fluorescentes/análise , Raios Ultravioleta , DNA/genética , Corantes Fluorescentes/química , Fatores de Tempo
20.
Int J Clin Exp Pathol ; 10(9): 9990-9997, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-31966888

RESUMO

Endoplasmic reticulum (ER) stress has been increasingly recognized to have an important role in various liver diseases. Sepsis-induced multi-organ failure remains to have a high mortality rate, and the liver plays a central pathophysiological role. This study aims to explore whether ER stress is involved in liver injury in septic rats. Sepsis was induced via cecal ligation and puncture (CLP). Rats were randomly divided into five groups as follows: sham, CLP 2 h, CLP 6 h, CLP 12 h, and CLP 24 h. They were monitored to record body weight (BW) and liver weight changes for every time point after surgery. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected via colorimetric activity assays. In addition, the morphological changes of the liver tissue were evaluated by staining the sections with hematoxylin and eosin and observing under light microscopy. The levels of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and cleaved caspase-12 were detected via Western blot analysis. Apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method. The results showed that septic rat serums ALT and AST were increased, with the increase being more obvious in the CLP 24 h group. In addition, septic rats appeared to have histopathological abnormalities in the liver. The liver weight and index increased after CLP. No differences were noted in the BW between septic groups. The level of GRP78, CHOP, and cleaved caspase-12 were upregulated after CLP. However, CHOP and caspase-12 were induced later than GRP78. The density of TUNEL-positive apoptotic hepatocytes was significantly increased after 12 h and 24 h CLP. It indicates that the unfolded protein response occurred in the early stage of sepsis-induced liver damage. The ER stress-mediated apoptosis signal pathway is among the mechanisms of septic liver injury and may be a target in clinical prevention and therapy of sepsis-induced liver injury.

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