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Find video protocols related to scientific articles indexed in Pubmed.
Preparation, characterization, and DNA interaction studies of cationic europium luminescent copolymer.
J Biomater Sci Polym Ed
PUBLISHED: 11-12-2014
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This paper proposed a simple synthetic strategy towards a novel cationic europium luminescent copolymer, poly(METAC-co-NIPAm-co-Eu(AA)3Phen) (PMNEu), and investigation about their complexation ability with DNA. In this approach, first, Eu(AA)3Phen complex monomer containing Eu(3+), acrylic acid (AA), and 1,10-phenanthroline (Phen) was synthesized, and subsequently, free radical copolymerization of Eu(AA)3Phen complex monomer with other two functional monomers, [2-(methacryloyloxy) ethyl] trimethylammonium chloride (METAC) and N-isopropylarylamide (NIPAm), was carried out in methanol using azodiisobutyronitrile (AIBN) as the initiator. (1)HNMR, GPC, fluorescence spectroscopy, UV-vis spectroscopy, and TEM were used to investigate the chemical structures, molecular weight and molecular weight distribution, fluorescence properties, UV spectra, and morphologies of PMNEu copolymer, respectively. Furthermore, the interaction of PMNEu with DNA was also studied with fluorescence spectroscopy, UV-vis spectroscopy, and agarose gel electrophoresis. These results indicated that PMNEu could interact with DNA via an electrostatic bonding mode and the bonding constant was 2.2 × 10(5) L/mol. Additionally, TEM observation showed that pure PMNEu formed micelles in water solution, while the size-controllable aggregations of PMNEu with DNA were obtained when PMNEu was mixed with DNA at various concentration ratios. A good biocompability of PMNEu was demonstrated through in vitro cytotoxicity assays.
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Organosilane-functionalized graphene quantum dots and their encapsulation into bi-layer hollow silica spheres for bioimaging applications.
Phys Chem Chem Phys
PUBLISHED: 09-26-2014
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Graphene quantum dots (GQDs) represent an important class of luminescent quantum dots owing to their low toxicity and superior biocompatibility. Chemical functionalization of GQDs and subsequent combination with other materials further provide attractive techniques for advanced bioapplications. Herein, we report the facile fabrication of fluorescent organosilane-functionalized graphene quantum dots (Si-GQDs) and their embedding into mesoporous hollow silica spheres as a biolabel for the first time. Well-proportioned Si-GQDs with bright and excitation dependent tunable emissions in the visible region were obtained via a simple and economical solvothermal route adopting graphite oxide as a carbon source and 3-(2-aminoethylamino)-propyltrimethoxysilane as a surface modifier. The as-synthesized Si-GQDs can be well dispersed and stored in organic solvents, easily manufactured into transparent film and bulk form, and particularly provide great potential to be combined with other materials. As a proof-of-principle experiment, we demonstrate the successful incorporation of Si-GQDs into hollow mesoporous silica spheres and conduct preliminary cellular imaging experiments. Interestingly, the Si-GQDs not only serve as fluorescent chromophores in the composite material, but also play a crucial role in the formation of mesoporous hollow silica spheres with a distinctive bi-layer architecture. The layer thickness and optical properties can be precisely controlled by simply adjusting the silane coupling agent addition procedure in the preparation process. Our demonstration of low-cost Si-GQDs and their encapsulation into multifunctional composites may expand the applications of carbon-based nanomaterials for future biomedical imaging and other optoelectronic applications.
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[Establishment of HLH-like mouse model with CPG-ODN and IFN-?].
Zhonghua Xue Ye Xue Za Zhi
PUBLISHED: 09-24-2014
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To establish a hemophagocytic lymphohistiocytosis (HLH)-like mouse model induced by CpG oligodeoxynucleotide (CpG-ODN1826) and interferon (IFN)-? for further study on therapy.
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Quercetin alleviates myocyte toxic and sensitizes anti-leukemic effect of adriamycin.
Hematology
PUBLISHED: 09-10-2014
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Objectives Derived from plants, flavonoids have been proven to possess anti-cancer activities. Adriamycin (ADM), an anthracycline antibiotic, is widely applied in the chemotherapy for leukemia; however, it has a side effect of heart damage. This study aims to explore potential anti-leukemia effects of quercetin (Que) and the underlying mechanism. Methods The P388 xenograft mice models were first established and then treated with Que alone or in combination with ADM. Subsequently, we evaluated their effects on cell proliferation and apoptosis by observing the cell cycle and detecting the Caspase-3 level, respectively. The underlying pro-apoptotic mechanism was further investigated by detecting the expression levels of NF-?B, Bcl-2, and Bax. The cardiomyocyte ultrastructural changes of P388 leukemic mice after drug treatment were also observed. The protective effect of Que on cardiomyocyte was evaluated by detecting enzymatic activity changes of glutathione peroxidase, superoxide dismutase, and malondialdehyde. Results Compared with ADM group, the combination of ADM and Que showed prolonged survival time and less peripheral white blood cells. Que could sensitize the anti-leukemic effect of ADM by inhibiting the proliferation of white blood cells through trapping the cells at the S phase; caspase-3 was activated via the expressional regulation of Bcl-2, Bax, and NF-?B. When applied in combination with ADM, Que could attenuate heart damage by cleaning the reactive oxygen species. Conclusion Our study may provide informative evidences for the underlying mechanism of anti-cancer effects of Que and sheds light on the clinical application of Que in leukemia treatment.
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[Expression, purification and characterization of a thermostable lactate dehydrogenase from Thermotoga maritima].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 09-09-2014
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The gene encoding thermostable lactate dehydrogenase (Tm-LDH) was cloned into the plasmid pHsh from Thermotoga maritima, and expressed in Escherichia coli JM 109. The recombinant protein was purified to homogeneity by a simple step, heat treatment. The recombinant enzyme had a molecular mass of 33 kDa. The optimal temperature and pH of Tm-LDH were observed 95 degrees C and 7.0. The purified enzyme had a half-life of 2 h at 90 degrees C, and exhibited better stability over a pH range from 5.5 to 8.0. The K(m) and V(max) values were 1.7 mmol/L, 3.8 x 10(4) U/mg of protein for pyruvate, and 7.2 mmol/L and 1.1 x 10(5) U/mg for NADH, respectively. The expression of Tm-LDH in T7 system could not obtain high efficiency, but it has been soluble over-expression in pHsh system and reached 340 mg/L. The superior stability and productivity of Tm-LDH will lay the foundation of its industrial-scale fermentation and application in the NAD regeneration.
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Near orthogonal launch of SPR modes in Au films.
Opt Lett
PUBLISHED: 08-29-2014
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We report the excitation of a surface plasmon resonance (SPR) close to the orthogonal axis of a gold (Au) film on borosilicate glass. Direct spectroscopic measurement of SPR shifts using different liquids are made at ?5° incidence within a reflection spectrophotometer. The proposed mechanism to establish coupling and plasmon localization is the scattering of light able to penetrate across the film at the interfaces.
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Unusual assembly of lacunary heteropolymolybdates with cyanometalate fragment.
Dalton Trans
PUBLISHED: 08-15-2014
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Two new cyanide-bridged heteropolymolybdate complexes have been synthesized by a facile self-assembly process in aqueous solution, which demonstrates a successful tactic to incorporate the cyanometalate fragment into lacunary heteropolymolybdate. Magnetic investigation showed that complex exhibits slow magnetic relaxation behavior.
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Modulation of cytokine expression in human macrophages by endocrine-disrupting chemical Bisphenol-A.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-13-2014
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Exposure to environmental endocrine-disrupting chemical Bisphenol-A (BPA) is often associated with dysregulated immune homeostasis, but the mechanisms remain unclear. In the present study, the effects of BPA on the cytokines responses of human macrophages were investigated. Treatment with BPA increased pro-inflammation cytokines tumor necrosis factor-? (TNF-?) and interleukin-6 (IL-6) production, but decreased anti-inflammation cytokines interleukin-10 (IL-10) and transforming growth factor-? (TGF-?) production in THP1 macrophages, as well as in primary human macrophages. BPA effected cytokines expression through estrogen receptor ?/? (ER?/?)-dependent mechanism with the evidence of ER?/? antagonist reversed the expression of cytokines. We also identified that activation of extracellular regulated protein kinases (ERK)/nuclear factor ?B (NF-?B) signal cascade marked the effects of BPA on cytokines expression. Our results indicated that BPA effected inflammatory responses of macrophages via modulating of cytokines expression, and provided a new insight into the link between exposure to BPA and human health.
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Proteomics analysis of Mahonia bealei leaves with induction of alkaloids via combinatorial peptide ligand libraries.
J Proteomics
PUBLISHED: 08-08-2014
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Alkaloids are one of the most attractive sources for obtaining active natural products. However, alkaloids exist in the plants as the secondary metabolites with tracing amount, and there is an enormous demand for a large production. In the present study, we aimed to profile the modification of benzylisoquinoline alkaloids in Mahonia bealei seedlings under the binary stress of ultraviolet-B irradiation and dark incubation. Comparative proteomics analysis was carried out to address the underlying proteome variations that accounted for the alkaloid induction under treatment. Thirteen differential proteins were identified in the leaves under binary stress. Of note, the abundance of S-adenosyl-l-methionine synthetase was highly increased to sustain a high concentration of S-adenosyl-l-methionine for the enhanced biosynthesis of alkaloids. Additionally, we presented the application of CPLL to M. bealei leaf proteins. Three new secondary metabolism proteins and 12 additional differential proteins were identified only after CPLL treatment. Six genes in the benzylisoquinoline alkaloid biosynthesis pathway were selected to verify their variable expression using quantitative real time polymerase chain reaction. The results suggest that the benzylisoquinoline alkaloids in M. bealei leaf were increased to eliminate the adverse effect of UV-B exposure. The suppression of photosynthesis and respiratory rate may save an extra energy for the secondary metabolites, and the enhanced N-metabolism may supply sufficient primary metabolite precursors. To our best knowledge, this is the first work aimed at the secondary metabolism proteomic characterization of M. bealei using the CPLL technique. It also presented an effective and innovative process to improve the contents of alkaloids in medicinal plants for industrial production.
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Construction of a novel expression system for use in Corynebacterium glutamicum.
Plasmid
PUBLISHED: 08-07-2014
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Corynebacterium glutamicum is an important microorganism for production of amino acids in industrial fermentation. Suitable vectors are needed for metabolic engineering in C. glutamicum. Most available vectors used in C. glutamicum carry antibiotic resistant genes as a genetic labeling for rapid identification of recombinant strains, and antibiotics have to be added to maintain the vector when growing the cells. These vectors, though excellent for laboratory use, are not preferable choices for industry-scale fermentation. In this work, we developed a novel expression system for use in C. glutamicum, which do not require antibiotics when used for industrial fermentation. This system includes two vectors: the shuttle vector pJYW-4 for expression of genes and the vector pJYW-6 for deletion of the essential gene alr in C. glutamicum. The vector pJYW-4 contains a large multiple cloning site for cloning multiple genes and two selective markers: one is the kanamycin-resistant gene kan and the other is an essential gene alr. The selective marker kan facilitates molecular manipulation or fermentations in the laboratory, and the selection marker alr is good for use in industry-scale fermentation, allowing in vivo maintenance of the expression vector through auxotrophic complementation; therefore, the two selection markers in pJYW-4 make it useful for both laboratory research and industrial fermentation, and convenient to transfer valuable laboratory-developed strains into industrial production. This newly-constructed expression system was successfully used to increase L-valine production in C. glutamicum ATCC 14067, indicating its potential on developing amino acid-producing C. glutamicum strains.
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Synthesis and biological evaluation of novel indoline-2,3-dione derivatives as antitumor agents.
Drug Discov Ther
PUBLISHED: 07-18-2014
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A new series of 1,5-disubstituted indolin-2,3-diones was synthesized and their inhibition of the growth of a human acute promyelocytic leukemia (HL-60) cell line was evaluated. These compounds had promising inhibition of HL-60 cell growth in vitro. Results indicated that compounds with a benzyl substituent at the N-1 position on the indolin-2,3-dione ring had more potent antiproliferative activity than those with a (4-fluorobenzyl) amino-2-oxoethyl substituent at the N-1 position. Among the compounds synthesized, compound 8l inhibited half of cell growth at a concentration of 0.07 ?M and compound 8p did so at a concentration of 0.14 ?M. These compounds may serve as lead compounds for further optimization in order to develop novel anticancer agents.
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[A method for primary culture of pulmonary microvascular endothelial cells].
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
PUBLISHED: 07-09-2014
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To explore a simple and practical method of primarily culturing rat pulmonary microvascular endothelial cells (PMECs) in vitro, and observe the cell growth status and identify the PMECs.
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Effects of transforming growth factor-?1 on the proliferation and invasion of the HTR-8/SVneo cell line.
Oncol Lett
PUBLISHED: 06-24-2014
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Transforming growth factor-?1 (TGF-?1) is involved in the regulation of trophoblast cell proliferation and invasion. However, the mechanism underlying this process remains unknown, which is predominantly due to the difficulty in obtaining and maintaining primary trophoblast cells in culture over a long period of time. The HTR-8/SVneo cell line is an immortalized trophoblast cell line, which has been reported to exhibit a number of similar characteristics to those of parental trophoblast cells. Therefore, the cell line has been a useful tool for the investigation of placental function and tumor progression. In the present study, the HTR-8/SVneo cell line was used as a model to investigate the TGF-?1/SMAD signaling pathway in the proliferation and invasion of trophoblast cells. The proliferation and invasion ability of HTR-8/SVneo cells was determined using the MTT and Transwell assays, respectively. In addition, reverse transcription polymerase chain reactions were performed to detect the mRNA expression of a panel of known downstream mediators of TGF-?1, including TGF-? receptor I (T?RI), SMAD4, SMAD3, SMAD7 and tissue inhibitor of metalloproteinases-1 (TIMP-1). The results indicated that TGF-?1 promotes the proliferation and invasion of the HTR-8/SVneo cell line at passage 90. Furthermore, the expression of T?RI, SMAD3 and SMAD4 were reduced following treatment with TGF-?1, while the expression of SMAD7 was increased and the expression of TIMP-1 remained unchanged following TGF-?1 treatment. These observations indicated that the effects of TGF-?1 on the proliferation and invasion of the HTR-8/SVneo cell line at passage 90 were different from those of parental trophoblasts, which is in contrast to the results of previous studies. It was concluded that the HTR-8/SVneo cell lines, which have been grown for over 90 passages, do not accurately represent parental trophoblast cells in studies of the TGF-?/SMAD signaling pathway.
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Syntheses and characterization of nearly monodispersed, size-tunable silver nanoparticles over a wide size range of 7-200 nm by tannic acid reduction.
Langmuir
PUBLISHED: 03-25-2014
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Nearly monodispersed spherical silver nanoparticles (Ag NPs) were synthesized by using tannic acid (TA) as both reductant and stabilizer in a 30 °C water bath. The size of the as-prepared Ag NPs could be tuned in a range of 7-66 nm by changing the molar ratio of TA to silver nitrate and pH of the reaction solutions. UV-vis spectra, TEM observations, and temporal evolution of the monomer concentrations for the reactions carried out at different experimental conditions showed that the improved size distribution and size tunability of the Ag NPs were mainly attributed to the use of TA, which could promote the balance of nucleation and growth processes of the NPs effectively. The size of the Ag NPs was extendable up to 200 nm in one-pot fashion by the multi-injection approach. The size-dependent surface-enhanced Raman scattering (SERS) activity of the as-prepared Ag NPs was evaluated, and the NPs with size around 100 nm were identified to show a maximum enhanced factor of 3.6 × 10(5). Moreover, the as-prepared TA-coated Ag NPs presented excellent colloidal stability compared to the conventional citrate-coated ones.
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A supramolecular microgel glutathione peroxidase mimic with temperature responsive activity.
Soft Matter
PUBLISHED: 03-21-2014
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Glutathione peroxidase (GPx) protects cells from oxidative damage by scavenging surplus reactive oxygen species (ROS). Commonly, an appropriate amount of ROS acts as a signal molecule in the metabolism. A smart artificial GPx exhibits adjustable catalytic activity, which can potentially reduce the amount of ROS to an appropriate degree and maintain its important physiological functions in metabolism. To construct an optimum and excellent smart artificial GPx, a novel supramolecular microgel artificial GPx (SM-Te) was prepared based on the supramolecular host-guest interaction employing the tellurium-containing guest molecule (ADA-Te-ADA) and the cyclodextrin-containing host block copolymer (poly(N-isopropylacrylamide)-b-[polyacrylamides-co-poly(6-o-(triethylene glycol monoacrylate ether)-?-cyclodextrin)], PPAM-CD) as building blocks. Subsequently, based on these building blocks, SM-Te was constructed and the formation of its self-assembled structure was confirmed by dynamic light scattering, NMR, SEM, TEM, etc. Typically, benefitting from the temperature responsive properties of the PNIPAM scaffold, SM-Te also exhibited similar temperature responsive behaviour. Importantly, the GPx catalytic rates of SM-Te displayed a noticeable temperature responsive characteristic. Moreover, SM-Te exhibited the typical saturation kinetics behaviour of a real enzyme catalyst. It was proved that the changes of the hydrophobic microenvironment and the pore size in the supramolecular microgel network of SM-Te played significant roles in altering the temperature responsive catalytic behaviour. The successful construction of SM-Te not only overcomes the insurmountable disadvantages existing in previous covalent bond crosslinked microgel artificial GPx but also bodes well for the development of novel intelligent antioxidant drugs.
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Induction of E-cadherin+ human amniotic fluid cell differentiation into oocyte-like cells via culture in medium supplemented with follicular fluid.
Mol Med Rep
PUBLISHED: 03-17-2014
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Pluripotent human amniotic fluid cells (HuAFCs) can differentiate into various types of somatic cell in vitro. However, their differentiation into oocyte-like cells has never been described to the best of our knowledge. In the present study, differentiation of E-cadherin+ and E-cadherin- HuAFC sub-populations into oocyte-like cells was induced via culture in medium containing bovine follicular fluid and ?-mercaptoethanol. The E-cadherin+ HuAFCs expressed DAZL highly. Post-induction, cells with an oocyte-like phenotype were found among the E-cadherin+ HuAFCs, expressing markers specific to germ cells and oocytes (VASA, ZP3 and GDF9) and meiosis (DMC1 and SCP3). When specific small interfering RNA (siRNA) was used to suppress E-cadherin in the E-cadherin+ HuAFCs, the levels of DAZL expression were reduced. Post-induction, the morphology of the siRNA?E?cadherin HuAFCs was poorer and the expression levels of germ cell-specific markers were lower compared with those of the siRNA-mock HuAFCs. Therefore, E-cadherin+ HuAFCs could be more easily induced to differentiate into oocyte-like cells by bovine follicular fluid and ?-mercaptoethanol. In addition, the E-cadherin+ HuAFCs exhibited potential characteristics of DAZL protein expression, and thus it was conjectured that bovine follicular fluid acts on DAZL protein and promotes E-cadherin+ HuAFC differentiation into oocyte-like cells.
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Iron-catalyzed carbonylative Suzuki reactions under atmospheric pressure of carbon monoxide.
Chem. Commun. (Camb.)
PUBLISHED: 03-04-2014
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The first highly effective iron-catalyzed carbonylative Suzuki reaction has been developed. Substrates with electron-donating or electron-withdrawing functionality, ortho-substitution, as well as active groups proceeded smoothly, affording desired products in high yields. This protocol is economical, environmentally benign and practical for the synthesis of biaryl ketones.
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The complete chloroplast genome sequence of Taxus chinensis var. mairei (Taxaceae): loss of an inverted repeat region and comparative analysis with related species.
Gene
PUBLISHED: 02-12-2014
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Taxus chinensis var. mairei (Taxaceae) is a domestic variety of yew species in local China. This plant is one of the sources for paclitaxel, which is a promising antineoplastic chemotherapy drugs during the last decade. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of T. chinensis var. mairei. The T. chinensis var. mairei cp genome is 129,513 bp in length, with 113 single copy genes and two duplicated genes (trnI-CAU, trnQ-UUG). Among the 113 single copy genes, 9 are intron-containing. Compared to other land plant cp genomes, the T. chinensis var. mairei cp genome has lost one of the large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperm such as Cycas revoluta and Ginkgo biloba L. Compared to related species, the gene order of T. chinensis var. mairei has a large inversion of ~110kb including 91 genes (from rps18 to accD) with gene contents unarranged. Repeat analysis identified 48 direct and 2 inverted repeats 30 bp long or longer with a sequence identity greater than 90%. Repeated short segments were found in genes rps18, rps19 and clpP. Analysis also revealed 22 simple sequence repeat (SSR) loci and almost all are composed of A or T.
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Complementary DNA cloning and functional characterization of cytochrome P450 3A138 in common carp (Cyprinus carpio L.).
J. Biochem. Mol. Toxicol.
PUBLISHED: 01-28-2014
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The full-length sequence of a cytochrome P450 3A 138 (CYP3A138) cDNA in common carp was cloned and sequenced. The transcriptional and microsome enzyme activities of CYP3A138 in the fish liver after rifampicin exposure were also determined in this study. The results showed that the full-length CYP3A138 cDNA is 1912 base pairs (bp) long and contains an open reading frame of 1551 bp encoding a protein of 517 amino acids. Sequence analysis revealed that CYP3A138 is highly conserved in fish. Furthermore, the results of quantitative real-time PCR revealed that CYP3A138 in common carp is constitutively expressed in all tissues, but mainly in the liver and intestine. Additionally, rifampicin exposure promoted both the expression of CYP3A138 at the transcriptional level and the activity of the protein, suggesting that CYP3A138 is a member of the CYP3A subfamily.
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Identification and refinement of two strong constitutive promoters for gene expression system of Schizosaccharomyces pombe.
World J. Microbiol. Biotechnol.
PUBLISHED: 01-12-2014
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Fission yeast Schizosaccharomyces pombe shares various important properties with higher eukaryotes and is now considered a useful host for elevated production of mammalian proteins for medicinal applications. The full-length nmt1 promoter has been widely used as a strong promoter in S. pombe expression system. In the present study, the promoters of the eno101 and gpd3 genes in S. pombe were identified as strong constitutive promoters. For convenient applications in the plasmids of S. pombe, these promoters were refined to 276-bp eno and 273-bp gpd promoters by deleting undesired sequences and examining the expression of reporter genes including lacZ and xynA. Both the refined eno and gpd promoters provided approximately 1.5-fold higher expression of LacZ than nmt1 promoter. Furthermore, gene expression under the control of the eno or gpd promoter was not repressed by the components of YES medium while nmt1 promoter was inhibited by thiamine in yeast extract. Therefore, both eno and gpd promoters offer opportunities for efficient production of recombinant proteins by S. pombe in high cell-density fermentation.
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The influence of goal-directed fluid therapy on the prognosis of elderly patients with hypertension and gastric cancer surgery.
Drug Des Devel Ther
PUBLISHED: 01-01-2014
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We aimed to investigate the influence of perioperative goal-directed fluid therapy (GDFT) on the prognosis of elderly patients with gastric cancer and hypertension.
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Influence of electrical resistivity and machining parameters on electrical discharge machining performance of engineering ceramics.
PLoS ONE
PUBLISHED: 01-01-2014
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Engineering ceramics have been widely used in modern industry for their excellent physical and mechanical properties, and they are difficult to machine owing to their high hardness and brittleness. Electrical discharge machining (EDM) is the appropriate process for machining engineering ceramics provided they are electrically conducting. However, the electrical resistivity of the popular engineering ceramics is higher, and there has been no research on the relationship between the EDM parameters and the electrical resistivity of the engineering ceramics. This paper investigates the effects of the electrical resistivity and EDM parameters such as tool polarity, pulse interval, and electrode material, on the ZnO/Al2O3 ceramic's EDM performance, in terms of the material removal rate (MRR), electrode wear ratio (EWR), and surface roughness (SR). The results show that the electrical resistivity and the EDM parameters have the great influence on the EDM performance. The ZnO/Al2O3 ceramic with the electrical resistivity up to 3410 ?·cm can be effectively machined by EDM with the copper electrode, the negative tool polarity, and the shorter pulse interval. Under most machining conditions, the MRR increases, and the SR decreases with the decrease of electrical resistivity. Moreover, the tool polarity, and pulse interval affect the EWR, respectively, and the electrical resistivity and electrode material have a combined effect on the EWR. Furthermore, the EDM performance of ZnO/Al2O3 ceramic with the electrical resistivity higher than 687 ?·cm is obviously different from that with the electrical resistivity lower than 687 ?·cm, when the electrode material changes. The microstructure character analysis of the machined ZnO/Al2O3 ceramic surface shows that the ZnO/Al2O3 ceramic is removed by melting, evaporation and thermal spalling, and the material from the working fluid and the graphite electrode can transfer to the workpiece surface during electrical discharge machining ZnO/Al2O3 ceramic.
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The effects of controlled release of neurotrophin-3 from PCLA scaffolds on the survival and neuronal differentiation of transplanted neural stem cells in a rat spinal cord injury model.
PLoS ONE
PUBLISHED: 01-01-2014
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Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2 ± 4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8 ± 15.1%) and neuronal differentiation (21.5 ± 5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.
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Cardiometabolic risk profiles associated with chronic complications in overweight and obese type 2 diabetes patients in South China.
PLoS ONE
PUBLISHED: 01-01-2014
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Type 2 diabetes is often accompanied by altered cardiometabolic risk profiles, including abdominal obesity, hypertension, and dyslipidaemia. The association of altered cardiometabolic risk profiles with chronic complications of diabetes is not well investigated.
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Ectopic TLX1 expression accelerates malignancies in mice deficient in DNA-PK.
PLoS ONE
PUBLISHED: 01-01-2014
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The noncluster homeobox gene HOX11/TLX1 (TLX1) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgH?-TLX1(Tg) mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgH?-TLX1(Tg) mice with mice deficient in the DNA repair enzyme DNA-PK (Prkdc(Scid/Scid) mice). IgHµ-TLX1(Tg)Prkdc(Scid/Scid) mice developed T-ALL and acute myeloid leukemia (AML) with reduced latency relative to control Prkdc(Scid/Scid) mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant IgHµ-TLX1(Tg)Prkdc(Scid/Scid) thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2) and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.
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Bending effect on modal interference in a fiber taper and sensitivity enhancement for refractive index measurement.
Opt Express
PUBLISHED: 11-13-2013
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We demonstrate the bending effect of microfiber on interference fringes in a compact taper-based modal interferometer and sensitivity for refractive index (RI) measurement. For the bend curvature ranging from 0 to 0.283 mm(-1), the measured RI sensitivity distinctively increases from 342.5 nm/RIU (refractive-index unit) to 1192.7 nm/RIU around RI = 1.333 and from 3847.1 nm/RIU to 11006.0 nm/RIU around RI = 1.430, respectively. Theoretical analysis reveals that such enhancement is determined by the dispersion property of the intermodal index rather than other parameters, such as the variation of the straightforward evanescent field. The magnitude of sensitivity varies as a function of the microfiber bend curvature. Approaching a critical curvature (the intermodal-index dispersion factor approaches zero), the sensitivity is significantly enhanced, exhibiting great potential in RI sensing areas.
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Different impacts of short-chain fatty acids on saturated and polyunsaturated fatty acid biosynthesis in Aurantiochytrium sp. SD116.
J. Agric. Food Chem.
PUBLISHED: 10-07-2013
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Aurantiochytrium is an important docosahexaenoic acid (DHA) producer containing two kinds of fatty acid synthesis pathways, that is, the fatty acid synthase pathway (FAS) for saturated fatty acid synthesis and the polyketide synthase pathway (PKS) for polyunsaturated fatty acid synthesis. To understand the regulation mechanism between the two pathways, the impacts of six short-chain fatty acids on the fatty acid synthesis of Aurantiochytrium sp. SD116 were studied. All short-chain fatty acids showed little effect on the cell growth, but some of them significantly affected lipid accumulation and fatty acid composition. Pentanoic acid and isovaleric acid greatly inhibited the synthesis of saturated fatty acids, whereas the polyunsaturated fatty acid synthesis was not affected. Analysis of malic enzyme activity, which supplied NADPH for saturated fatty acids biosynthesis, indicated that the two fatty acid synthesis pathways can utilize different substrates and possess independent sources of NADPH.
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A novel pH-controlled immunosensor using hollow mesoporous silica and apoferritin combined system for target virus assay.
Biosens Bioelectron
PUBLISHED: 08-21-2013
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A novel pH-controlled immunosensor using hollow mesoporous silica and apoferritin combined system has been reported for the first time. The goal of this study was to introduce more electroactive probes into the electrochemical immunosensor. Under such background, we focused our attention on hollow mesoporous silica and apoferritin, both of which have admirable accommodating performances and can be applied for encapsulating electroactive probes. Based on the pH-controlled disassociation-reconstitution process of apoferritin and the mesoporous channels of silica, after the appropriate chemical modification, apoferritin could be fabricated with silica. The results showed that the hollow mesoporous silica and apoferritin combined system was successfully assembled. The excellent performance of the combined system can be applied for ultrasensitive detection of a target virus.
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Tlx3 controls cholinergic transmitter and Peptide phenotypes in a subset of prenatal sympathetic neurons.
J. Neurosci.
PUBLISHED: 06-28-2013
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The embryonic sympathetic nervous system consists of predominantly noradrenergic neurons and a very small population of cholinergic neurons. Postnatal development further allows target-dependent switch of a subset of noradrenergic neurons into cholinergic phenotype. How embryonic cholinergic neurons are specified at the prenatal stages remains largely unknown. In this study, we found that the expression of transcription factor Tlx3 was progressively restricted to a small population of embryonic sympathetic neurons in mice. Immunostaining for vesicular acetylcholine transporter (VAChT) showed that Tlx3 was highly expressed in cholinergic neurons at the late embryonic stage E18.5. Deletion of Tlx3 resulted in the loss of Vacht expression at E18.5 but not E12.5. By contrast, Tlx3 was required for expression of the cholinergic peptide vasoactive intestinal polypeptide (VIP), and somatostatin (SOM) at both E12.5 and E18.5. Furthermore, we found that, at E18.5 these putative cholinergic neurons expressed glial cell line-derived neurotrophic factor family coreceptor Ret but not tyrosine hydroxylase (Ret(+)/TH(-)). Deletion of Tlx3 also resulted in disappearance of high-level Ret expression. Last, unlike Tlx3, Ret was required for the expression of VIP and SOM at E18.5 but not E12.5. Together, these results indicate that transcription factor Tlx3 is required for the acquisition of cholinergic phenotype at the late embryonic stage as well as the expression and maintenance of cholinergic peptides VIP and SOM throughout prenatal development of mouse sympathetic neurons.
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Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum.
Plasmid
PUBLISHED: 05-30-2013
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Gene deletion techniques are important for modifying Corynebacterium glutamicum, the bacterium for industrial production of amino acids. In this study, a novel multiple-gene-deletion system for C. glutamicum was developed. The system is composed of three plasmids pDTW109, pDTW201 and pDTW202. pDTW109 is a temperature-sensitive vector which harbors a cat gene under the tacM promoter, a cre gene under the tac promoter, an origin oriE for replicating in Escherichia coli, and another origin rep(TS) for replicating in C. glutamicum only at low temperatures; it has high transformation efficiency in C. glutamicum and can be easily eliminated by growing at 37°C. pDTW201 and pDTW202 carry loxp-flanked or mutant lox-flanked kan, respectively. This deletion system combines homologous recombination and Cre/lox site-specific recombination, could efficiently delete the aceE gene from the chromosome of C. glutamicum ATCC13032, ATCC13869 or ATCC14067, respectively, and could also delete both genes of aceE and ilvA from the chromosome of C. glutamicum ATCC14067. The system is simple and efficient, and can be easily implemented for multiple-gene-deletion in C. glutamicum.
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Design and synthesis of novel pyrimidone analogues as HIV-1 integrase inhibitors.
Bioorg. Med. Chem. Lett.
PUBLISHED: 05-11-2013
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A series of novel pyrimidone analogues have been designed and synthesized as HIV-1 integrase (IN) inhibitors. This study demonstrated that introducing a substituent in the N1-position of the pyrimidone scaffold does not significantly influence IN inhibitory activity. Molecular docking studies showed these compounds could occupy the IN active site and form pi-pi interactions with viral DNA nucleotides DC16 and DA17 to displace reactive viral DNA 3OH and block intasome activity.
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An integrative approach for the large-scale identification of human genome kinases regulating cancer metastasis.
Nanomedicine
PUBLISHED: 04-19-2013
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Kinases become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis. We first employed self-assembled cell microarray to screen functional genes that regulate cancer cell migration using a human genome kinase siRNA library. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases.
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Sertraline and periodic limb movements during sleep: an 8-week open-label study in depressed patients with insomnia.
Sleep Med.
PUBLISHED: 04-16-2013
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Previous studies have reported that selective serotonin reuptake inhibitors (SSRIs) might induce or exacerbate periodic limb movements during sleep (PLMS). However, most of these studies were retrospective and cross-sectional studies with small sample sizes on a selective SSRI, fluoxetine. Because different SSRIs have different pharmacologic profiles, it was not certain if other SSRIs also might lead to PLMS.
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Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase.
BMC Mol. Biol.
PUBLISHED: 04-16-2013
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Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5 and 3 junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.
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Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells.
Genomics
PUBLISHED: 04-03-2013
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We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.
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[Advanced oxidation protein products inhibit proliferation and differentiation of rat osteoblasts through oxidative stress].
Nan Fang Yi Ke Da Xue Xue Bao
PUBLISHED: 03-27-2013
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To investigate the effect of advanced oxidation protein products (AOPP) on proliferation, differentiation, and oxidative stress in rat osteoblasts.
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[Progress of the study on DNA electrochemical biosensor].
Sheng Wu Yi Xue Gong Cheng Xue Za Zhi
PUBLISHED: 03-16-2013
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With its rapid development, the electrochemical biosensor has recently been widely used in gene diagnosis, environmental monitoring, and medical sciences. More and more attention has been focused on how to improve the sensitivity and selectivity of biosensor. In this review, the principle and composition of DNA electrochemical biosensor is simply introduced, the preparation of biological membrane, the application of indicator are specially emphasized, and the future prospect for the development in this field is given.
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Development of intravenous lipid emulsion of vinorelbine based on drug-phospholipid complex technique.
Int J Pharm
PUBLISHED: 02-25-2013
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In order to reduce the severe venous toxicity, we developed an intravenous lipid emulsion of vinorelbine and investigated its preclinical stability, toxicity and antitumor efficacy. Vinorelbine-phospholipid complex was prepared to enhance the lipophilicity of vinorelbine thus facilitating the encapsulation into lipid emulsion. After complexation more than 70% of vinorelbine was encapsulated into the oil phase. Meanwhile, the lipid emulsion showed good stability without drug leakage. Local irritation after injection of the lipid emulsion was investigated in rabbits and compared with Navelbine(®) (the commercial product). The antitumor therapeutic efficacies were evaluated in tumor-bearing mouse models inoculated with A549 human lung cancer cells and BCAP-37 human breast cancer cells and compared as well. Results showed that the lipid emulsion significantly reduced the injection irritation compared with that of Navelbine(®), while maintained the antitumor activity in A549 and BCAP-37 cells xenograft tumor mouse models. Taken together, lipid emulsion loaded with vinorelbine-phospholipid complex is a promising vinorelbine intravenous injection with reduced venous irritation.
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Comparative proteomic analysis of the sun- and freeze-dried earthworm Eisenia fetida with differentially thrombolytic activities.
J Proteomics
PUBLISHED: 02-25-2013
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The dried earthworm is a traditional thrombolytic medicine in East Asia. Its thrombolytic mechanism has been extensively studied. However, the effects of drying process on thrombolysis were rarely investigated. Herein, we compared the thrombolytic activity of earthworm Eisenia fetida processed by sun-drying to that by freeze-drying. Fibrin plate and blood clot lysis assays showed that freeze-dried earthworms gave dramatically higher fibrinolytic and thrombolytic activities than the sun-dried earthworms. To address the thrombolytic difference, comparative proteomic analysis was carried out using fibrin zymography and two-dimensional gel electrophoresis (2-DE). The freeze- and sun-dried earthworms generated remarkably different 2-DE protein spot patterns. A total of 126 differential protein spots were detected, 83 of them were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching with 13 quantitative changes and 70 qualitative changes. Five of these differential proteins were identified as fibrinolytic proteases (lumbrokinases), responsible for dissolving fibrin, the main protein component of thrombus. The total abundance of these fibrinolytic proteases in the freeze-dried earthworms was significantly higher, consistent with the results of fibrin zymography. Therefore, the higher concentration of fibrinolytic enzymes along with their broad substrate specificity explained the stronger fibrinolytic and thrombolytic activities of the freeze-dried earthworms. This study suggests that freeze-drying represents an improved processing method for earthworm as the thrombolytic therapy in the future.
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Ectopic expression of wheat TaCIPK14, encoding a calcineurin B-like protein-interacting protein kinase, confers salinity and cold tolerance in tobacco.
Physiol Plant
PUBLISHED: 02-22-2013
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Calcineurin B-like protein-interacting protein kinases (CIPKs) are components of Ca(2+) signaling in responses to abiotic stresses. In this work, the full-length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT-PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H2 O2 . Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H2 O2 and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na(+) content in the transgenic lines under salt stress. Higher expression of stress-related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress-responsive gene in plants.
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Microfiber Mach-Zehnder interferometer based on long period grating for sensing applications.
Opt Express
PUBLISHED: 02-08-2013
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A Mach-Zehnder interferometer (MZI) composed by a pair of long period gratings (LPGs) fabricated in silica microfiber for sensing applications is demonstrated. Each LPG is fabricated with a pulsed CO2 laser by creating six periodical deformations along fiber length with only one scanning cycle. The length of the MZI can reach as short as 8.84 mm when the diameter of the microfiber is 9.5 ?m. Compared with the ones fabricated in single-mode fibers, the present MZI is much shorter owing to the large effective-index difference between the fundamental and higher order modes. The microfiber MZI exhibits a sensitivity to surrounding refractive index (RI) of 2225 nm per refractive index unit and the temperature sensitivity of only 11.7 pm/°C. Theoretical analysis suggests that the performances of the MZI sensor can be improved by using thinner microfibers with a diameter down to 3.5 ?m: The sensitivity can be greatly enhanced due to the stronger evanescent-field interaction and reduced dispersion factor; the transmission dips become narrower which benefits high-resolution measurement; the thinner fiber also allows further reduction in device length. The present device has great potential in biochemical and medical sensing due to the advantages including easy fabrication, excellent compactness and high sensitivity.
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Phylogenetic analysis of the genus Cylicocyclus (Nematoda: Strongylidae) based on nuclear ribosomal sequence data.
Acta Parasitol.
PUBLISHED: 02-07-2013
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Seven species of Cylicocyclus Ihle, 1922 (Nematoda: Strongylidae) were collected from donkeys from Henan Province, China. Five samples of each species were selected for sequencing. Sixteen different internal transcribed spacer (ITS) sequences representing the seven species of Cylicocyclus were obtained. Sequence differences in the first internal transcribed spacer (ITS-1) among species was lower than that of the second internal transcribed spacer (ITS-2). Phylogenetic analyses were conducted using the combined ITS-1 and ITS-2 data sets from the present study and using reference sequences from the GenBank database. The MP and ML trees were similar in topology. The phylogenetic trees were divided into two clades. Clade I included 8 species of Cylicocyclus; within this group, Cylicocyclus leptostomus (Kotlan, 1920) is nested between different samples of Cylicocyclus ashworthi (LeRoux, 1924), suggesting C. ashworthi may represent a species complex. Clade II included Cylicocyclus elongatus (Looss, 1900) and Cylicocyclus ultrajectinus (Ihle, 1920); however, these two species always clustered with the comparative species (Petrovinema poculatum (Looss, 1900) and Poteriostomum imparidentatum Quiel, 1919), suggesting that C. elongatus and C. ultrajectinus represent members of other genera.
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Salinity increases the mobility of Cd, Cu, Mn, and Pb in the sediments of Yangtze Estuary: relative role of sediments properties and metal speciation.
Chemosphere
PUBLISHED: 01-29-2013
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Batch leaching experiments, Freudlich isotherm, correlation analysis (CA) and principal component analysis (PCA) were undertaken to explore the mechanisms that govern the release of sediment-associated metals (i.e. Cd, Cu, Mn and Pb) under the salinity stress in the Yangtze River Estuary. Special attention has been paid to the role of sediments physico-chemical properties and metal chemical speciation. The increase of salinity promoted the metal mobility which followed the order: Cd>Mn>Cu>Pb. Sediments properties (e.g., particle component and organic carbon) governed the mobility of Cd; metal chemical speciation controlled the release of Mn, while the mobility of Cu and Pb were simultaneously affected by the two factors. Different metal release mechanisms caused by salinity changes might be explained by: the chloro-complexation for Cd, the encouragement of acidity changes for Mn, and the high affinity to Fe-Mn oxides, organic substances and specific sorption sites in the sediments for Cu and Pb. Under salinity effect, Cd and Mn exhibited higher ecological risk and mobility, while Cu and Pb seemed to be more conservative.
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Investigation of the charging characteristics of micrometer sized droplets based on parallel plate capacitor model.
Langmuir
PUBLISHED: 01-23-2013
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The charging characteristics of micrometer sized aqueous droplets have attracted more and more attentions due to the development of the microfluidics technology since the electrophoretic motion of a charged droplet can be used as the droplet actuation method. This work proposed a novel method of investigating the charging characteristics of micrometer sized aqueous droplets based on parallel plate capacitor model. With this method, the effects of the electric field strength, electrolyte concentration, and ion species on the charging characteristics of the aqueous droplets was investigated. Experimental results showed that the charging characteristics of micrometer sized droplets can be investigated by this method.
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Construction of monophosphoryl lipid A producing Escherichia coli mutants and comparison of immuno-stimulatory activities of their lipopolysaccharides.
Mar Drugs
PUBLISHED: 01-15-2013
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The lipid A moiety of Escherichia coli lipopolysaccharide is a hexaacylated disaccharide of glucosamine phosphorylated at the 1- and 4-positions. It can be recognized by the TLR4/MD-2 complex of mammalian immune cells, leading to release of proinflammatory cytokines. The toxicity of lipid A depends on its structure. In this study, two E. coli mutants, HW001 and HW002, were constructed by deleting or integrating key genes related to lipid A biosynthesis in the chromosome of E. coli W3110. HW001 was constructed by deleting lacI and replacing lacZ with the Francisella novicida lpxE gene in the chromosome and only synthesizes monophosphoryl lipid A. HW002 was constructed by deleting lpxM in HW001 and synthesizes only the pentaacylated monophosphoryl lipid A. The structures of lipid A made in HW001 and HW002 were confirmed by thin layer chromatography and electrospray ionization mass spectrometry. HW001 and HW002 grew as well as the wild-type W3110. LPS purified from HW001 or HW002 was used to stimulate murine macrophage RAW264.7 cells, and less TNF-? were released. This study provides a feasible way to produce interesting lipid A species in E. coli.
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Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits.
Cell
PUBLISHED: 01-11-2013
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The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell-lineage-specific transcription factors. Here, we report that repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby derepressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in nonneuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage.
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Formation of nanoscale networks: selectively swelling amphiphilic block copolymers with CO2-expanded liquids.
Nanoscale
PUBLISHED: 01-10-2013
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Polymeric films with nanoscale networks were prepared by selectively swelling an amphiphilic diblock copolymer, polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP), with the CO(2)-expanded liquid (CXL), CO(2)-methanol. The phase behavior of the CO(2)-methanol system was investigated by both theoretical calculation and experiments, revealing that methanol can be expanded by CO(2), forming homogeneous CXL under the experimental conditions. When treated with the CO(2)-methanol system, the spin cast compact PS-b-P4VP film was transformed into a network with interconnected pores, in a pressure range of 12-20 MPa and a temperature range of 45-60 °C. The formation mechanism of the network, involving plasticization of PS and selective swelling of P4VP, was proposed. Because the diblock copolymer diffusion process is controlled by the activated hopping of individual block copolymer chains with the thermodynamic barrier for moving PVP segments from one to another, the formation of the network structures is achieved in a short time scale and shows "thermodynamically restricted" character. Furthermore, the resulting polymer networks were employed as templates, for the preparation of polypyrrole networks, by an electrochemical polymerization process. The prepared porous polypyrrole film was used to fabricate a chemoresistor-type gas sensor which showed high sensitivity towards ammonia.
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Reduction of graphene oxide by thiourea.
J Nanosci Nanotechnol
PUBLISHED: 11-15-2011
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Various compounds with sulfur or diamine structure have been served as efficient reducing agents to convert graphene oxide to reduced graphene oxide. In this work, we used thiourea with both sulfur and diamine structure to synthesize reduced graphene oxide by the general wet chemical reduction method. The effective deoxygenation of graphene oxide was confirmed by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy analysis and Raman spectroscopy. The as-prepared reduced graphene oxide is consisted of few-layered (no more than seven) and over 60% single-layered graphene sheet determined by atomic force microscopy and transmission electron microscopy. It also exhibits good dispersion in organic solvents such as ethanol and N,N-dimethylformamide, which is useful for the further modification of graphene and preparation of novel nanocomposites. This newly found reducing agent is of low toxicity and nonvolatile, which makes the reduction much safer.
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[Effect of recombinant trichosanthin on proliferation of human cevical cancer Caski cells].
Zhongguo Zhong Yao Za Zhi
PUBLISHED: 10-01-2011
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To observe the effects of high expression of recombinant trichosanthin (rTCS) on the cell proliferation and cell cycle of human cervical cancer Caski cells.
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A spin-frustrated cobalt(II) carbonate pyrochlore network.
Acta Crystallogr C
PUBLISHED: 09-17-2011
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The crystal structure of the cobalt(II) carbonate-based compound cobalt(II) dicarbonate trisodium chloride, Co(CO(3))(2)Na(3)Cl, grown from a water-ethanol mixture, exhibits a three-dimensional network of corner-sharing {Co(4)(?(3)-CO(3))(4)} tetrahedral building blocks, in which the Co(II) centres define a pyrochlore lattice and reside in a slightly distorted octahedral Co(O-CO(2))(6) environment. The space outside the hexagonal framework defined by these interlinked groups is occupied by Na(+) and Cl(-) ions. Antiferromagnetic coupling between adjacent Co(II) centres, mediated by carbonate bridges, results in geometric spin frustration which is typical for pyrochlore networks. The Co and Cl atoms reside on the special position 3, one Na atom on position 2 and a carbonate C atom on position 3.
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Biosorptive dehydration of tert-butyl alcohol using a starch-based adsorbent: characterization and thermodynamics.
Bioresour. Technol.
PUBLISHED: 09-16-2011
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Biosorptive dehydration of tert-butyl alcohol (TBA) using a specially formulated compound starch-based adsorbent was investigated. The net retention time and separation factor of TBA and water were obtained using inverse gas chromatography (IGC), which demonstrated the feasibility of this biosorptive separation process, with low temperature propitious to the process. Through orthogonal experimental design, the optimum adsorption condition was determined from different bed depths, bed temperatures and kettle temperatures. Thermal regeneration experiments showed no change in biosorption capacity after five biosorption/regeneration cycles. Field emission scanning electron microscopy (FESEM) and mercury porosimetry were used to investigate the change in surface morphology and microstructure of the biosorbent before and after adsorption. Finally, the thermodynamic parameters ?H(s) and ?G(s) were determined, which indicated that the process occurred by physisorption and was spontaneous and exothermic. The results indicated that the biosorbent can be used as an effective low-cost sorbent to dehydrate TBA.
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Silica nanorattle-doxorubicin-anchored mesenchymal stem cells for tumor-tropic therapy.
ACS Nano
PUBLISHED: 08-24-2011
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Low targeting efficiency is one of the biggest limitations for nanoparticulate drug delivery system-based cancer therapy. In this study, an efficient approach for tumor-targeted drug delivery was developed with mesenchymal stem cells as the targeting vehicle and a silica nanorattle as the drug carrier. A silica nanorattle-doxorubicin drug delivery system was efficiently anchored to mesenchymal stem cells (MSCs) by specific antibody-antigen recognitions at the cytomembrane interface without any cell preconditioning. Up to 1500 nanoparticles were uploaded to each MSC cell with high cell viability and tumor-tropic ability. The intracellular retention time of the silica nanorattle was no less than 48 h, which is sufficient for cell-directed tumor-tropic delivery. In vivo experiments proved that the burdened MSCs can track down the U251 glioma tumor cells more efficiently and deliver doxorubicin with wider distribution and longer retention lifetime in tumor tissues compared with free DOX and silica nanorattle-encapsulated DOX. The increased and prolonged DOX intratumoral distribution further contributed to significantly enhanced tumor-cell apoptosis. This strategy has potential to be developed as a robust and generalizable method for targeted tumor therapy with high efficiency and low systematic toxicity.
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Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum.
Plasmid
PUBLISHED: 08-15-2011
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Bacillus subtilis sacB gene with its 463bp upstream region including its native promoter has been used for marker-free gene deletion in Corynebacterium glutamicum, but the role of this upstream region is not clear. In this study, it was demonstrated that the upstream region of sacB failed to efficiently promote its expression in C. glutamicum, and the native promoter of sacB is weak in C. glutamicum. The expression level of sacB under its native promoter in C. glutamicum is not high enough for cells to confer sucrose sensitivity. Therefore, a new promoter PlacM and a novel vector pDXW-3 were constructed. PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum. The pDXW-3 contains B. subtilissacB with the PlacM fused at the 5-end, a general Escherichia coli replicon oriE for easy cloning, a kanamycin resistance marker for selection, and a multiple unique restriction sites for XhoI, NotI, EagI, SalI, SacI, BamHI, and NheI, respectively. By using pDXW-3, the aceE gene in the chromosome of C. glutamicum was deleted. This sacB-based system should facilitate gene disruption and allelic exchange by homologous recombination in many bacteria.
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Likelihood-based concordance tests for analysis of ascertained twin pair multinomial data.
Stat Med
PUBLISHED: 08-02-2011
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The classic twin model design has a wide application in human genetics. Under the assumption that nongenetic effects are shared to the same degree by monozygotic (MZ) and dizygotic (DZ) twin pairs, a test of the equality of casewise concordances between MZ and DZ twins provides a clue to the influence of genetic and environmental factors on a disease. The casewise concordance is the conditional probability that given that one member of a twin pair is affected, the other is also affected. When disease prevalence is low or cost-effectiveness is considered, collection of twin pairs by ascertainment for performing casewise concordance analysis is required. In this article, by defining an overall casewise concordance parameter, several likelihood-based tests, such as likelihood ratio test LR, score test Score, the usual Wald test Wald and an alternative Wald test WaldA are investigated for a test of the equality of concordances between ascertained MZ and DZ twin pairs under multinomial models. Simulation studies were conducted for data with small sample sizes. The results show that the type I error rates and power of LR and Score are stable only when the overall casewise concordances are not extremely small or large. The Wald has higher power performance in most cases but would slightly inflate type I error rates; the WaldA is the most robust and recommended approach.
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Enhanced performance and mechanism study of microbial electrolysis cells using Fe nanoparticle-decorated anodes.
Appl. Microbiol. Biotechnol.
PUBLISHED: 06-10-2011
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Anode properties are critical for the performance of microbial electrolysis cells (MECs). In the present study, Fe nanoparticle-modified graphite disks were used as anodes to investigate the effects of nanoparticles on the performance of Shewanella oneidensis MR-1 in MECs. Results demonstrated that the average current densities produced with Fe nanoparticle-decorated anodes up to 5.89-fold higher than plain graphite anodes. Whole genome microarray analysis of the gene expression showed that genes encoding biofilm formation were significantly up-regulated as a response to nanoparticle-decorated anodes. Increased expression of genes related to nanowires, flavins, and c-type cytochromes indicates that enhanced mechanisms of electron transfer to the anode may also have contributed to the observed increases in current density. The majority of the remaining differentially expressed genes associated with electron transport and anaerobic metabolism demonstrate a systemic response to increased power loads.
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Efficient Power-Transfer Capability Analysis of the TET System Using the Equivalent Small Parameter Method.
IEEE Trans Biomed Circuits Syst
PUBLISHED: 06-01-2011
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Transcutaneous energy transfer (TET) enables the transfer of power across the skin without direct electrical connection. It is a mechanism for powering implantable devices for the lifetime of a patient. For maximum power transfer, it is essential that TET systems be resonant on both the primary and secondary sides, which requires considerable design effort. Consequently, a strong need exists for an efficient method to aid the design process. This paper presents an analytical technique appropriate to analyze complex TET systems. The systems steady-state solution in closed form with sufficient accuracy is obtained by employing the proposed equivalent small parameter method. It is shown that power-transfer capability can be correctly predicted without tedious iterative simulations or practical measurements. Furthermore, for TET systems utilizing a current-fed push-pull soft switching resonant converter, it is found that the maximum energy transfer does not occur when the primary and secondary resonant tanks are "tuned" to the nominal resonant frequency. An optimal turning point exists, corresponding to the systems maximum power-transfer capability when optimal tuning capacitors are applied.
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Short-term serum-free culture reveals that inhibition of Gsk3? induces the tumor-like growth of mouse embryonic stem cells.
PLoS ONE
PUBLISHED: 05-31-2011
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Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3?. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3?, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3? in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3? governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.
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Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3.
Hum. Mol. Genet.
PUBLISHED: 05-10-2011
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SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan-McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4-9. Isoform-specific Shank3(e4-9) homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3(e4-9) male mice display more severe impairments than females in motor coordination. Shank3(e4-9) mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3(e4-9) mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.
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Phylogenetic analysis of C, M, N, and U genomes and their relationships with Triticum and other related genomes as revealed by LMW-GS genes at Glu-3 loci.
Genome
PUBLISHED: 04-16-2011
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Phylogenetic relationships between the C, U, N, and M genomes of Aegilops species and the genomes of common wheat and other related species were investigated by using three types of low-molecular-weight glutenin subunit (LMW-GS) genes at Glu-3 loci. A total of 20 LMW-GS genes from Aegilops and Triticum species were isolated, including 11 LMW-m type and 9 LMW-i type genes. Particularly, four LMW-m type and three LMW-i type subunits encoded by the genes on the C, N, and U genomes possessed an extra cysteine residue at conserved positions, which could provide useful information for understanding phylogenetic relationships among Aegilops and Triticum genomes. Phylogenetic trees constructed by using either LMW-i or the combination of LMW-m and LMW-s, as well as analysis of all the three types of LMW-GS genes together, demonstrated that the C and U genomes were closely related to the A genome, whereas the N and M genomes were closely related to the D genome. Our results support previous findings that the A genome was derived from Triticum uratu, the B genome was from Aegilops speltoides, and the D genome was from Aegilops tauschii. In addition, phylogenetic relationships among different genomes analysed in this study support the concept that Aegilops is not monophyletic.
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Development of an automatic subsea blowout preventer stack control system using PLC based SCADA.
ISA Trans
PUBLISHED: 02-21-2011
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An extremely reliable remote control system for subsea blowout preventer stack is developed based on the off-the-shelf triple modular redundancy system. To meet a high reliability requirement, various redundancy techniques such as controller redundancy, bus redundancy and network redundancy are used to design the system hardware architecture. The control logic, human-machine interface graphical design and redundant databases are developed by using the off-the-shelf software. A series of experiments were performed in laboratory to test the subsea blowout preventer stack control system. The results showed that the tested subsea blowout preventer functions could be executed successfully. For the faults of programmable logic controllers, discrete input groups and analog input groups, the control system could give correct alarms in the human-machine interface.
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Migration of cells from the yolk sac to hematopoietic tissues after in utero transplantation of early and mid gestation canine fetuses.
Transplantation
PUBLISHED: 02-18-2011
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In utero hematopoietic cell transplantation offers a means of early intervention for the treatment of diseases before birth. Delivery of cells to the yolk sac is a minimally invasive approach that results in low levels of chimerism. However, there is little information on the optimal doses, timing of delivery, and migration of transplanted cells from the yolk sac into the fetus.
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Construction of a hyperbranched supramolecular polymer as a bifunctional antioxidative enzyme model.
Macromol Biosci
PUBLISHED: 01-25-2011
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A HBSP has been designed as a novel bifunctional enzyme model with SOD and GPx activity by host/guest-directed self-assembly of MnTPyP-M-Ad and 6-Te-diCD. The structure of the host/guest complex was elucidated by (1) H NMR spectra, and the HBSP was characterized by SEM, DLS and measurement of catalytic properties. In the bifunctional enzyme model, the Mn(III) porphyrins act as efficient SOD active sites and the tellurol moieties endow GPx activity. The SOD-like activity (IC(50) ) of this new supramolecular catalyst was found to be 1.05?×?10(-7) M, which corresponds to 2.82% of the activity of the native SOD enzyme. Besides this, the hyperbranched supramolecular polymer also shows a higher GPx activity (?(0?) =?21.7?×?10(-6) M?·?min(-1) ) than other supramolecular enzyme models.
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Phylogenetic relationship of a new class of LMW-GS genes in the M genome of Aegilops comosa.
Theor. Appl. Genet.
PUBLISHED: 01-14-2011
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A new class of low molecular weight glutenin subunit (LMW-GS) genes was isolated and characterized from Aegilops comosa (2n = 2x = 14, MM). Although their DNA structure displayed high similarity to LMW-i type genes, there are some key differences. The deduced amino acid sequences of their mature proteins showed that the first amino acid residue of each gene was leucine and therefore they were designated as LMW-l type subunits. An extra cysteine residue was present in the signal peptide and the first cysteine residue of mature proteins located at the end of repetitive domain. Additionally, a long insertion of 10-22 residues (LGQQPQ(5-17)) occurred in the end of the C-terminal II. Comparative analysis demonstrated that LMW-l type glutenin genes possessed a great number of single-nucleotide polymorphisms and insertions/deletions. A new classification system was proposed according to the gene structure and phylogenetic analysis. In this new system, LMW-GS is classified into two major classes, LMW-M and LMW-I, with each including two subclasses. The former included LMW-m and LMW-s types while the latter contained LMW-l and LMW-i types. Analysis of their evolutionary origin showed that the LMW-l genes diverged from the group 2 of LMW-m type genes at about 12-14 million years ago (MYA) while LMW-i type evolved from LMW-l type at approximately 8-12 MYA. The LMW-s type was a variant form of group 1 of LMW-m type and their divergence occurred about 4-6 MYA. In addition to homologous recombination, non-homologous illegitimate recombination could be an important molecular mechanism for the origin and evolution of LMW-GS gene family. The secondary structure prediction suggested that the novel LMW-l type subunits, such as AcLMW-L1 and AcLMW-L2, may have positive effects on dough properties.
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Emx1-expressing neural stem cells in the subventricular zone give rise to new interneurons in the ischemic injured striatum.
Eur. J. Neurosci.
PUBLISHED: 01-11-2011
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Neural stem cells from different regions within the subventricular zone (SVZ) are able to produce several different subtypes of interneurons in the olfactory bulb throughout life. Previous studies have shown that ischemic stroke induces the production of new neurons in the damaged striatum from the SVZ. However, the origins and genetic profiles of these newborn neurons remain largely unknown as SVZ neural stem cells are heterogeneous. In the present study, using a mouse model of perinatal hypoxic-ischemic (H/I) brain injury combined with BrdU labeling methods, we found that, as in rat brains, virtually all newborn neuroblasts that migrate from the SVZ into the ischemic injured striatum exclusively express the transcription factor Sp8. Furthermore, although newborn neuroblasts are plentiful in the damaged striatum, only a few can differentiate into calretinin-expressing (CR+) interneurons that continuously express Sp8. Genetic fate mapping reveals that newly born CR+ interneurons are generated from Emx1-expressing neural stem cells in the dorsal-lateral SVZ. These results suggest that the fate of the Emx1-expressing lineage in the ischemic damaged striatum is restricted. However, when Sp8 was conditionally inactivated in the Emx1-lineage cells, Pax6 was ectopically expressed by a subpopulation of Emx1-derived CR+ cells in the normal and damaged striatum. Interestingly, these cells possessed large cell bodies and long processes. This work identifies the origin of the newly born CR+ interneurons in the damaged striatum after ischemic brain injury.
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Comparative proteome analysis of glutenin synthesis and accumulation in developing grains between superior and poor quality bread wheat cultivars.
J. Sci. Food Agric.
PUBLISHED: 01-02-2011
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Wheat glutenins are the major determinants of wheat quality. In this study, grains at the development stage from three wheat cultivars (Jimai 20, Jin 411 and Zhoumai 16) with different bread-making quality were harvested based on thermal times from 150 °C(d) to 750 °C(d) , and were used to investigate glutenin accumulation patterns and their relationships with wheat quality.
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Electroacupuncture protected pyramidal cells in hippocampal CA1 region of vascular dementia rats by inhibiting the expression of p53 and Noxa.
CNS Neurosci Ther
PUBLISHED: 10-15-2010
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Clinically electroacupuncture (EA) is proved an effective therapy for vascular dementia (VD), but their mechanisms remain uncertain. The aim of this study was to determine whether EA protects pyramidal cells from apoptosis in hippocampus of a VD rat model by inhibiting the expression of p53 and Noxa.
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Layer-by-layer assembly of poly(L-glutamic acid)/chitosan microcapsules for high loading and sustained release of 5-fluorouracil.
Eur J Pharm Biopharm
PUBLISHED: 10-08-2010
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Hollow polyelectrolyte microcapsules based on poly(l-glutamic acid) (PLGA) and chitosan (CS) with opposite charges were fabricated by layer-by-layer (LbL) assembly technique using melamine formaldehyde (MF) microparticles as sacrificial templates. The LbL assembly of polyelectrolytes and the resultant PLGA/CS microcapsules were characterized. A hydrophilic anticancer drug, 5-fluorouracil (5-FU), was chosen to investigate the loading and release properties of the microcapsules. The PLGA/CS microcapsules show high loading capacity of 5-FU under conditions of high drug concentration and salt adding. The high loading can be ascribed to spontaneous deposition of 5-FU induced by hydrogen bonding between 5-FU and PLGA/CS microcapsules. The PLGA/CS microcapsules show sustained release behavior. The release rate of 5-FU drastically slows down after loading in PLGA/CS microcapsules. The 5-FU release from PLGA/CS microcapsules can be best described using Ritger-Peppas or Baker-Londale models, indicating the diffusion mechanism of 5-FU release from the PLGA/CS microcapsules. In vitro cytotoxicity evaluation by the MTT assay shows good cell viability over the entire concentration range of PLGA/CS microcapsules. Therefore, the novel PLGA/CS microcapsules are expected to find application in drug delivery systems because of the properties of biodegradability, high loading, sustained release and cell compatibility.
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Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.
PLoS ONE
PUBLISHED: 08-24-2010
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Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.
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Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3.
PLoS ONE
PUBLISHED: 07-19-2010
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Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11-q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11-q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m-/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m-/p+), but not paternal (m+/p-), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.
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