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Find video protocols related to scientific articles indexed in Pubmed.
Sensitive detection of DNA methyltransferase activity based on exonuclease-mediated target recycling.
Anal. Chem.
PUBLISHED: 10-30-2014
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DNA methylation plays vital roles in various biological processes in both prokaryotes and eukaryotes. In bacteria, modification of adenine at N6 can protect bacterial DNA against cleavage by restriction enzymes, and bacterial DNA adenine methyltransferases are essential for bacterial virulence and viability. DNA adenine methyltransferase (DAM) targets the sequence of 5'-GATC-3' and can convert adenine into N(6)-methyladenine (m(6)A). Because mammals do not methylate DNA at adenine, bacterial DAM represents an excellent candidate for antibiotic development. Here, we developed an exonuclease III-aided target recycling strategy to sensitively assay activity of DAM. In this method, a hairpin probe labeled with a donor fluorophore (FAM) at the 5' end and a quencher (BHQ) close to the 3' end (FQ probe) was employed as reporter. Another hairpin substrate containing sequence of GATC was used as the methylation substrate of DAM. Once the hairpin substrate was methylated by DAM, it could be recognized and cleaved by Dpn I, which allows the release of a single-stranded oligodeoxynucleotide (ssODN). The ssODN can then hybridize to the 3' protruding terminus of FQ probe, which subsequently triggers the exonuclease III-mediated target recycling reaction and therefore can significantly improve the detection sensitivity of DAM. The exonuclease-mediated target recycling strategy is extremely sensitive and as low as 0.01 U/mL DAM can be distinctly determined. Using this developed method, we evaluated DAM activity in different growth stages of E. coli cells, and we also demonstrated that the assay has the potential to screen suitable inhibitor drugs for DAM for disease(s) treatment.
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In-syringe dispersive solid phase extraction: a novel format for electrospun fiber based microextraction.
Analyst
PUBLISHED: 10-16-2014
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A novel in-syringe dispersive solid phase extraction (dSPE) system using electrospun silica fibers as adsorbents has been developed in the current work. A few milligrams of electrospun silica fibers were incubated in sample solution in the barrel of a syringe for microextraction assisted by vortex. Due to the benefit of dispersion and the high mass transfer rate of the sub-microscale electrospun silica fibers, the extraction equilibrium was achieved in a very short time (less than 1 min). Moreover, thanks to the long fibrous properties of electrospun fibers, the separation of the adsorbent from sample solution was easily achieved by pushing out the sample solution which therefore simplified the sample pretreatment procedure. Besides, the analytical throughput was largely increased by using a multi-syringe plate to perform the extraction experiment. The performance of the in-syringe dSPE device was evaluated by extraction of endogenous cytokinins from plant tissue samples based on the hydrophilic interaction. Six endogenous cytokinins in 20 mg of Oryza sativa L. (O. sativa) leaves were successfully determined under optimized conditions using in-syringe dSPE combined with liquid chromatography-mass spectrometry analysis. The results demonstrated that the in-syringe dSPE method was a rapid and high-throughput strategy for the extraction of target compounds, which has great potential in microscale sample pretreatment using electrospun fibers.
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Decreased N(6)-methyladenosine in peripheral blood RNA from Diabetic Patients Is Associated with FTO Expression Rather than ALKBH5.
J. Clin. Endocrinol. Metab.
PUBLISHED: 10-11-2014
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Context: N(6)-methyladenosine (m(6)A) modification plays a fundamental role in epigenetic regulation of the mammalian transcriptome. m(6)A can be demethylated by fat mass and obesity associated (FTO) protein and ALKBH5 protein. However, the importance of m(6)A alteration in type 2 diabetes mellitus (T2DM) has not been explored. Objective: To investigate whether m(6)A content was reduced in T2DM patients and whether m(6)A content was correlated with the mRNA expression levels of FTO and ALKBH5 genes. Methods: In this case-control study, peripheral blood samples were obtained from 88 T2DM patients and 92 healthy controls. For the diabetic animal model experiment, blood samples were obtained from 7 diabetic and 8 nondiabetic rats. A sensitive liquid chromatography- electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the determination of m(6)A content in RNA, quantitative real-time PCR (qPCR) was used to examine the mRNA expression levels of FTO and ALKBH5 genes, and high-resolution melting (HRM) and DNA sequencing were used to detect FTO single nucleotide polymorphisms (SNPs). Results: Our results showed that m(6)A contents in RNA from T2DM patients and diabetic rats were significantly lower compared to the control groups (p = 2.6×10(-24) for T2DM patients; p = 0.001 for diabetic rats, respectively) and T2DM can be characterized by the content of m(6)A. The mRNA expression level of FTO was significantly higher in T2DM patients than that of the controls (p=0.0007), and associated with the risk of T2DM (odds ratio (OR) 2.797, 95% confidence interval (CI) 1.452 - 5.389, p= 0.002). Moreover, m(6)A contents were correlated with FTO mRNA expression. Conclusions: These data suggest that the increased mRNA expression of FTO could be responsible for the reduction of m(6)A in T2DM, which may further increase the risk of complications of T2DM. Low m(6)A should be investigated further as a novel potential biomarker of T2DM.
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Facile Preparation of Biocompatible Sulfhydryl Cotton Fiber-Based Sorbents by "Thiol-ene" Click Chemistry for Biological Analysis.
ACS Appl Mater Interfaces
PUBLISHED: 10-10-2014
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Sulfhydryl cotton fiber (SCF) has been widely used as adsorbent for a variety of metal ions since 1971. Thanks to the abundant thiols on SCF, in this study, we reported a universal method for the facile preparation of SCF-based materials using "thiol-ene" click chemistry for the first time. With the proposed method, two types of SCF-based materials, phenylboronic acid grafted sulfhydryl cotton fiber (SCF-PBA) and zirconium phosphonate-modified sulfhydryl cotton fiber (SCF-pVPA-Zr(4+)), were successfully prepared. The grafted functional groups onto the thiol group of SCF were demonstrated by X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDX). The prepared fibrous materials exhibited excellent fiber strength, good stability in aqueous or nonaqueous solutions, and great biocompatibility. Moreover, we developed filter-free in-pipet-tip SPE using these SCF-based materials as adsorbent for the enrichment of ribonucleosides, glycopeptides and phosphopeptides. Our results showed that SCF-PBA adsorbent can selectively capture ribonucleosides and glycopeptides from complex biological samples. And SCF-pVPA-Zr(4+) adsorbent exhibited high selectivity and capacity in the enrichment of phosphopeptides from the digestion mixture of ?-casein and bovine serum albumin (BSA), as well as human serum and nonfat milk digest. Generally, the preparation strategy can be a universal method for the synthesis of other functionalized cotton-based adsorbents with special requirement in microscale biological analysis.
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An Aqueous Rechargeable Formate-Based Hydrogen Battery Driven by Heterogeneous Pd Catalysis.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 09-27-2014
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The formate-based rechargeable hydrogen battery (RHB) promises high reversible capacity to meet the need for safe, reliable, and sustainable H2 storage used in fuel cell applications. Described herein is an additive-free RHB which is based on repetitive cycles operated between aqueous formate dehydrogenation (discharging) and bicarbonate hydrogenation (charging). Key to this truly efficient and durable H2 handling system is the use of highly strained Pd nanoparticles anchored on graphite oxide nanosheets as a robust and efficient solid catalyst, which can facilitate both the discharging and charging processes in a reversible and highly facile manner. Up to six repeated discharging/charging cycles can be performed without noticeable degradation in the storage capacity.
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Profiling of thiol-containing compounds by stable isotope labeling double precursor ion scan mass spectrometry.
Anal. Chem.
PUBLISHED: 09-24-2014
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Here we developed a novel strategy of isotope labeling in combination with high-performance liquid chromatography-double precursor ion scan mass spectrometry (IL-LC-DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of isotope labeling reagents (?-bromoacetonylquinolinium bromide, BQB; ?-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-d7 labeled thiol-containing compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiol-containing compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, offering good identification and accurate quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Using the IL-LC-DPIS-MS strategy, we profiled the thiol-containing compounds in beer and human urine, and 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with thiol standards or tandem mass spectrometry analysis. Taken together, the IL-LC-DPIS-MS method is demonstrated to be a promising strategy in the profiling of compounds with identical groups in metabolomics study.
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Magnetic solid phase extraction coupled with in situ derivatization for the highly sensitive determination of acidic phytohormones in rice leaves by UPLC-MS/MS.
Analyst
PUBLISHED: 08-23-2014
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A simple, rapid and sensitive method based on magnetic solid phase extraction (MSPE) coupled with in situ derivatization (ISD) was developed for the determination of endogenous acidic phytohormones in rice leaves by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. With this method, acidic phytohormones were extracted onto the surface of a TiO2/magnetic hollow mesoporous silica sphere (MHMSS) through hydrophilic interaction, and then in situ derivatization was performed by the subsequent addition of 3-bromoactonyltrimethylammonium bromide (BTA). Thus, the process integrated extraction, purification, and derivatization into one step. Additionally, the permanent positively charged moiety from BTA significantly improved the ionization efficiencies of the acidic phytohormones. Several parameters affecting the efficiencies of the extraction, derivatization, and desorption were evaluated. The signal intensities of acidic phytohormones increased by 2 to 481 fold after treatment with MSPE-ISD. Under the optimized conditions, several endogenous acidic phytohormones, including GA4, GA9, GA20, JA, IAA, and ABA, were identified and quantified in rice leaves by the MSPE-ISD method. The limits of detection (LODs) were in the range of 1.03-91.21 pg mL(-1). The relative recoveries ranged from 71.6-112.8%, with the intra- and interday relative standard deviations (RSDs) being less than 14.9% and 16.2%, respectively. Taken together, the proposed method provides a novel approach of combining magnetic solid phase extraction and in situ derivatization for the highly sensitive determination of endogenous acidic phytohormones.
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"Old" metal oxide affinity chromatography as "novel" strategy for specific capture of cis-diol-containing compounds.
J Chromatogr A
PUBLISHED: 08-07-2014
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The metal oxide affinity chromatography (MOAC) materials have been extensively used for extraction of phosphate compounds in the past decade. Actually, some of these materials also possess adsorption affinity towards cis-diol-containing compounds, which was seldom explored in separation field so far. Here we present the proof-of-concept study to evaluate the feasibility of expanding MOAC for specific capture of cis-diol biomolecules. Benefitting from the high commercialisation of the metal oxide materials, such MOAC strategy possesses several advantages, like synthesis-free, low cost and high expandability. Firstly, the recognition of adenosine against 2'-deoxyadenosine was performed using zirconium oxide and cerium oxide, two typical commercial MOAC materials. The results showed that efficient adsorption and elution could be achieved easily by pH switching from basic to acidic. The isotherm curves demonstrated the adsorption process fitted well with Freundlich isotherm model and was spontaneous at room temperature (?G(0)<0) with an exothermic nature (?H(0)<0). Afterwards, the highly efficient and selective enrichment of various model cis-diol biomolecules, including ribonucleosides, glycopeptides and glycoproteins, was achieved using this MOAC strategy. Finally, the endogenous ribonucleosides and modified ribonucleosides were successfully purified from human urine sample, which demonstrated the potential application of MOAC materials in the enrichment of target compounds from complex biological samples. Besides the excellent performance of extraction for cis-diol-containing compounds, equally important is that these materials are commercially available with low cost, which makes the MOAC a promising strategy for the study of cis-diol biomolecules in metabolomics and proteomics.
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Facile one-pot synthesis of a aptamer-based organic-silica hybrid monolithic capillary column by "thiol-ene" click chemistry for detection of enantiomers of chemotherapeutic anthracyclines.
Analyst
PUBLISHED: 07-30-2014
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In the current study, we developed a facile strategy for the one-pot synthesis of an aptamer-based organic-silica hybrid monolithic capillary column. A 5'-SH-modified aptamer, specifically targeting doxorubicin, was covalently modified in the hybrid silica monolithic column by a sol-gel method combined with "thiol-ene" click reaction. The prepared monolithic column had good stability and permeability, large specific surface, and showed excellent selectivity towards chemotherapeutic anthracyclines of doxorubicin and epirubicin. In addition, the enantiomers of doxorubicin and epirubicin can be easily separated by aptamer-based affinity monolithic capillary liquid chromatography. Furthermore, doxorubicin and epirubicin spiked in serum and urine were also successfully determined, which suggested that the complex biological matrix had a negligible effect on the detection of doxorubicin and epirubicin. Finally, we quantified the concentration of epirubicin in the serum of breast cancer patients treated with epirubicin by intravenous injection. The developed analytical method is cost-effective and rapid, and biological samples can be directly analyzed without any tedious sample pretreatment, which is extremely useful for monitoring medicines in serum and urine for pharmacokinetic studies.
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Determination of oxidation products of 5-methylcytosine in plants by chemical derivatization coupled with liquid chromatography/tandem mass spectrometry analysis.
Anal. Chem.
PUBLISHED: 07-08-2014
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Cytosine methylation (5-methylcytosine, 5-mC) in DNA is an important epigenetic mark that has regulatory roles in various biological processes. In plants, active DNA demethylation can be achieved through direct cleavage by DNA glycosylases, followed by replacement of 5-mC with cytosine by base excision repair (BER) machinery. Recent studies in mammals have demonstrated 5-mC can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC) by Ten-eleven translocation (TET) proteins. The consecutive oxidations of 5-mC constitute the active DNA demethylation pathway in mammals, which raised the possible presence of oxidation products of 5-mC (5-hmC, 5-foC, and 5-caC) in plant genomes. However, there is no definitive evidence supporting the presence of these modified bases in plant genomic DNA, especially for 5-foC and 5-caC. Here we developed a chemical derivatization strategy combined with liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to determine 5-formyl-2'-deoxycytidine (5-fodC) and 5-carboxyl-2'-deoxycytidine (5-cadC). Derivatization of 5-fodC and 5-cadC by Girard's reagents (GirD, GirT, and GirP) significantly increased the detection sensitivities of 5-fodC and 5-cadC by 52-260-fold. Using this method, we demonstrated the widespread existence of 5-fodC and 5-cadC in genomic DNA of various plant tissues, indicating that active DNA demethylation in plants may go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with BER. Moreover, we found that environmental stresses of drought and salinity can change the contents of 5-fodC and 5-cadC in plant genomes, suggesting the functional roles of 5-fodC and 5-cadC in response to environmental stresses.
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Partially reduced iridium oxide clusters dispersed on titania as efficient catalysts for facile synthesis of dimethylformamide from CO2, H2 and dimethylamine.
Chem. Commun. (Camb.)
PUBLISHED: 07-04-2014
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A novel bifunctional catalyst based on partially reduced iridium oxide supported on TiO2 was found to be exceedingly efficient for the organic-solvent-free synthesis of dimethylformamide from CO2, H2 and dimethylamine.
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Isotope labelling--paired homologous double neutral loss scan-mass spectrometry for profiling of metabolites with a carboxyl group.
Analyst
PUBLISHED: 05-21-2014
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We developed a novel method for non-targeted screening of metabolites by high performance liquid chromatography-mass spectrometry with paired homologous double neutral loss scan mode after in vitro isotope labelling (IL-HPLC-PHDNL-MS). As a proof of concept, we investigated the carboxylic acid metabolite profiling in plant samples by the IL-HPLC-PHDNL-MS method. To this end, N,N-dimethylaminobutylamine (DMBA) and d(4)-N,N-dimethylaminobutylamine (d(4)-DMBA) were synthesized and utilized to label carboxylic acids. Our results show the MS response of carboxylic acids was enhanced by 20- to 40-fold after labelling. As for the IL-HPLC-PHDNL-MS analysis, DMBA and d(4)-DMBA labelled samples were mixed equally before MS analysis. Because the isotope labelled moieties (dimethylamino moiety, Me2N) of DMBA and d(4)-DMBA are easily ruptured and lost as neutral fragments (NL 45 and NL 49) under collision induced dissociation (CID), two neutral loss scans can be carried out simultaneously to record the signals of DMBA and d(4)-DMBA labelled samples, respectively. In this respect, the metabolites from two samples labelled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, which can eliminate the MS response fluctuation and mutual interference. Using this method, six potential biomarkers involved in wounded tomato leaves were identified, and their structures were further elucidated by product ion scan and high resolution mass spectrometry analysis. Taken together, the IL-HPLC-PHDNL-MS method demonstrated good performance on the identification as well as relative quantification of metabolites with a carboxyl group in biological samples.
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Pyridostigmine ameliorates cardiac remodeling induced by myocardial infarction via inhibition of the transforming growth factor-?1/TGF-?1-activated kinase pathway.
J. Cardiovasc. Pharmacol.
PUBLISHED: 05-09-2014
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Autonomic imbalance characterized by sympathetic predominance coinciding with diminished vagal activity is an independent risk factor in cardiovascular diseases. Several studies show that vagus nerve stimulation exerted beneficial effects on cardiac function and survival. In this study, we investigated the vagomimetic effect of pyridostigmine on left ventricular (LV) remodeling in rats after myocardial infarction. After myocardial infarction, surviving rats were treated with or without pyridostigmine (31 mg·kg?¹·d?¹) for 2 weeks, and hemodynamic parameters were measured. LV tissue was used to assess infarct size and interstitial fibrosis by Masson's trichrome and 0.1% picrosirius red staining. Protein expression of heart tissues was used to assess the efficacy of the treatment. Pyridostigmine markedly reduced myocardial infarct size and improved cardiac diastolic function. These improvements were accompanied with a significant decrease in matrix metalloproteinase-2 expression and collagen deposition. Additionally, pyridostigmine inhibited both transforming growth factor-?1 (TGF-?1) and TGF-?1-activated kinase expression in hearts postmyocardial infarction. Thus, pyridostigmine reduces collagen deposition, attenuates cardiac fibrosis, and improves LV diastolic function after myocardial infarction via TGF-?1/TGF-?1-activated kinase pathway inhibition.
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[Analysis of monosaccharide compositions in polysaccharides from exopleura of Ginkgo biloba].
Zhongguo Zhong Yao Za Zhi
PUBLISHED: 04-26-2014
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A high-performance liquid chromatography method of pre-column derivatization with 1-phenyl-3-methyl-5 -pyrazolone (PMP) has been established for determination of 6 kinds of monosaccharides simultaneously. A special Agilent HC-C18 column (4. 6 mm x 250 mm, 5 microm), optimized for the separation of PMP derivatives, was used at ambient temperature of 40 degrees C. The PMP derivatives elution was performed with a mixture of 0.1 mol x L(-1) phosphate buffer (pH 6. 8) and acetonitrile in a ratio of 84: 16 at a flow rate of 1 mL x min(-1), and UV absorbance of the effluent was monitored at 245 nm. The results showed that the polysaccharides from exopleura of Ginkgo biloba were acidic heteropolysaccharides mainly containing mannose, rhamnose, D-galacturonic acid, glucose, galactose, arabinose, with the molar ratio of 0.032: 0.14: 0.296: 0.403:0.106: 0.046.
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Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells.
J Chromatogr A
PUBLISHED: 04-25-2014
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As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.
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A facile approach for the polymer grafting of silica based on tandem reversible addition fragmentation chain transfer/click chemistry and its application in high performance liquid chromatography.
J Chromatogr A
PUBLISHED: 04-08-2014
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In this work, a tandem reversible addition fragmentation chain transfer (RAFT)/click chemistry method was developed to prepare amide-polystyrene-silica (NHCO-Psty-silica) stationary phase. Styrene was immobilized on amino-silica surface via an azide functionalized RAFT agent in a one-pot procedure. The resultant NHCO-Psty-silica column demonstrates better performance for shielding of residue silanols than traditional ODS column, which was ascertained by Engelhardt test (E test), Tanaka test (T test), Galushko test (G test), and Walters test (W test). Our results showed lower values of silanol activity calculated according to the formula in these standard tests for NHCO-Psty-silica column compared to the ODS column we tested. As a result, the NHCO-Psty-silica is suitable for the separation of basic compounds. The hydrophobic, anion exchanging and ?-? interaction of the column toward analytes were also evaluated. Moreover, the NHCO-Psty-silica column also showed excellent stability with pure water as mobile phase.
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Preparation and sonodynamic activities of water-soluble tetra-?-(3-carboxyphenoxyl) zinc(II) phthalocyanine and its bovine serum albumin conjugate.
Ultrason Sonochem
PUBLISHED: 04-01-2014
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Sonodynamic therapy (SDT) is a new approach for cancer treatment, involving the synergistic effect of ultrasound and certain chemical compounds termed as sonosensitizers. A water-soluble phthalocyanine, namely tetra-?-(3-carboxyphenoxyl) zinc(II) phthalocyanine (ZnPcC4), has been prepared and characterized. The interactions between ZnPcC4 and bovine serum albumin (BSA) were also investigated by absorption and fluorescence spectroscopy. It was found that there were strong interactions between ZnPcC4 and BSA with a binding constant of 6.83×10(7)M(-1). A non-covalent BSA conjugate of ZnPcC4 (ZnPcC4-BSA) was prepared. Both ZnPcC4 and ZnPcC4-BSA exhibited efficient sonodynamic activities against HepG2 human hepatocarcinoma cells. Compared with ZnPcC4, conjugate ZnPcC4-BSA showed a higher sonodynamic activity with an IC50 value of 7.5?M. Upon illumination with ultrasound, ZnPcC4-BSA can induce an increase of intracellular reactive oxygen species (ROS) level, resulting in cellular apoptosis. The results suggest that the albumin conjugates of zinc(II) phthalocyanines functionalized with carboxyls can serve as promising sonosensitizers for sonodynamic therapy.
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Rapid and high-throughput determination of endogenous cytokinins in Oryza sativa by bare Fe3O4 nanoparticles-based magnetic solid-phase extraction.
J Chromatogr A
PUBLISHED: 03-10-2014
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A rapid method was developed for determination of endogenous cytokinins (CKs) based on magnetic solid-phase extraction (MSPE) followed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). We illustrated the hydrophilic character of bare Fe3O4 nanoparticles that were directly used as a MSPE sorbent for rapid enrichment of endogenous CKs from complex plant extract. To the best of our knowledge, this is the first report of bare Fe3O4 directly used as efficient extraction sorbent to enrich target CKs based on hydrophilic interaction. Under the optimized conditions, a rapid, sensitive and high-throughput method for the determination of 16 CKs was established by combination of MSPE with UPLC-MS/MS. Good linearity was obtained with correlation coefficients (r) from 0.9902 to 0.9998. The limits of detection (LODs) and quantification (LOQs) ranged from 1.2 pg mL(-1) to 391.3 pg mL(-1) and 4.1 pg mL(-1) to 1304.3 pg mL(-1), respectively. 16 CKs could be successfully determined in spiked sample with 80.6-117.3% recoveries and the relative standard deviations (RSDs) were less than 16.6%. Finally, 10 endogenous CKs were successfully quantified in 50mg Oryza sativa sample using the developed MSPE-UPLC-MS/MS method.
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Acetylcholine Inhibits Tumor Necrosis Factor ? Activated Endoplasmic Reticulum Apoptotic Pathway via EGFR-PI3K Signaling in Cardiomyocytes.
J. Cell. Physiol.
PUBLISHED: 02-25-2014
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Previous findings have shown that acetycholine (ACh) decreased hypoxia-induced tumor necrosis factor alpha (TNF ?) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF ?-induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF ? stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF ?-induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh-elicited anti-apoptotic property. Together, these data indicate that EGFR-PI3K/Akt signaling is involved in M2 AChR-mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
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Electrospun polystyrene/oxidized carbon nanotubes film as both sorbent for thin film microextraction and matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
J Chromatogr A
PUBLISHED: 02-24-2014
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In the current study, polystyrene/oxidized carbon nanotubes (PS/OCNTs) film was prepared and applied as both an adsorbent of thin film microextraction (TFME) and matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the first time. The uniform size of PS/OCNTs film with OCNTs evenly and firmly immobilized in PS was obtained by electrospinning. And a novel TFME device was developed using the prepared PS/OCNTs film to enrich benzo[a]pyrene (BaP) from water, and also BaP and 1-hydroxypyrene (1-OHP) from urine sample. Then the extracted analytes on the PS/OCNTs film were directly applied to MALDI-MS analysis with PS/OCNTs film as the MALDI matrix. Our results show that PS/OCNTs film is a good TFME adsorbent toward the analytes and an excellent matrix for the sensitive determination of BaP and 1-OHP using MALDI-TOF-MS. The employment of PS/OCNTs as the matrix for MALDI can effectively avoid the large variation of signal intensity normally resulting from heterogeneous distribution of the adsorbed analyte on matrix layer, which therefore significantly improve spot-to-spot reproducibility. The introduction of PS in the film can prevent OCNTs from flying out of MALDI plate to damage the equipment. In addition, PS/OCNTs film also largely extended the duration of ion signal of target analyte compared to OCNTs matrix. The developed method was further successfully used to quantitatively determine BaP in environmental water and 1-OHP in urine samples. The results show that BaP and 1-OHP could be easily detected at concentrations of 50pgmL(-1) and 500pgmL(-1), respectively, indicating the high detection sensitivity of this method. For BaP analysis, the linear range was 0.1-20ngmL(-1) with a correlation coefficient of 0.9970 and the recoveries were in the range of 81.3 to 123.4% with the RSD?8.5% (n=3); for urinary 1-OHP analysis, the linear range was 0.5-20ngmL(-1) with a correlation coefficient of 0.9937 and the recoveries were in the range of 79.2 to 103.4% with the RSD?7.6% (n=3). Taken together, the developed method provides a simple, rapid, cost-effective and high-throughput approach for the analysis of BaP in environmental water and endogenous 1-OHP in urine samples.
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TiO2-based solid phase extraction strategy for highly effective elimination of normal ribonucleosides before detection of 2-deoxynucleosides/low-abundance 2-O-modified ribonucleosides.
Anal. Chem.
PUBLISHED: 10-09-2013
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A novel TiO2-based SPE strategy was developed for eliminating normal ribonucleosides before mass spectrometry (MS) analysis of 2-deoxynucleosides and 2-O-modified ribonucleosides. The chromatographic research for the retention behavior of ribonucleosides and 2-deoxynucleosides on TiO2 materials was investigated using TiO2 separation column. The results indicated a specific affinity interaction mechanism between TiO2 and cis-diol-containing ribonucleosides, and the interaction was proved effective even under a wide range of pH conditions and salt concentrations. Benefiting from these features, a TiO2-based solid phase extraction (SPE) method was developed for highly efficient elimination of RNA contamination from genomic DNA. Compared with the widely used enzymatic digestion method, the proposed TiO2-based SPE method showed much more efficiency for the removal of RNA as well as provided high recoveries for the 2-deoxynucleosides. In addition, the sample processing time is dramatically shortened using the TiO2-based SPE method (~5 min) compared to the traditional enzymatic digestion method (~12 h). Finally, the purification of 2-O-methylated ribonucleosides from RNA was successfully achieved in HeLa cells by the TiO2-based SPE method, which provided a proof-of-concept for the purification of relevant modified ribonucleosides from bulky normal ribonucleosides. Taken together, this strategy developed in the current study offers a promising option to purify 2-deoxynucleosides/2-O-modified ribonucleosides for their sensitive and accurate determination by eliminating normal ribonucleosides in biological samples.
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[Effects of sediment on the early settlement stage of Sargassum horneri on rocky subtidal reefs].
Ying Yong Sheng Tai Xue Bao
PUBLISHED: 09-11-2013
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By using sediment trap and suction pump to measure the relative sediment levels across different sites and water depths, and through the in situ measurements of Sargassum horneri density, this paper assessed the relationships between the distribution of S. horneri and the sediment levels and wave exposure on the rocky subtidal platforms around Gouqi Island, China. The laboratory-based experiments were also conducted to test the effects of different sediment levels on the attachment of S. horneri zygote and the survival rate of S. horneri germling after the attachment. S. horneri predominated at the sites with lesser sediment and wave exposure, but less distributed in the sites with high level sediment and wave-exposure. At different water depths, the distribution of S. horneri was negatively correlated with the amount of sediment. A medium dusting (dry mass 10.47 mg x cm(-2), approximate 0.543 mm deep) of sediment on the plate reduced the percentage of S. horneri zygotes attached to the substratum by 4.4%, and a heavy dusting (dry mass 13.96 mg x cm(-2), approximate 0.724 mm deep) of sediment on the plate completely prevented the attachment. One week after the settlement of the zygotes, there were 24% of the germlings still survived when the dry mass sediment coverage was 13.96 mg x cm(-2). However, when the dry mass sediment coverage was up to 34.9 mg x cm(-2) (approximate 1.81 mm deep), 100% of the germlings died. Overall, the distribution of S. horneri was not only related to sediment level, but also restricted by wave exposure to some extent. Sediment level was a critical factor affecting the distribution of S. horneri, particularly at its zygote attachment stage.
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Rapid enrichment of phosphopeptides by SiO2-TiO2 composite fibers.
Analyst
PUBLISHED: 07-29-2013
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SiO2-TiO2 composite fibers, prepared by electrospinning, were successfully applied to the rapid enrichment of phosphopeptides using a lab-in-syringe approach for the first time. Because of their large surface area, mesoporous structure, extraordinary length and appropriate Lewis acidity, the as-prepared SiO2-TiO2 composite fibers exhibited high selectivity and capacity in the enrichment of phosphopeptides from the digestion mixture of ?-casein and bovine serum albumin (BSA), as well as human blood serum and nonfat milk. The targeted phosphopeptides could be easily enriched and detected even when the total amount of ?-casein was decreased to only 10 fmol, indicating the high detection sensitivity of this method. In addition, the whole enrichment extraction procedure can be finished in less than 3 min, which can avoid or decrease the degradation of endogenous phosphoproteins by proteases released ex vivo during time-consuming treatments. The developed method is rapid, cost-effective, selective, sensitive, operationally simple, and does not require any harsh conditions and intricate equipment, providing an ideal candidate for the enrichment of phosphopeptides from complex biological samples either in the lab or in the field.
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A simple sample preparation approach based on hydrophilic solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry for determination of endogenous cytokinins.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 07-20-2013
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Cytokinins (CKs), a vital family of phytohormones, play important roles in the regulation of shoot and root development. However, the quantification of CKs in plant samples is frequently affected by the complex plant matrix. In the current study, we developed a simple, rapid and efficient hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE) method for CKs purification. CKs were extracted by acetonitrile (ACN) followed by HILIC-SPE (silica as sorbents) purification. The extraction solution of plant samples could be directly applied to HILIC-SPE without solvent evaporation step, which simplified the analysis process. Moreover, with HILIC chromatographic retention mechanism, the hydrophobic co-extracted impurities were efficiently removed. Subsequently, CKs were separated by RPLC, orthogonal to the HILIC pretreatment process, and detected by tandem mass spectrometry. The method exhibits high specificity and recovery yield (>77.0%). Good linearities were obtained for all eight CKs ranging from 0.002 to 100ngmL(-1) with correlation coefficients (r) higher than 0.9927. The limits of detection (LODs, signal/noise=5) for the CKs were between 1.0 and 12.4pgmL(-1). Reproducibility of the method was evaluated by intra-day and inter-day measurements and the results showed that relative standard deviations (RSDs) were less than 10.5%. Employing this method, we successfully quantified six CKs in 20mg Oryza sativa leaves and the method was also successfully applied to Brassica napus (flower and leaves).
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Preparation of titanium-grafted magnetic mesoporous silica for the enrichment of endogenous serum phosphopeptides.
J Chromatogr A
PUBLISHED: 07-20-2013
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As one of the most important post-translational modifications, reversible phosphorylation of protein plays crucial roles in a large number of biological processes. Moreover, endogenous phosphopeptides are also associated with certain human diseases. An efficient enrichment and separation method is the premise for successful identification and quantification of phosphopeptides. In this work, titanium grafted magnetic mesoporous silica (Fe3O4@Ti-mSiO2) was developed and applied for the enrichment of endogenous phosphopeptides. Fe3O4@Ti-mSiO2 particles were prepared by grafting titanocene dichloride on the inner walls of magnetic mesoporous silica and then being calcined to remove cyclopentadienyl ligand. The physicochemical properties of the prepared materials were characterized by energy dispersive X-ray spectroscopy (EDX), nitrogen adsorption-desorption analysis, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and vibrating sample magnetometer (VSM). For selective enrichment of phosphopeptides, the prepared Fe3O4@Ti-mSiO2 particles were applied for tryptic digests of ?-casein, mixtures of ?-casein and bovine serum albumin (BSA), and low-fat milk. Finally, Fe3O4@Ti-mSiO2 was successfully applied for the enrichment of endogenous phosphopeptides from human serum.
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Rational modification of protein stability by targeting surface sites leads to complicated results.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-24-2013
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The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions.
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The first silicon(IV) phthalocyanine-nucleoside conjugates with high photodynamic activity.
Dalton Trans
PUBLISHED: 06-19-2013
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A series of novel silicon(IV) phthalocyanines conjugated axially with different nucleoside moieties (uridine, 5-methyluridine, cytidine, and 5-N-cytidine derivatives) have been synthesized and evaluated for their photodynamic activities. The uridine-containing compound 1 exhibits the highest photocytotoxicity against HepG2 human hepatocarcinoma cells with an IC50 value as low as 6 nM, which can be attributed to its high cellular uptake and non-aggregated nature in the biological media. This compound shows high affinity toward the mitochondria of HepG2 cells and causes cell death mainly through apoptosis upon illumination. The result indicates that 1 is a highly promising photosensitizer for photodynamic therapy.
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Hydrophilic material for the selective enrichment of 5-hydroxymethylcytosine and its liquid chromatography-tandem mass spectrometry detection.
Anal. Chem.
PUBLISHED: 05-28-2013
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5-Methylcytosine (5-mC), an important epigenetic modification involved in development, can be converted enzymatically to 5-hydroxymethylcytosine (5-hmC). 5-hmC is considered an intermediate of active DNA cytosine demethylation and makes itself serve as an epigenetic mark. 5-hmC content in most mammalian cells is low and the quantification of 5-hmC by liquid chromatography-mass spectrometry (LC-MS) frequently suffers from ion suppression by the presence of unmodified nucleosides. To circumvent this problem, we developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of 5-hmC and form a more hydrophilic residue (?-glucosyl-5-hydroxymethyl-2-deoxycytidine, 5-gmdC) by using T4 ?-glucosyltransferase. The more hydrophilic 5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which eliminates the ion suppression and significantly improves the detection sensitivity and accuracy. Using this method, we successfully quantified 5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of 5-hmC in the model organism of yeast. In addition, the contents of 5-hmC in two yeast strains of Schizosaccharomyces pombe are even higher than those of 5-mC, indicating that 5-hmC may play important roles on the physiological functions of yeast.
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Preparation of mesoporous silica embedded pipette tips for rapid enrichment of endogenous peptides.
J Chromatogr A
PUBLISHED: 04-09-2013
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Mesoporous silica embedded pipette tips (mSiO2-Tips) were successfully prepared by a simple method and applied to rapid enrichment of endogenous peptides for the first time. The prepared mSiO2-Tips showed low back pressure when solvent was pipetted up and down. As a result, mSiO2-Tip could selectively trap peptides and exclude high-MW proteins simultaneously based on size-exclusion mechanism due to the uniform mesopore structure of the sorbent bed. The in-pipette-tip SPE approach was proved to be easy-operation, sensitive and fast (less than 2 min) for the analysis of peptides, which was further successfully applied in the efficient enrichment of peptides from human plasma.
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Preparation and chromatographic evaluation of a novel phosphate ester-bonded stationary phase with complexation and hydrophobic interactions retention mechanism.
J Chromatogr A
PUBLISHED: 03-28-2013
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The present study described the preparation of a novel phosphate ester-bonded silica (PES) stationary phase based on "thiol-ene" click chemistry. The composition of the surface grafts of PES stationary phase was determined by elemental analysis and solid state (31)P MAS NMR. Due to its hydrophilic phosphate-ester groups and short hydrophobic alkyl chains, the PES stationary phase exhibited dual retention mechanism via complexation and hydrophobic interactions with phenols. Benefiting from this special interaction mechanism, the newly synthesized PES stationary phase showed better selectivity in the separation of phenols compared to commercial octadecylsilyl-bonded silica (C18) columns. Furthermore, the separations of 10 nucleosides and nucleobases on the PES stationary phase were achieved in both reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode.
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Facile fabrication of reduced graphene oxide-encapsulated silica: a sorbent for solid-phase extraction.
J Chromatogr A
PUBLISHED: 03-16-2013
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In this study, a facile hydrothermal reduction strategy was developed for the preparation of reduced graphene oxide-encapsulated silica (SiO2@rGO). Compared with other conventional methods for the synthesis of SiO2@rGO, the proposed strategy endowed the obtained SiO2@rGO with larger amount of immobilized rGO. The prepared functionalized silica shows remarkable adsorption capacity toward chlorophenols (CPs) and peptides. When it was used as solid-phase extraction (SPE) sorbent, a superior recovery could be obtained compared to commercial sorbents, such as C18 silica, graphitized carbon black and carbon nanotubes. Based on these, the prepared material was used as SPE sorbent for the enrichment of CPs, and a method for the analysis of CPs in water samples was established by coupling SPE with high performance liquid chromatography-ultra violet detection (SPE-HPLC/UV). In addition, the obtained SiO2@rGO was further successfully extended to the enrichment of peptides in bovine serum albumin (BSA) digests.
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Vagal stimulation triggers peripheral vascular protection through the cholinergic anti-inflammatory pathway in a rat model of myocardial ischemia/reperfusion.
Basic Res. Cardiol.
PUBLISHED: 03-06-2013
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Myocardial ischemia/reperfusion (I/R) induces inflammatory response that may lead to remote vascular injury. Vagal nerve elicits the cholinergic anti-inflammatory pathway by activating ?7 nicotinic acetylcholine receptors (?7nAChR). Nevertheless, the role of vagal nerve-mediated anti-inflammatory pathway in the vasculature has not been studied previously. Therefore, we aimed to clarify the potential role of vagal stimulation (VNS) in regulating remote vascular injury after myocardial I/R. Adult male Sprague-Dawley rats were subjected to VNS starting 15 min prior to ischemia until the end of reperfusion. VNS not only reduced infarct size and improved cardiac function, but also ameliorated myocardial I/R-induced dysfunctional vasoconstriction and vasodilatation and degradation of endothelial structure in mesenteric arteries. VNS decreased serum and vascular levels of tumor necrosis factor-? and IL-1?. Interestingly, in vivo microdialysis studies demonstrated that VNS increased ACh concentration in the mesenteric circulation. Furthermore, VNS up-regulated expressions of muscarinic ACh receptors-3 (M3AChR) and ?7nAChR in mesenteric arteries. Preserved endothelial relaxations by VNS were inhibited by atropine or methyllycaconitine, indicating that functional protection was associated with M3 and ?7nAChR activation. Finally, VNS increased STAT3 phosphorylation and inhibited NF-?B activation in mesenteric arteries, and these effects were abolished by ?7nAChR shRNA treatment, indicating VNS-mediated anti-inflammatory effect mainly involved ?7nAChR. These results demonstrated for the first time that VNS protected against remote vascular dysfunction, through the cholinergic anti-inflammatory pathway which is dependent on ?7nAChR. Our findings represent a significant addition to the understanding of vagal nerve-mediated pathways and the potential roles they play in regulating the vasculature.
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Determination of endogenous brassinosteroids in plant tissues using solid-phase extraction with double layered cartridge followed by high-performance liquid chromatography-tandem mass spectrometry.
Phytochem Anal
PUBLISHED: 02-25-2013
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Brassinosteroids (BRs) are a group of important phytohormones that play vital roles in plant growth, development and a series of physiological phenomena. In order to understand biosynthesis, degradation and metabolic pathways of BRs, a reliable analytical method of BRs with effective sample pre-treatment process is favourable.
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Facile preparation of organic-silica hybrid monolith for capillary hydrophilic liquid chromatography based on "thiol-ene" click chemistry.
J Chromatogr A
PUBLISHED: 02-02-2013
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In this work, a one-step approach to facile preparation of organic-inorganic hybrid monoliths was successfully developed. After vinyl-end organic monomers and azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethoxysilane (TMOS) and 3-mercaptopropyltrimethoxysilane (MPTMS), the homogeneous mixture was introduced into a fused-silica capillary for simultaneous polycondensation and "thiol-ene" click reaction to form the organic-silica hybrid monoliths. By employing this strategy, two types of organic-silica hybrid monoliths with positively charged quaternary ammonium and amide groups were prepared, respectively. The functional groups were successfully introduced onto the monoliths during the sol-gel process with "thiol-ene" click reaction, which was demonstrated by ?-potential assessment, energy dispersive X-ray spectroscopy (EDX), and Fourier transform infrared (FT-IR) spectroscopy. The porous structure of the prepared monolithic columns was examined by scanning electron microscopy (SEM), nitrogen adsorption-desorption measurement, and mercury intrusion porosimetry. These results indicate the prepared organic-silica hybrid monoliths possess homogeneous column bed, large specific surface area, good mechanical stability, and excellent permeability. The prepared monolithic columns were then applied for anion-exchange/hydrophilic interaction liquid chromatography. Different types of analytes, including benzoic acids, inorganic ions, nucleosides, and nucleotides, were well separated with high column efficiency around 80,000-130,000 plates/m. Taken together, we present a facile and universal strategy to prepare organic-silica hybrid monoliths with a variety of organic monomers using one-step approach.
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Role of endothelial nitric oxide synthase and vagal activity in the endothelial protection of atorvastatin in ischemia/reperfusion injury.
J. Cardiovasc. Pharmacol.
PUBLISHED: 02-01-2013
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Vascular endothelial dysfunction plays a pivotal role in the development and maintenance of ischemia/reperfusion (I/R) injury. Statins, developed as lipid-lowering drugs, partially restore vagal activity and exhibit pleiotropic effects. This study was aimed at determining the effect of atorvastatin (ATV) on endothelial dysfunction in peripheral resistance arteries after I/R injury. After pretreatment with ATV (10 mg·kg·d) or its vehicle for 3 days, the superior mesenteric artery was occluded for 60 minutes and reperfusion for 90 minutes or the rats were anesthetized without being subjected to ischemia. In the ATV-treated I/R group, the increased contractions to KCl and 5-hydroxytryptamine induced by I/R were ameliorated, and attenuated endothelium-dependent relaxations to acetylcholine (ACh) were normalized. The restored relaxation to ACh was abolished by N-nitro-L-arginine methyl ester. ATV prevented the structural damage of vascular endothelial cells. Furthermore, the activities of phosphatidylinositol-3-kinase, Akt, and endothelial nitric oxide synthase were elevated in mesenteric arteries after ATV treatment. In addition, I/R-induced increment of endothelial cells apoptosis was also attenuated by ATV. Intriguingly, ATV also increased baroreflex sensitivity and serum ACh content after I/R. In conclusion, the endothelial protective effect of ATV in peripheral arteries is associated with the activated phosphatidylinositol-3-kinase/Akt/endothelial nitric oxide synthase pathway and restored vagal activity.
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Quantification of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from hepatocellular carcinoma tissues by capillary hydrophilic-interaction liquid chromatography/quadrupole TOF mass spectrometry.
Clin. Chem.
PUBLISHED: 01-23-2013
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5-Methylcytosine (5-mC) is an important epigenetic modification involved in development and is frequently altered in cancer. 5-mC can be enzymatically converted to 5-hydroxymethylcytosine (5-hmC). 5-hmC modifications are known to be prevalent in DNA of embryonic stem cells and neurons, but the distribution of 5-hmC in human liver tumor and matched control tissues has not been rigorously explored.
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Association of 5-methylcytosine and 5-hydroxymethylcytosine with mitochondrial DNA content and clinical and biochemical parameters in hepatocellular carcinoma.
PLoS ONE
PUBLISHED: 01-01-2013
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Increasing epidemiological evidence has indicated that inherited variations of mitochondrial DNA (mtDNA) copy number affect the genetic susceptibility of many malignancies in a tumour-specific manner and that DNA methylation also plays an important role in controlling gene expression during the differentiation and development of hepatocellular carcinoma (HCC). Our previous study demonstrated that HCC tissues showed a lower 5-hydroxymethylcytosine (5-hmC) content when compared to tumour-adjacent tissues, but the relationship among 5-hmC, 5-methylcytosine (5-mC) and mtDNA content in HCC patients is still unknown. This study aimed to clarify the correlation among mtDNA content, 5-mC and 5-hmC by quantitative real-time PCR and liquid chromatography tandem mass spectrometry analysis. We demonstrated that 5-hmC correlated with tumour size [odds ratio (OR) 0.847, 95% confidence interval (CI) 0.746-0.962, P = 0.011], and HCC patients with a tumour size ? 5.0 cm showed a lower 5-hmC content and higher levels of fasting plasma aspartate aminotransferase, the ratio of alanine aminotransferase to aspartate aminotransferase, ?-glutamyltransferase, alpha-fetoprotein than those with a tumour size <5 cm (all P<0.05). We further revealed that the mtDNA content of HCC tumour tissues was 225.97(105.42, 430.54) [median (25th Percentile, 75th Percentile)] and was negatively correlated with 5-mC content (P = 0.035), but not 5-hmC content, in genomic DNA from HCC tumour tissues.
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Mutagenic and cytotoxic properties of oxidation products of 5-methylcytosine revealed by next-generation sequencing.
PLoS ONE
PUBLISHED: 01-01-2013
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5-methylcytosine (5-mC) can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and finally to 5-carboxylcytosine (5-caC), which is thought to function in active DNA cytosine demethylation in mammals. Although the roles of 5-mC in epigenetic regulation of gene expression are well established, the effects of 5-hmC, 5-foC and 5-caC on DNA replication remain unclear. Here we report a systematic study on how these cytosine derivatives (5-hmC, 5-foC and 5-caC) perturb the efficiency and accuracy of DNA replication using shuttle vector technology in conjugation with next-g sequencing. Our results demonstrated that, in Escherichia coli cells, all the cytosine derivatives could induce CT transition mutation at frequencies of 0.17%-1.12%, though no effect on replication efficiency was observed. These findings provide an important new insight on the potential mutagenic properties of cytosine derivatives occurring as the intermediates of DNA demethylation.
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Assessing gibberellins oxidase activity by anion exchange/hydrophobic polymer monolithic capillary liquid chromatography-mass spectrometry.
PLoS ONE
PUBLISHED: 01-01-2013
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Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography--mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA1, GA4, GA9, GA20). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62-0.90 fmol. We determined the kinetic parameters (K m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA9 to GA4 in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.
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A selective pretreatment method for determination of endogenous active brassinosteroids in plant tissues: double layered solid phase extraction combined with boronate affinity polymer monolith microextraction.
Plant Methods
PUBLISHED: 01-01-2013
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BACKGROUND: Brassinosteriods (BRs), a group of important phytohormones, have various effects on plant growth and development. However, their physiological functions in plants have not been fully understood to date. Endogenous BRs in plant tissue are extremely low and the elucidation of BRs functions relies on sensitive detection method. Reported methods for the determination of BRs required large amount of plant tissue, tedious pretreatment process, and were lack of selectivity. Therefore, development of a simple and selective method for the sensitive quantification of BRs is highly needed. RESULTS: We established a pretreatment method of BRs in plant tissues by employing double layered solid phase extraction (DL/SPE) combined with boronate affinity polymer monolith microextraction (BA/PMME). After the initial depigmentation with DL/SPE cartridge, boronate affinity polymer BA/PMME was employed to selectively extract BRs from sample matrix. Uniquely, most sample matrix was successfully removed by BA monolith purification. Using this method, BRs was determined by liquid chromatography-mass spectrometry (LC-MS). Endogenous active BRs could be detected in only 1 g fresh weigh (FW) leaves or 0.5 g FW flower tissues. CONCLUSION: A DL/SPE-BA/PMME pretreatment method for the determination of endogenous brassinosteroids in plant tissues was developed and validated. The proposed method was sensitive and selective. The proposed method may be further developed for the determination of other BRs including their precursors and conjugates.
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[Effect of ischemia/hypoxia on mesenteric vasomotor function in spontaneously hypertensive rats and its possible mechanism].
Sheng Li Xue Bao
PUBLISHED: 12-24-2011
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Hypertension is a common cardiovascular disease and can induce many complications, such as stroke and coronary heart disease. The purpose of the present study was to investigate the effect of ischemia/hypoxia on mesenteric artery vasomotor function in spontaneously hypertensive rats (SHR). Rat mesenteric arterial rings were cultured in modified ischemia-mimetic solution in a hypoxia incubator for a certain time period. Isometric tension changes of isolated mesenteric arterial rings were recorded continuously by a myograph system. The results obtained were as follows: In SHR group, the maximum contractions to KCl and phenylephrine (PE) were increased, and the maximum relaxation to acetylcholine (ACh) was decreased, compared to those in Wistar-Kyoto (WKY) rats group. Compared with SHR group and WKY with acute ischemia/hypoxia (WKY+H) group, SHR with acute ischemia/hypoxia (SHR+H) increased the maximum contractions induced by KCl and PE and inhibited the maximum relaxations by ACh. In SHR+H and SHR groups, the vasodilation induced by ACh was unaffected by N(G)-nitro-L-arginine methylester (L-NAME), whereas in WKY group, the relaxation to ACh was attenuated by L-NAME. CaCl2-induced contraction in depolarized rings in SHR+H group significantly shifted to the left compared with SHR group. In Ca(2+)-free K-H solution, the maximum contractions induced by PE and caffeine were increased in SHR+H group compared to those in WKY+H group; the PE- and caffeine-induced contractions were also enhanced in SHR group versus WKY group; the maximum contraction induced by PE was significantly increased in SHR+H group versus SHR group. These findings suggest that acute ischemia/hypoxia aggravates mesenteric artery dysfunction in SHR. The mechanism may be related to the decreased NO generation and increased sarcoplasmic reticulum Ca(2+) release.
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Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides.
Analyst
PUBLISHED: 12-20-2011
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Phosphorylation, one of the most important post-translational modifications of protein, plays a crucial role in a large number of biological processes. Large-scale identification of protein phosphorylation by mass spectrometry is still a challenging task because of the low abundance of phosphopeptides and sub-stoichiometry of phosphorylation. In this work, a novel strategy based on the specific affinity of zirconium arsenate to the phosphate group has been developed for the effective enrichment of phosphopeptides. Zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe(3)O(4)@SiO(2)) were prepared by covalent immobilization of zirconium arsenate on Fe(3)O(4)@SiO(2) magnetic nanoparticles under mild conditions, and characterized by transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometer (VSM). The prepared ZrAs-Fe(3)O(4)@SiO(2) was applied for the selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA). Our results demonstrated that the ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles possess higher selectivity for phosphopeptides and better capture capability towards multiply-phosphorylated peptides than commercial zirconium dioxide (ZrO(2)), which has been widely employed for the enrichment of phosphopeptides. In addition, endogenous phosphopeptides from human serum can be effectively captured by ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles. It is the first report, to the best of our knowledge, in which the zirconium arsenate-modified magnetic nanoparticles were successfully applied to the enrichment of phosphopeptides, which offers the potential application of this new material in phosphoproteomics study.
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[Copy-number variations of SHANK3 and related clinical phenotypes in children with autism].
Zhonghua Er Ke Za Zhi
PUBLISHED: 11-19-2011
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To explore possible relationship between copy-number variations (CNVs) in 15q11-13, 16p11 and SHANK3 gene by using multiplex ligation-dependent probe amplification (MLPA) and the phenotypes in children with autism and to further explore the clinical application of MLPA to make an etiological diagnosis of Autism.
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Pseudomorphic synthesis of monodisperse magnetic mesoporous silica microspheres for selective enrichment of endogenous peptides.
J Chromatogr A
PUBLISHED: 10-16-2011
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In this work, we describe a novel synthetic strategy of magnetic mesoporous silica spheres (Fe3O4@mSiO2) for the selective enrichment of endogenous peptides. Fe3O4 particles were coated with silica shell by a sol-gel method, followed by pseudomorphic synthesis to transform nonporous silica shell into ordered mesoporous silica shell. The core/shell structure and mesostructure were individually fabricated in two steps, which can be expedient to independently optimize the properties of monodispersion, magnetization and mesostructure. Actually, it was confirmed that the produced Fe3O4@mSiO2 particles possess good monodispersion, high magnetization, superparamagnetism, uniform accessible mesopores, and large surface area and pore volume. With these good properties, Fe3O4@mSiO2 spheres were applied to the rapid enrichment of peptides. Based on the size-exclusion mechanism and hydrophobic interaction with siloxane bridge group mainly on the surface of inside pores, Fe3O4@mSiO2 can selectively capture peptides and exclude high-MW proteins and salts. Furthermore, peptides in human plasma were successfully enriched by Fe3O4@mSiO2.
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Clinical outcomes of fertility-sparing treatments in young patients with epithelial ovarian carcinoma.
J Zhejiang Univ Sci B
PUBLISHED: 10-01-2011
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To assess the clinical outcomes of fertility-sparing treatments in young patients with epithelial ovarian carcinoma (EOC).
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Conversion of biomass-derived levulinate and formate esters into ?-valerolactone over supported gold catalysts.
ChemSusChem
PUBLISHED: 08-23-2011
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The utilization of biomass has recently attracted tremendous attention as a potential alternative to petroleum for the production of liquid fuels and chemicals. We report an efficient alcohol-mediated reactive extraction strategy by which a hydrophobic mixture of butyl levulinate and formate esters, derived from cellulosic biomass, can be converted to valuable ?-valerolactone (GVL) by a simple supported gold catalyst system without need of an external hydrogen source. The essential role of the supported gold is to facilitate the rapid and selective decomposition of butyl formate to produce a hydrogen stream, which enables the highly effective reduction of butyl levulinate into GVL. This protocol simplifies the recovery and recycling of sulfuric acid, which is used for cellulose deconstruction.
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A magnetite/oxidized carbon nanotube composite used as an adsorbent and a matrix of MALDI-TOF-MS for the determination of benzo[a]pyrene.
Chem. Commun. (Camb.)
PUBLISHED: 08-08-2011
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A magnetite/oxidized carbon nanotube composite, Fe(3)O(4)@SiO(2)/OCNT, was fabricated in a simple way, and it was successfully used as a magnetic solid-phase extraction sorbent and a significant matrix of the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the detection of benzo[a]pyrene (BaP).
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Grafting of silica with a hydrophilic triol acrylamide polymer via surface-initiated "grafting from" method for hydrophilic-interaction chromatography.
J Sep Sci
PUBLISHED: 07-01-2011
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A novel hydrophilic polymer-coated silica sorbent has been prepared using the radical "grafting from" polymerization method through surface-bound azo initiators for hydrophilic-interaction chromatography (HILIC). The azo groups were introduced to the surface of silica gel through the reaction with amino groups on the surface of silica gel with 4,4-azobis(4-cyanopentanoic acid chloride) (ACVC). The resultant azo-immobilized silica gel served as surface initiator to polymerize hydrophilic triol acrylamide monomer N-acryloyltris(hydroxymethyl) aminomethane (NA) in methanol to get hydrophilic polymer-coated silica sorbent. The obtained poly(NA)-coated silica (pNA-sil) was characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), and nitrogen sorption porosimetry (NSP). Then the pNA-sil was packed into the stainless-steel column and evaluated in high-performance liquid chromatography (HPLC). Good chromatographic performance for the separation of peptides and nucleosides was obtained under HILIC mode. The results indicated that the pNA-sil stationary phase behaved as mixed-mode retention mechanisms of hydrophilic and ionic interactions. Furthermore, the pNA-sil phase was used to separate tryptic digest of ?-casein and our results showed that more than 12 peptides peaks were resolved and well distributed within the elution window. Finally, the pNA-sil stationary phase was demonstrated to possess remarkable reproducibility and stability. Taken together, the pNA-sil stationary phase prepared in the current study offers a potential application in proteomics study.
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Preparation of magnetic polymer material with phosphate group and its application to the enrichment of phosphopeptides.
J Chromatogr A
PUBLISHED: 04-15-2011
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As one of the most important post-translational modifications (PTM), reversible phosphorylation of protein is involved in many cellular processes. Enrichment and separation of phosphopeptides have become essential for large-scale identification of protein phosphorylation by mass spectrometry. In this work, five magnetic polymer materials with different numbers of phosphate groups were fabricated using a simple polymeric method and their abilities to enrich phosphopeptides were investigated. Our results showed that the enrichment efficiency is closely related to the number of phosphate groups attached to magnetic polymer sorbent. Under optimized condition (3% trifluoroacetic acid and 80% acetonitrile), magnetic polymer-particles with appropriate proportion of phosphate groups (Fe(3)O(4)@p(VPA-EDMA-1)-Zr(4+)) showed high performance for extracting phosphopeptides from complex peptides mixture of standard protein digestion. In this regard, a total of 988 unique phosphopeptides were successfully identified from proteolytic digestion of HeLa cell extracts by employing magnetic polymer-particles combined with nano-RPLC-MS/MS analysis.
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Preparation of methacrylate-based monolith for capillary hydrophilic interaction chromatography and its application in determination of nucleosides in urine.
J Chromatogr A
PUBLISHED: 03-19-2011
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A novel poly(N-acryloyltris(hydroxymethyl)aminomethane-co-pentaerythritol triacrylate) (NAHAM-co-PETA) monolith was prepared in the 100 ?m i.d. capillary and investigated for capillary liquid chromatography (cLC). The polymer monolith was synthesized by in situ polymerization of NAHAM and PETA in the presence of polyethylene glycol (PEG) in dimethyl sulfoxide (DMSO) as the porogen. The porous structure of monolith was optimized by changing the ratio of NAHAM to PETA, the molecular weight and amount of PEG. To evaluate the separation performance of the resultant polymer monolith, several groups of model compounds (including nucleosides, benzoic acids and anilines) were selected to perform cLC separation. Our results showed that these model compounds can be baseline separated on the resultant poly(NAHAM-co-PETA) monolithic column with the optimized mobile phases. The column efficiency was estimated to be 87,000 plates/m for acrylamide. In addition, this monolithic column was coupled with on-line solid-phase microextraction (SPME) for the analysis of four nucleosides (uridine, adenosine, cytidine, guanosine) in urine. The limit of detection of the proposed method was in the range from 40 to 52 ng/mL. The method reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 8.3% and 10.2%, respectively. Recoveries of the target analytes from spiked urine samples were ranged from 86.5% to 106.8%.
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Highly sensitive profiling assay of acidic plant hormones using a novel mass probe by capillary electrophoresis-time of flight-mass spectrometry.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 02-26-2011
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Plant hormones play crucial roles in plant growth and development. However, up to date, identification and quantification of acidic plant hormones with trace amount in complicated plant matrix is still a challenge. In current study, we developed a high sensitive assay for the determination of acidic plant hormones in rice by combining capillary electrophoresis and electrospray ionization-time of flight-mass spectrometry (CE-ESI-TOF-MS). To improve the detection sensitivity of acidic plant hormones, 3-bromoactonyltrimethylammonium bromide (BTA) was synthesized as a new mass probe, which can react efficiently with acidic plant hormones in acetonitrile containing triethylamine (TEA). The positively charged BTA-derivatives were separated by CE using amino-coated capillary, which provided a reversed electroosmotic flow (EOF) at low pH, as well as reduced the adsorption of BTA-derivatives on the inner wall of capillary. Using the CE-ESI-TOF-MS method developed in current study, 15 acidic plant hormones, including 10 gibberellins (GAs), were identified and quantified with good linearities from 1.3 to 850 ng/mL with linear coefficient R(2) values of >0.99. The limits of detection (LODs) were in the range of 0.34-4.59 ng/mL. Recoveries of compounds from spiked beverage samples ranged from 84.6 to 112.2%. And a good reproducibility was obtained by evaluating the intra and inter-day precisions with relative standard deviations (RSDs) less than 6.7 and 9.9%, respectively.
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[Screening and characterization of peptides specifically binding to the schistosomulum tegument of Schistosoma japonicum].
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi
PUBLISHED: 09-02-2010
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To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum tegument of Schistosoma japonicum.
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A method for obtaining peri-implant soft-tissue contours by using screw-retained provisional restorations as impression copings: a clinical report.
J Oral Implantol
PUBLISHED: 06-16-2010
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In the esthetic zone, remodeling of the peri-implant soft-tissue contours through the use of provisional restorations is one of the imperative key factors for optimizing outcomes. Several methods have been described to produce the desired peri-implant soft-tissue contours using customized impression copings or cement-retained provisional crowns. The aim of this article is to present an alternative method for obtaining the desired peri-implant soft-tissue contours by using screw-retained provisional restorations as impression copings, which facilitates the definitive prosthesis fabrication.
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Spontaneous formation of hierarchically structured curly films of nickel carbonate hydrate through drying.
Langmuir
PUBLISHED: 03-06-2010
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Novel curly nickel carbonate hydrate film superstructures can be prepared for the first time via a facile drying process of the films formed on air/solution interface in the presence of double hydrophilic copolymer or polyelectrolyte additives. As-prepared curly film patches with average edge sizes of several hundred micrometers display adjustable curly features along different orientation. The coiling up degree of the film edges is strongly dependent on the polymer concentration in bulk solution. Most of these curly structures have a relatively smooth outer surface; however, the microstructures of the outer surface of curly films formed show porous network-like features. In addition, using different kinds of nickel salts can produce distinct curly film samples. A possible formation mechanism of the curly film structure has been proposed. The multiple interaction modes between nickel salt precursors and polymer can favor the self-organization of the film formed at the air/solution interface. This approach is expected to be extended for producing a variety of curly hierarchical structures.
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Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing.
Biochem. Biophys. Res. Commun.
PUBLISHED: 10-29-2009
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Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. DQ316984 and DQ320011), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.
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[Effectiveness and significance of prostatic ischemia in rabbit].
Zhonghua Yi Xue Za Zhi
PUBLISHED: 10-19-2009
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To evaluate the effect of prostatic ischemia in rabbit and approach the significance of ischemia in the development of benign prostatic hyperplasia.
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Effect of cholesterol lowering on stiffness of aortic and femoral arterial walls in rabbits on a high fat diet.
Chin. Med. J.
PUBLISHED: 07-02-2009
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Researches in arterial elasticity have increased over the past few years. We investigated the effects of simvastatin on vascular stiffness in fat fed rabbits by ultrasonography.
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[Effect of alexandrite laser treatment for hair removal in Tibet mini-pigs].
Nan Fang Yi Ke Da Xue Xue Bao
PUBLISHED: 05-01-2009
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To observe the histological and ultrastructural changes of the skin and hair follicles following hair removal by alexandrite laser in Tibet mini-pigs.
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Analysis of core packing in a cooperatively folded miniature protein: the ultrafast folding villin headpiece helical subdomain.
Biochemistry
PUBLISHED: 04-10-2009
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The helical subdomain of the villin headpiece is the smallest naturally occurring cooperatively folded protein. Its small size, simple three-helix topology, and very rapid folding have made it an extremely popular model system for computational and theoretical studies of protein folding. The domain has a well-packed hydrophobic core comprised in part of an unusual set of three closely packed phenylalanine residues, F47, F51, and F58 (denoted using the numbering of the larger headpiece protein). Aromatic-aromatic interactions have been thought to play a critical role in specifying the subdomain fold and have been proposed to play a general role in stabilizing small proteins. The modest stability of the subdomain has hindered studies of core packing since multiple mutations can lead to constructs which fail to fold, and even single mutants can result in poorly folded variants. Using a previously characterized hyperstable mutant of the domain, generated by targeting surface residues, a complete set of single, double, and triple core Phe to Leu mutants were characterized. A highly conserved surface Trp which is part of a Trp-Pro interaction was also examined. All mutants are well-folded as judged by CD and NMR, and all exhibit sigmoidal urea and thermally induced unfolding transitions, thus proving that aromatic-aromatic, aromatic-proline, or aromatic-hydrophobic interactions are not required for specifying the subdomain fold. Double mutant cycle analysis demonstrates that F47 and F51 make the strongest pairwise interaction. Mutations which lack F58 are the most destabilized, although even the triple mutant is folded. Interestingly, mutation of the central Phe, F51, has the smallest effect on stability even though it makes contact with both F47 and F58 and appears to form the strongest pairwise interaction.
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Preparation of a hyper-cross-linked polymer monolithic column and its application to the sensitive determination of genomic DNA methylation.
Chemistry
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A hyper-cross-linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium-co-vinylbenzene chloride-co-divinylbenzene) (MATE-co-VBC-co-DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE-co-VBC-co-DVB) monolithic column was demonstrated to have strong anion exchange/reversed-phase (SAX/RP) mixed-mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5-methylcytosine (5-mC)) can be baseline separated within 15?min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise = 3) of 5-mC is 0.014?pmol and endogenous 5-mC can be distinctly detected by using only 10?ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5-azacytidine (5-aza-C) and 5-aza-2-deoxycytidine (5-aza-CdR) could induce a significant decrease of genome-wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).
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Borated titania, a new option for the selective enrichment of cis-diol biomolecules.
Chemistry
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As low abundance cis-diol biomolecules are of great significance in biological organisms, preparation of materials for the selective enrichment of such compounds is highly favorable for the development of the related proteomics and metabolomics. To this end, we have prepared monolithic borated titania by a non-aqueous sol-gel strategy as a new inorganic affinity material for the specific capture of nucleosides, glycopeptides and glycoproteins. Benefiting from the inorganic framework, this material prevented the hydrophobic interference, which was somewhat inevitable for the mainstream organic-based boronate affinity materials. The prepared material was carefully characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and nitrogen-sorption experiments to investigate the morphology and elemental composition. The excellent performance of borated titania on enrichment of cis-diol biomolecules was demonstrated by extracting the glycopeptides from horseradish peroxidase (HRP) digestion, standard glycoproteins, and nucleosides from a human-urine matrix. This kind of inorganic affinity material offers a new option for selective enrichment or separation of cis-diol biomolecules.
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Delayed preconditioning prevents ischemia/reperfusion-induced endothelial injury in rats: role of ROS and eNOS.
Lab. Invest.
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Ischemic preconditioning (IPC) strongly protects against ischemia/reperfusion (I/R) injury; however, the molecular mechanism involved in delayed preconditioning-induced endothelial protection in peripheral arteries is unknown. Therefore, we examined using functional, morphologic and molecular biologic studies whether delayed IPC decreases formation of reactive oxygen species and upregulates endothelial nitric oxide synthase (eNOS) that in turn contributes to vascular endothelial protection. Adult male Sprague-Dawley rats were subjected to 30-min ischemia induced by mesenteric artery occlusion followed by 60-min reperfusion 24 h after sham surgery or preconditioning (three cycles of 5-min ischemia/5-min reperfusion). Delayed preconditioning prevented the I/R-induced impairment of endothelium-dependent relaxations to acetylcholine (maximal relaxation: sham 91.4±2.2%; I/R 54.0±4.0%; IPC 80.2±6.3%). This protective effect was abolished by NOS inhibitor N(G)-nitro-L-arginine methyl ester and not changed by ascorbic acid. Electron microscopy showed marked endothelial damage after I/R and the ultrastructural changes were prevented by delayed preconditioning. Following I/R, the impairment of eNOS phosphorylation and expression was observed in mesenteric vessels. Furthermore, phosphatidylinositol 3-kinase (PI3K) and Akt phosphorylation were reduced, although total PI3K and Akt remained unchanged. IPC restored I/R-induced impairment of eNOS expression and activity. This was possibly the result of the recovery of PI3K/Akt phosphorylation. Furthermore, I/R increased serum level of malondialdehyde, intravascular superoxide and nitrotyrosine generation, which were abrogated by IPC. These results suggest that delayed preconditioning prevented I/R-induced endothelial injury in peripheral resistance vasculature, both in terms of functional and structural changes. Endothelial protection afforded by delayed IPC is associated with inhibition of oxidative stress and upregulation of PI3K/Akt/eNOS pathway.
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Substrateless graphene fiber: a sorbent for solid-phase microextraction.
J Chromatogr A
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In current study, a substrateless graphene fiber was successfully prepared by a simple hydrothermal strategy and used as solid-phase microextraction (SPME) sorbent. Five organochlorine pesticides (OCPs) were employed as model analytes to evaluate the performance of as-prepared graphene fiber. The results showed that the graphene fiber exhibited higher extraction efficiencies, higher thermal stability (up to 310°C), better reproducibility, and longer service life (more than 180 times reuse) than commercial fibers. In addition, the method for the determination of OCPs was proposed by coupling headspace (HS)-SPME technique with gas chromatography/electron capture detector (HS-SPME-GC/ECD). The proposed HS-SPME-GC/ECD method showed low limits of detection (0.83-11.5 ng/L), wide linear dynamic ranges (more than 2 orders of magnitude), and acceptable reproducibility (RSD<10.9%). Finally, the proposed method was successfully applied to the analysis of OCPs in environmental water samples with good recoveries (81-121%) and satisfactory precisions (RSD<9%).
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Preparation of magnetic poly(diethyl vinylphosphonate-co-ethylene glycol dimethacrylate) for the determination of chlorophenols in water samples.
J Chromatogr A
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In this work, a novel type of magnetic polymer particle, magnetic poly(diethyl vinylphosphonate-co-ethylene glycol dimethacrylate) [Fe(3)O(4)@p(DEVP-co-EDMA)], was successfully synthesized and applied for the extraction and determination of chlorophenols in water samples by coupling with high-performance liquid chromatography (HPLC). Fe(3)O(4)@p(DEVP-co-EDMA) was synthesized by a simple seeded radical polymerization method and exhibited well-defined core-shell structure and good magnetic response ability. In addition, the magnetic polymer had the advantages of abundant adsorption sites and high enrichment efficiency. Due to the presence of PO group in the skeleton of polymer, the magnetic polymer material displayed excellent extraction performance for chlorophenols, such as 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Hydrophobic skeleton of the magnetic polymer also provided strong interaction with the target analytes, especially pentachlorophenol (PCP) which is a kind of non-polar chlorophenol. Desorption solution, pH of water sample, extraction time and desorption time, the amount of adsorbent, and the volume of desorption solution were optimized. Under the optimized conditions, the linear ranges of four chlorophenols were 2-500 ng/mL with the limits of detection (S/N=3) ranging from 0.20 to 0.34 ng/mL. The repeatability was investigated by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) lower than 15.0%. The recoveries for real water samples were in the range of 92.7-108.0%. Collectively, the results indicated that the novel Fe(3)O(4)@p(DEVP-co-EDMA) was successfully applied in the extraction and detection of chlorophenols from water samples, and the magnetic polymer particle showed potential applications in the analysis of polar compounds.
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[Sensitivity to chemotherapeutic drugs of polyploid tumor cells induced by a spindle poison nocodazole].
Zhonghua Zhong Liu Za Zhi
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To investigate the changes of drug sensitivity of spindle poison-induced polyploid tumor cells to chemotherapeutic agents and its possible mechanism.
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Highly sensitive and quantitative profiling of acidic phytohormones using derivatization approach coupled with nano-LC-ESI-Q-TOF-MS analysis.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
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In current study, we developed a highly sensitive method for the quantitative profiling of acidic phytohormones. Tandem solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was employed to efficiently purify acidic phytohormones, which were further derived by 3-bromoactonyltrimethylammonium bromide (BTA) to increase the ionization efficiency in electrospray ionization-mass spectrometry detection. Additionally, fifteen BTA-derived acidic phytohormones, including ten gibberellins (GAs), were well separated with a salt gradient on poly(methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-co-EDMA) monolithic column. By employing online trapping system, the signal intensities of the analytes were significantly improved. The limits of detection (LODs, Signal/Noise=3) of targeted phytohormones ranged from 1.05 to 122.4 pg/mL, which allowed the highly sensitive determination of low abundant acidic phytohormones with tiny amount plant sample. Good reproducibility was obtained by evaluating the intra- and inter-day precisions with relative standard deviations (RSDs) less than 10.9 and 11.9%, respectively. Recoveries of the target analytes from spiked rice leave samples ranged from 88.3 to 104.3%. By employing the method developed here, we were able to simultaneously determine 11 endogenous acidic phytohormones from only 5mg of rice leave sample, which dramatically decreased the required sample amount (three orders of magnitude lower) for the profiling of low abundant acidic phytohormones compared to previous reports. Taken together, the method provided a good solution for the highly sensitive and quantitative profiling of endogenous acidic phytohormones.
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Facile preparation of SiO2/TiO2 composite monolithic capillary column and its application in enrichment of phosphopeptides.
Anal. Chem.
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A novel SiO(2)/TiO(2) composite monolithic capillary column was prepared by sol-gel technology and successfully applied to enrich phosphopeptides as a metal oxide affinity chromatography (MOAC) material. For the monolith preparation, tetramethoxysilane (TMOS) and tetrabutoxytitanium (TBOT) were used as silica and titania source, respectively, and glycerol was introduced to attenuate the activity of titanium precursor, which provided a mild synthetic condition. The prepared monolith was characterized by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results revealed an approximate 1/2 molar ratio of titanium to silica as well as an atom-scale homogeneity in the framework. The scanning electron microscopy (SEM) results demonstrated an excellent anchorage between the column and the inner capillary wall, and nitrogen adsorption-desorption experiments showed a bimodal porosity with a narrow mesopore distribution around 3.6 nm. The prepared monolith was then applied for selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA) as well as human blood serum, nonfat milk, and egg white using an in-tube solid phase microextraction (SPME) system. Our results showed that SiO(2)/TiO(2) composite monolithic capillary column could efficiently enrich the phosphopeptides from complex matrixes. To the best of our knowledge, this is the first attempt for preparing the silica-metal composite monolithic capillary column, which offers the promising application of the monolith on phosphoproteomics study.
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Sample preparation and direct electrospray ionization on a tip column for rapid mass spectrometry analysis of complex samples.
Analyst
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A handheld pipette tip column electrospray ionization source (PTC-ESI source) was developed for rapid mass spectrometry analysis at ambient pressure. The PTC-ESI source was made up of three main component parts including a micro DC high voltage (HV) power supply, a micropipette and a disposable micropipette tip filled with a plug of adsorbent. A DC high voltage was applied to the sharp point of the micropipette tip column to induce electrospray ionization. The PTC-ESI source was successfully used for direct analysis of basic organic compounds, organic acids and peptides in a simple matrix. In the case of complex samples, micro-extraction based on the adsorbent phase filled in the pipette tip was used to remove impurities and concentrate target analytes prior to ionization. The eluting solution was not pipetted out, but directly dispersed in the form of electrospray from the pipette tip for ionization. The effectiveness of the PTC-ESI source has been further demonstrated by fast analysis of therapeutic compounds and endogenous bioactive chemicals in complex biological samples.
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Electrospinning-based synthesis of highly ordered mesoporous silica fiber for lab-in-syringe enrichment of plasma peptides.
Chem. Commun. (Camb.)
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Silica fiber with highly ordered mesoporous structure and continuously long fibrous property was synthesized on a large-scale for the first time. It can be applied to the rapid (less than 3 min) and effective enrichment of endogenous peptides with a novel lab-in-syringe approach.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.