ABSTRACT
This study aimed to explore the mechanism of Shexiang Tongxin Dropping Pills(STDP) in treating diabetic cardiomyopathy(DCM) based on network pharmacology, molecular docking, and animal experiments. BATMAN, TCMSP, and GeneCards were searched for the active ingredients and targets of STDP against DCM. STRING and Cytoscape were used to build the protein-protein interaction(PPI) network and "drug-active ingredient-target" network. Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis of the targets were carried out based on DAVID. The molecular docking of key receptor proteins with corresponding active ingredients was performed using AutoDock Vina. The rat model of DCM was established by a high-fat diet combined with intraperitoneal injection of streptozotocin. Rats were assigned into control, model, low-(20 mg·kg~(-1)) and high-dose(40 mg·kg~(-1)) STDP, and metformin(200 mg·kg~(-1)) groups. After 8 weeks of continuous administration, the cardiac function, myocardial pathological changes, and myocardial collagen fiber deposition of rats in each group were detected by echocardiography, hematoxylin-eosin(HE) staining, and Sirius red staining, respectively. The myocardial hypertrophy was detected by WGA staining. The expression levels of p38 mitogen-activated protein kinase(p38), phosphorylation-p38(p-p38), c-Jun N-terminal kinase(JNK), phosphorylation-JNK(p-JNK), caspase-3, and C-caspase-3 in the myocardial tissue of rats in each group were measured by Western blot. The network pharmacology predicted 199 active ingredients and 1 655 targets of STDP and 463 targets of DCM. One hundred and thirty-four potential targets of STDP for treating DCM were obtained, and the AGE-RAGE signaling pathway in diabetic complications was screened out. Molecular docking results showed that miltirone, dehydromiltirone, and tryptanthrin had strong binding affinity with RAGE. The results of animal experiments confirmed that STDP effectively protected the cardiac function of DCM rats. Compared with the DCM model group, the STDP groups showed significantly down-regulated protein levels of p-p38, p-JNK, and C-caspase-3. To sum up, STDP may protect the cardiac function of DCM rats by regulating the AGE-RAGE signaling pathway.
Subject(s)
Diabetic Cardiomyopathies , Drugs, Chinese Herbal , Molecular Docking Simulation , Network Pharmacology , Animals , Diabetic Cardiomyopathies/drug therapy , Diabetic Cardiomyopathies/metabolism , Diabetic Cardiomyopathies/genetics , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/administration & dosage , Rats , Male , Rats, Sprague-Dawley , HumansABSTRACT
OBJECTIVE: To study the effect of Shexiang Tongxin Dropping Pill (STDP) on angiogenesis in diabetic cardiomyopathy mice with coronary microcirculation dysfunction (CMD). METHODS: According to a random number table, 6 of 36 SPF male C57BL/6 mice were randomly selected as the control group, and the remaining 30 mice were injected with streptozotocin intraperitoneally to replicate the type 1 diabetes model. Mice successfully copied the diabetes model were randomly divided into the model group, STDP low-dose group [15 mg/(kg·d)], medium-dose group [30 mg/(kg·d)], high-dose group [60 mg/(kg·d)], and nicorandil group [15 mg/(kg·d)], 6 in each group. The drug was given by continuous gavage for 12 weeks. The cardiac function of mice in each group was detected at the end of the experiment, and coronary flow reserve (CFR) was detected by chest Doppler technique. Pathological changes of myocardium were observed by hematoxylin-eosin staining, collagen fiber deposition was detected by masson staining, the number of myocardial capillaries was detected by platelet endothelial cell adhesion molecule-1 staining, and the degree of myocardial hypertrophy was detected by wheat germ agglutinin staining. The expression of the vascular endothlial growth factor (VEGF)/endothelial nitric oxide synthase (eNOS) signaling pathway-related proteins in myocardial tissue was detected by Western blot. RESULTS: Compared with the model group, medium- and high-dose STDP significantly increased the left ventricular ejection fraction and left ventricular fraction shortening (P<0.01), obviously repaired the disordered cardiac muscle structure, reduced myocardial fibrosis, reduced myocardial cell area, increased capillary density, and increased CFR level (all P<0.01). Western blot showed that high-dose STDP could significantly increase the expression of VEGF and promote the phosphorylation of vascular endothelial growth factor receptor 2, phosphoinositide 3-kinase, protein kinase B, and eNOS (P<0.05 or P<0.01). CONCLUSION: STDP has a definite therapeutic effect on diabetic CMD, and its mechanism may be related to promoting angiogenesis through the VEGF/eNOS signaling pathway.
ABSTRACT
Brassinosteroids (BRs) are plant steroid hormones that are involved in the regulation of plant growth and development as well as environmental adaptation. The discovery of new BR derivatives is beneficial to their biosynthesis and regulation mechanisms research. However, there are few reports on the methods for exploring new BRs, and the existing methods tend to lack coverage. In this work, we established a comprehensive, highly sensitive and selective structure-oriented method for the screening and structural identification of potential BRs in plants using chemical isotope labeling-assisted liquid chromatography-high resolution mass spectrometry (CL-LC-HRMS). The potential BRs were speculated according to the structural features of the reported BRs, and those speculated BRs containing cis-diol groups were labeled by isotope reagents of 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) and 2-methyl-4-PAMBA-d5 to improve the sensitivity and selectivity of MS detection. In addition, the fragmentation of 2-methyl-4-PAMBA-labeled BRs via collision-induced dissociation (CID) led to the generation of reporter ions, which contributed to specific screening of potential BRs. In-depth structure of potential BRs was elucidated by analyzing multistage MS (MSn) fragmentation patterns. Using our proposed method, a total of 16 potential BRs were detected from plant samples including 5 new ones, of which one new BR derivative was identified as 2-deoxy-3-epi-6-deoxy-dolichosterone, and this new BR may be involved in the biosynthesis of BRs as precursor of 6-deoxo-dolichosterone. The method developed in this work is promising for screening and identifying new BR derivatives in plants, thereby supplementing the biosynthesis pathway of BRs.
Subject(s)
Brassinosteroids , Tandem Mass Spectrometry , Brassinosteroids/analysis , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Tandem Mass Spectrometry/methods , Plants/chemistry , Indicators and ReagentsABSTRACT
In this study, a screening strategy was established based on ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry assisted by chemical isotope labeling (CIL-UPLC-HRMS) for screening and identifying abscisic acid (ABA) catabolites. Based on the structures of known ABA catabolites, this strategy first proposed the structures of catabolites to be discovered. Afterward, a pair of isotope reagents N,N-2-dimethylaminoethylamine (DMED) and d4-DMED were used as labeling reagents to label the carboxyl groups in ABA and its catabolites. Then, the mass-to-charge ratio (m/z) of DMED- and d4-DMED-labeled ABA catabolites was calculated based on the labeling schema. In light of the characteristic fragmentation patterns of the DMED-labeled standards of ABA and its catabolites, screening criteria were formulated. Using our strategy, ABA, t-ABA, and 18 ABA catabolites were identified from seven plant samples. Of the identified catabolites, 16 were known, and to our knowledge, 2 were previously unidentified. Our findings contribute to ABA catabolic network improvement.
Subject(s)
Abscisic Acid , Abscisic Acid/metabolism , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Isotope Labeling , Mass Spectrometry/methodsABSTRACT
Abscisic acid (ABA) hydroxylation is an important pathway for ABA inactivation and homeostasis maintenance. Here, we discover a new downstream catabolite of neophaseic acid (neoPA) in the ABA 9'-hydroxyl pathway and identify it as epi-neodihydrophaseic acid (epi-neoDPA) by comparing its accurate mass, retention time, and MSn spectra with those of our chemically synthesized epi-neoDPA. Analyses of Arabidopsis seed germination and ABA-related gene expression reveal that neoPA rather than epi-neoDPA possesses ABA-like hormonal activity. In vitro enzyme activity tests of prokaryotic recombinant protein reveal that NeoPAR1 (neoPA reductase 1) identified from Arabidopsis converts neoPA into epi-neoDPA. Site-directed mutation at Tyr163 in the conserved motif of NeoPAR1 abolishes the catalytic activity of NeoPAR1. Accelerated seed germination was observed in NeoPAR1 knockdown and knockout mutants, whereas retarded seed germination and the accumulation of epi-neoDPA and ABA were observed in NeoPAR1 overexpression lines, suggesting that NeoPAR1 is involved in seed germination and maintenance of ABA homeostasis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Abscisic Acid/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Germination/genetics , Hydroxylation , Seeds/geneticsABSTRACT
Retinoic acids (RAs), an important class of fatty acid derived from vitamin A, are closely associated with various human diseases including cancer. Hence, determination of endogenous RAs would help to uncover the mechanisms underlying RAs-related diseases. However, accurate quantification of RAs is still a challenge due to their high structure similarity, low abundance in biological samples and the lack of isotope internal standards (ISs). In this study, a liquid chromatography-mass spectrometry (LC-MS) method with high-sensitivity and high-throughput was developed to simultaneously determine 5 RAs (all-trans-retinoic acid, 13-cis-retinoic acid, 9,13-cis-retinoic acid, all-trans-4-oxoretinoic acid and 4-hydroxy-retinoic acid) in human serum. In the method, three derivatization reagents, N, N-dimethylethylenediamine (DMED), d4-N,N-dimethylethylenediamine (d4-DMED) and 13C2-N,N-dimethylethylenediamine (13C2-DMED), were used for triple chemical derivatization of RAs, thus the detection sensitivity and analysis throughput of endogenous RAs could be achieved. Benefiting from the developed strategy, the analysis throughput was enhanced and the detection sensitivity of RAs was increased by 14-398 folds. The limits of quantification (LOQs) of RAs were found to be between 8.4 and 130 pg/mL, which were better than previously reported methods. Good linearities of RAs were obtained with determination coefficient (R2) ranging from 0.9774 to 0.9999. The intra- and inter-day relative standard deviations (RSDs) were below 11.7% and 12.7%, respectively, indicating the acceptable reproducibility of the method. Using the developed method, we successfully quantified 4 RAs (all-trans-retinoic acid, 13-cis-retinoic acid, 9,13-cis-retinoic acid and all-trans-4-oxoretinoic acid) in health controls and cancer patient serum samples. Furthermore, t-test analysis showed that the concentration of three RAs (all-trans-retinoic acid, 13-cis-retinoic acid and all-trans-4-oxoretinoic acid) in cancer patient serum samples were significantly decreased compared with health controls, which indicated that RAs might play an important role in the formation and development of cancer.
Subject(s)
Isotretinoin , Tretinoin , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid , Humans , Mass Spectrometry , Reproducibility of ResultsABSTRACT
Melatonin is known to involve multiple physiological actions in plants. Herein, we found that exogenous melatonin inhibited the Arabidopsis seedling growth through the elevated abscisic acid (ABA) levels, and the elevated ABA was ascribed to the upregulation of 9-cis-epoxycarotenoid dioxygenase genes (NCEDs) in the ABA biosynthesis pathway. We also found that the overexpression lines of the melatonin receptor gene PMTR1 (also known as Cand2) yielded smaller seeds and germinated slower than the wild type, whereas PMTR1-knockout mutants produced larger seeds and germinated faster than the wild type. During the seed development, the accumulation peak of ABA was higher in the PMTR1-knockout mutant, while it was lower in the PMTR1-overexpression line than that in the wild type. In the dry seeds and imbibed seeds, the PMTR1-overexpression line accumulated higher ABA levels, while the PMTR1-knockout contained less ABA than the wild type. In summary, our findings suggest that PMTR1 is involved in ABA-mediated seed development and germination in Arabidopsis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Melatonin , Abscisic Acid/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Germination , Homeostasis , Melatonin/metabolism , SeedsABSTRACT
In this study, a highly sensitive method for analysis of 4 cyanogenic glycosides (CNGs) in cold-pressed flaxseed oil was developed by using cigarette filter fiber-based SPE and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The cold-pressed flaxseed oil was diluted with 5% (v/v) isopropanol/n-hexane solution and loaded to a cigarette filters fiber-based SPE column for CNG enrichment and purification. Under optimized conditions, four CNGs could be detected with limits of detection ranging from 1.3 to 4.4 pg/mL. The linear range was 0.05-50 ng/ml with a linear correlation coefficient (r) > 0.9935. CNG recovery ranged from 113% to 133%, and the relative standard deviation was between 0.8% and 20.5%. Finally, the proposed method was applied to the determination of CNGs in nine cold-pressed flaxseed oils.
Subject(s)
Tandem Mass Spectrometry , Tobacco Products , Chromatography, High Pressure Liquid , Chromatography, Liquid , Glycosides , Linseed Oil , Plant Oils , Solid Phase ExtractionABSTRACT
Bile acids (BAs) are a type of gut microbiota-host cometabolites with abundant structural diversity, and they play critical roles in maintaining host-microbiota homeostasis. In this study, we developed a new N-(4-aminomethylphenyl) pyridinium (AMPP) derivatization-assisted alternating dual-collision energy scanning mass spectrometry (AMPP-dual-CE MS) method for the profiling of BAs derived from host-gut microbiota cometabolism in mice. Using the proposed method, we discovered two new types of amino acid conjugations (alanine conjugation and proline conjugation) and acetyl conjugation with host BAs, for the first time, from mouse intestine contents and feces. Additionally, we also determined and identified nine new leucine- and phenylalanine-conjugated BAs. These findings broaden our knowledge of the composition of the BA pool and provide insight into the mechanism of host-gut microbiota cometabolism of BAs.
Subject(s)
Bile Acids and Salts , Gastrointestinal Microbiome , Animals , Bile , Bile Acids and Salts/analysis , Feces/chemistry , Mass Spectrometry , MiceABSTRACT
This study aims to investigate the effect of Shexiang Tongxin Dropping Pills(STDP) on angiogenesis in type 2 diabetic rats. Thirty-two healthy male SD rats were randomized into a control group(n=6) and a type 2 diabetes mellitus(T2 D) group(n=26). The T2 DM rat model was established with a high-fat diet combined with intraperitoneal injection of streptozotocin(STZ). The model rats were randomized into a model group, a metformin group(200 mg·kg~(-1)·d~(-1)), a high-dose STDP group(40 mg·kg~(-1)·d~(-1)), and a low-dose STDP group(20 mg·kg~(-1)·d~(-1)). The corresponding agents were administered continuously for 8 weeks. The cardiac function indexes were detected by ultrasound at the experiment endpoint, and the myocardial pathological changes were observed via hematoxylin-eosin(HE) staining. The myocardial collagen fiber deposition was detected by sirius red staining, the myocardial microvascular density by immunohistochemistry, and the myocardial hypertrophy by immunofluorescence assay. Western blot was employed to measure the protein levels of vascular endothelial growth factor(VEGF), phosphorylated vascular endothelial growth factor receptor 2(p-VEGFR2), angiogenin-1(Ang1), and TEK tyrosine kinase endothelial(Tie2) in the myocardial tissue. Compared with the model group, STDP improved the left ventricular ejection fraction(EF) and fractional shortening(FS)(P<0.05), alleivated the arrangement disorder of myocardial cells and local myocardial fiber rupture. Sirius red staining showed that STDP, especially the high-dose group(P<0.01), repaired the deposition of myocardial collagen fibers in the model group. The results of immunohistochemistry showed that the cluster of differentiation 31(CD31) expression in myocardial tissue of the STDP groups was higher than that of the model group(P<0.01). The immunofluorescence results showed that STDP mitigated the hypertrophy of myocardial cells(P<0.01). Furthermore, STDP up-regulated the protein levels of VEGF, p-VEGFR2, Angl, and Tie2 compared with the model group(P<0.01). In conclusion, STDP can effectively promote angiogenesis and improve cardiac function in type 2 diabetic rats by up-regulating the expression of VEGF, p-VEGFR2, Ang1, and Tie2.
Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Rats , Male , Animals , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Rats, Sprague-Dawley , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/drug therapy , Stroke Volume , Ventricular Function, Left , HypertrophyABSTRACT
Phaseic acid (PA), a main catabolite of abscisic acid (ABA), is structurally related to ABA and possesses ABA-like hormonal activity. However, the comprehensive metabolism pathway and roles of PA are not well understood. Here, using homologous alignment and expression pattern analysis, we identified in Arabidopsis the previously named CRL1 (Cinnamoyl coA: NADP oxidoreductase-like 1) as a PA reductase that catalyses PA to dihydrophaseic acid. The function of CRL1 and the potential role of PA were studied in transgenic CRL1 plants. Overexpression of CRL1 resulted in decreased ABA sensitivity in seed germination and attenuated drought tolerance. In contrast, increased ABA sensitivity and elevated drought tolerance was observed in down-regulated and loss-of-function crl1 mutants. Tyr162 in the conserved motif is the key residue in CRL1 to catalyse PA. Accelerated seed germination and earlier flowering phenotype were also observed in overexpressing lines, while retarded seed germination and delayed flowering occurred in crl1 mutants which accumulated more PA, but less dihydrophaseic acid than the wild type. This study demonstrates that PA plays diverse functions in drought tolerance, seed germination and flowering in an ABA-like manner, which may increase the adaptive plasticity of plants.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Abscisic Acid/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Coenzyme A/genetics , Coenzyme A/metabolism , Droughts , Gene Expression Regulation, Plant , Germination/genetics , Homeostasis , NADP/metabolism , Oxidoreductases/metabolism , Plants, Genetically Modified/genetics , Seeds/metabolism , SesquiterpenesABSTRACT
In this paper, a novel two-stage epidemic model with a dynamic control strategy is proposed to describe the spread of Corona Virus Disease 2019 (COVID-19) in China. Combined with local epidemic control policies, an epidemic model with a traceability process is established. We aim to investigate the appropriate control strategies to minimize the control cost and ensure the normal operation of society under the premise of containing the epidemic. This work mainly includes: (i) propose the concept about the first and the second waves of COVID-19, as well as study the case data and regularity of four cities; (ii) derive the existence and stability of the equilibrium, the parameter sensitivity of the model, and the existence of the optimal control strategy; (iii) carry out the numerical simulation associated with the theoretical results and construct a dynamic control strategy and verify its feasibility.
ABSTRACT
BACKGROUND: This study aimed to access the efficacy and safety of integrated Traditional Chinese and Western medicine treatment for patients with ulcerative colitis (UC) combined diabetes. METHODS: This protocol adheres to the preferred reporting items for systematic reviews and meta-analysis protocol statement. We plan to search 8 electronic databases to identify qualifying studies published from database inception until December 1, 2020. The software of EndNote reference manager (X9) will be used to study selection. A pre-developed standardized data collection form will be used to extract from all eligible studies. For included studies, the quality will be assessed by Cochrane Risk of bias tool. The RevMan 5.3 software (Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) developed by the Cochrane Collaboration will be used for all statistical analysis. If possible, meta-analysis will be undertaken for each of the outcomes. For continuous variable data, we will used mean differences with 95% confidence intervals (CIs) as summary statistics. For dichotomous variable data, we will calculate Mantel-Haenszel odds ratio with 95% CIs as summary statistics from the numbers of events in control and intervention groups. We will consider a result to be statistically significant if Pâ<â.05. If outcomes cannot be meta-analyzed, we will performer a descriptive analysis. RESULTS: This study will be performed to test the efficacy and safety of integrated Traditional Chinese and Western medicine treatment for patients with UC combined diabetes. CONCLUSION: The results of our study will be published in a peer-reviewed journals, and we will promotion results in domestic and foreign conferences. REGISTRATION NUMBER: INPLASY2020120087. ETHICS AND DISSEMINATION: As a systematic review and meta-analysis which based on previously published literature, ethical approval, and informed consent from patients are not required.
Subject(s)
Colitis, Ulcerative/therapy , Delivery of Health Care, Integrated/methods , Diabetes Mellitus, Type 1/therapy , Diabetes Mellitus, Type 2/therapy , Medicine, Chinese Traditional/methods , Adolescent , Adult , Aged , Colitis, Ulcerative/complications , Combined Modality Therapy , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 2/complications , Female , Humans , Male , Meta-Analysis as Topic , Middle Aged , Randomized Controlled Trials as Topic , Research Design , Systematic Reviews as Topic , Treatment Outcome , Young AdultABSTRACT
cis-Diol-containing metabolites are widely distributed in living organisms, and they participate in the regulation of various important biological activities. The profiling of cis-diol-containing metabolites could help us in fully understanding their functions. In this work, based on the characteristic isotope pattern of boron, we employed a boronic acid reagent as the isotope tag to establish a sensitive and selective liquid chromatography-high-resolution mass spectrometry method for the screening and annotation of cis-diol-containing metabolites in biological samples. Boronic acid reagent 2-methyl-4-phenylaminomethylphenylboronic acid was used to label the cis-diol-containing metabolites in biological samples to improve the selectivity and MS sensitivity of cis-diol-containing metabolites. Based on the characteristic 0.996 Da mass difference of precursor ions and the peak intensity ratio of 1:4 originating from 10B and 11B natural isotopes, the potential cis-diol-containing metabolites were rapidly screened from biological samples. Potential cis-diol-containing metabolites were annotated by database searching and analysis of fragmentation patterns obtained by multistage MS (MSn) via collision-induced dissociation. Importantly, the cis-diol position could be readily resolved by the MS3 spectrum. With this method, a total of 45 cis-diol-containing metabolites were discovered in rice, including monoglycerides, polyhydroxy fatty acids, fatty alcohols, and so forth. Furthermore, the established method showed superiority in avoiding false-positive results in profiling cis-diol-containing metabolites.
Subject(s)
Boron , Tandem Mass Spectrometry , Alcohols , Chromatography, Liquid , Isotope Labeling , IsotopesABSTRACT
Brassinosteroids (BRs) play crucial roles in a variety of physiological processes in plants. The full elucidation of the functions of RBs relies on sensitive detection and accurate measurement of BRs in plants. However, the identification and quantification of BRs are challenging due to their low abundance as well as poor ionization efficiencies during mass spectrometry-based analysis. Herein, we developed a highly sensitive and selective strategy for profiling potential BRs in plants by stable isotope labeling liquid chromatography multiple reaction monitoring scan mass spectrometry (SIL-LC-MRM-MS) analysis. In the strategy, we used a pair of stable isotope labeling reagents 4-phenylaminomethyl-benzeneboronic acid (4-PAMBA) and d5-4-phenylaminomethyl-benzeneboronic acid (4-PAMBA-d5) that can react with C22-C23 cis-diol on BRs for profiling potential BRs in plant tissues. The 4-PAMBA and 4-PAMBA-d5 labeled BRs could generate two characteristic neutral loss under collision induced dissociation (CID), respectively, which is used to establish the MRM-based detection and screening. The precursor ions of BRs labeled with 4-PAMBA and 4-PAMBA-d5 were set according to the reported structures of BRs, and the corresponding product ions were predicted by subtracting the lost neutral loss. In this respect, corresponding precursor ions and product ions in MRM transitions are formed. The peak pairs with a fixed mass difference, similar retention times and intensities were assigned as potential BRs. Using the developed SIL-LC-MRM-MS strategy, we successfully found 13 potential BR in different tissues of rape flower. Taken together, the SIL-LC-MRM-MS analytical strategy is promising for profiling potential BRs as well as other compounds that have the same functional moiety from complex biological samples.
Subject(s)
Brassica napus/chemistry , Brassinosteroids/analysis , Flowers/chemistry , Chromatography, Liquid , Isotope Labeling , Mass Spectrometry , Molecular ConformationABSTRACT
An efficient and selective pretreatment method of one-step hydrophilic interaction chromatography-based solid phase extraction (HILIC SPE) was developed using silica as the sorbent to quickly and sensitively detect endogenous ABA and its five catabolites in fresh Oryza sativa tissues. The extracted analytes were sensitively quantified with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Under the optimized conditions, good linearity of the developed analytical method was obtained in the range of 0.2-1000 ng/mL with linear correlation coefficients ( r) greater than 0.9987. The limits of detection (LODs, signal/noise = 3) ranged from 0.01 to 0.74 ng/mL. The relative recoveries were between 83.3% and 112.0% with the relative standard deviations (RSDs) ranging from 0.5 to 15.0%. Using the proposed method, the concentration variations of ABA and its catabolites were monitored in the salt-stressed rice tissues.
Subject(s)
Abscisic Acid/analysis , Abscisic Acid/metabolism , Plant Extracts/chemistry , Solid Phase Extraction/methods , Tandem Mass Spectrometry/methods , Adsorption , Chromatography, High Pressure Liquid/methods , Hydrophobic and Hydrophilic Interactions , Limit of Detection , Oryza/chemistry , Seedlings/chemistry , Silicon Dioxide/chemistryABSTRACT
BACKGROUND: To evaluate the diagnostic performances of detecting circulating tumor cells (CTCs) and tumor cells in bronchoalveolar lavage fluid (BALF) for peripheral lung cancer. METHODS: A total of 247 patients with lung cancer and 70 cases with benign lung disease were recruited in this study. Peripheral blood and BALF samples were collected, in which the tumor cells were enriched by negative immunomagnetic selection and detected by fluorescence in situ hybridization (FISH) of chromosome enumeration probe 8 (CEP8). The levels of tumor-associated markers (e.g., CEA, CA125, and NSE) in peripheral blood plasma were measured by using electrochemiluminescence. RESULTS: The numbers of CTCs detected in peripheral blood were significantly higher in patients with lung cancer than those with benign lung disease (5.78±0.57 vs. 1.13±0.39, Z=-8.64, P<0.01). Similarly, tumor cells count in BALF of malignancy were higher than that of benign lesions (6.76±0.89 vs. 0.89±0.23, Z=-6.254, P<0.01). However, as for patients with lung cancer and benign lung disease, the numbers of tumor cells in peripheral blood were comparable with those in BALF (both P>0.05). Detecting CTCs and tumor cells in BALF had similar areas under curves (AUC =0.871 and 0.963, respectively; P>0.05) in discriminating benign lesions from lung cancer (sensitivity 83.8% and 92.6%, specificity 86.5% and 99.9%, respectively), both of which were larger than those of NSE, CEA, and CA125 (AUC =0.564, 0.512 and 0.554, respectively; all P<0.05). The diagnostic performances of discriminating benign lesions and lung cancer in BALF and peripheral blood were both in concordance with that of histopathology (kappa values 0.662 and 0.569, respectively; both P<0.001). CONCLUSIONS: Detecting tumor cells in peripheral blood and BALF may sensitive to identify benign and malignant peripheral lung lesions and be of value for early diagnosis of lung cancer.
ABSTRACT
A novel strategy was developed for the application of Rh carbenes generated from readily accessible N-sulfonyl 1,2,3,-triazoles or diazocompouds in the high order cycloadditions, which offered an efficient route to a variety of N-containing medium-sized rings. The process provided a wide range of cyclohepta[b]pyrazine and cyclohepta[b]pyrrolone derivatives with high yields.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the roles of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in host defense against Streptococcus pneumoniae infection in the middle ear.</p><p><b>METHODS</b>Wild-type (WT) C57BL/6J, TLR2-deficient (TLR2(-/-)) and TLR4-deficient (TLR4(-/-)) mice were inoculated with Streptococcus pneumoniae (1 × 10(6)CFU) through the tympanic membrane. All animals were tested the mouse ABR thresholds and tympanometry measurement before, and 1 day, 3 days and 7 days following pneumococcal challenge. Blood bacterial titer were determined by plating 50 µl volumes of 10-fold diluted blood. Histological analysis of middle ear and inner ear were performed by fixation, decalcification, embedded section, and counterstained with hematoxylin/eosin and toluidine blue staining. Semi-quantitative RT-PCR was applied to determine mRNA accumulation of TLR2 and TLR4 related genes.</p><p><b>RESULTS</b>Forty of 68 TLR2(-/-) mice and twenty-one of 59 TLR4(-/-) mice showed bacteremia and died within 3 days after the pneumococcal challenge, however, only 9 of 52 WT mice died. The survive mice were shown have more severe hearing loss in the TLR2(-/-) and TLR4(-/-) mice than in the WT mice, indicated by ABR thresholds, at 3 or 7 days postinoculation. The histological pathology was characterized by effusion and tissue damage in the middle ear, and in the TLR2(-/-) and TLR4(-/-) mice, the outcome of infection became more severe at 7 days. At both 3 and 7 days after challenge, the TLR2(-/-) mice had higher blood bacterial titers than WT mice (P < 0.05). Temporal bone histopathologic change indicated that 3 days after the pneumococcal challenge, the TLR2(-/-) and TLR4(-/-) mice showed effusion and tissue damage in the middle ear, and the infection became more severe at 7 days postinoculation. TLR2(-/-) mice showed severe inflammatory cell infiltration in the cochlear, the organ of Corti showed the outer hair cells damage, the tectorial membrane swelling, degeneration of the stria vascularis, and severe loss of spiral ganglion cells; However, the WT mice was not found the cell infiltration and tissue damage in the cochlear, the organ of Corti shown normal of outer hair cells. Mast cells were not found in the middle ear mucosa of TLR2(-/-) mice, but in the TLR4(-/-) and WT mice, more mast cells were found in the middle ear mucosa of effusion ear by 3 and 7 days postchallenge. Moreover, by 3 days postchallenge, the mRNA accumulation levels of NF-κB, tumor necrosis factor alpha (TNFα), interleukin1β, MIP-1α, MUC5AC and MUC5B were significantly lower in the ears of TLR2(-/-) mice than that in WT and TLR4(-/-) mice.</p><p><b>CONCLUSIONS</b>TLR2(-/-) mice may produce relatively low levels of proinflammatory cytokines following pneumococcal challenge, thus hindering the clearance of bacteria from the middle ear and leading to sepsis and high mortality rate. This study indicated that TLR2 and TLR4 are important in the molecular pathogenesis and host response to otitis media.</p>