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Find video protocols related to scientific articles indexed in Pubmed.
The specification and global reprogramming of histone epigenetic marks during gamete formation and early embryo development in C. elegans.
PLoS Genet.
PUBLISHED: 10-01-2014
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In addition to the DNA contributed by sperm and oocytes, embryos receive parent-specific epigenetic information that can include histone variants, histone post-translational modifications (PTMs), and DNA methylation. However, a global view of how such marks are erased or retained during gamete formation and reprogrammed after fertilization is lacking. To focus on features conveyed by histones, we conducted a large-scale proteomic identification of histone variants and PTMs in sperm and mixed-stage embryo chromatin from C. elegans, a species that lacks conserved DNA methylation pathways. The fate of these histone marks was then tracked using immunostaining. Proteomic analysis found that sperm harbor ?2.4 fold lower levels of histone PTMs than embryos and revealed differences in classes of PTMs between sperm and embryos. Sperm chromatin repackaging involves the incorporation of the sperm-specific histone H2A variant HTAS-1, a widespread erasure of histone acetylation, and the retention of histone methylation at sites that mark the transcriptional history of chromatin domains during spermatogenesis. After fertilization, we show HTAS-1 and 6 histone PTM marks distinguish sperm and oocyte chromatin in the new embryo and characterize distinct paternal and maternal histone remodeling events during the oocyte-to-embryo transition. These include the exchange of histone H2A that is marked by ubiquitination, retention of HTAS-1, removal of the H2A variant HTZ-1, and differential reprogramming of histone PTMs. This work identifies novel and conserved features of paternal chromatin that are specified during spermatogenesis and processed in the embryo. Furthermore, our results show that different species, even those with diverged DNA packaging and imprinting strategies, use conserved histone modification and removal mechanisms to reprogram epigenetic information.
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Large-scale organic single-crystal thin films and transistor arrays via the evaporation-controlled fluidic channel method.
ACS Appl Mater Interfaces
PUBLISHED: 05-07-2014
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We report a facile and versatile approach for fabricating large-area organic thin film transistor (OTFTs) arrays via a fluidic channel method. Evaporation-controlled fluidic channel-containing organic semiconductors easily produce large-area organic single-crystalline thin films in a quite uniform manner. The unidirectional movement of the meniscus and the subsequent film growth via solvent evaporation inside the fluidic channel correspond to the simulation based on the finite element method. Utilizing this fluidic channel method, we fabricated high-performance 6,13-bis(triisopropylsilylethynyl)pentacene OTFT arrays with average and maximal mobilities of 0.71 and 2.18 cm(2) V(-1) s(-1), respectively, while exhibiting current on:off ratios of >1 × 10(6). We claim that this scalable fluidic channel method offers a competitive way to fabricate high-performance and large-area organic semiconductor devices for a variety of applications.
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Census 2: isobaric labeling data analysis.
Bioinformatics
PUBLISHED: 03-28-2014
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We introduce Census 2, an update of a mass spectrometry data analysis tool for peptide/protein quantification. New features for analysis of isobaric labeling, such as Tandem Mass Tag (TMT) or Isobaric Tags for Relative and Absolute Quantification (iTRAQ), have been added in this version, including a reporter ion impurity correction, a reporter ion intensity threshold filter and an option for weighted normalization to correct mixing errors. TMT/iTRAQ analysis can be performed on experiments using HCD (High Energy Collision Dissociation) only, CID (Collision Induced Dissociation)/HCD (High Energy Collision Dissociation) dual scans or HCD triple-stage mass spectrometry data. To improve measurement accuracy, we implemented weighted normalization, multiple tandem spectral approach, impurity correction and dynamic intensity threshold features.
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R-CHOP chemoimmunotherapy followed by autologous transplantation for the treatment of diffuse large B-cell lymphoma.
Blood Res
PUBLISHED: 03-01-2014
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We investigated factors that influence outcomes in diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab combined with the CHOP regimen (R-CHOP) followed by upfront autologous stem cell transplantation (Auto-SCT).
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Interference-free proteome quantification with MS/MS-based isobaric isotopologue detection.
J. Proteome Res.
PUBLISHED: 02-11-2014
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Chemical labeling of peptides prior to shotgun proteomics allows relative quantification of proteins in biological samples independent of sample origin. Current strategies utilize isobaric labels that fragment into reporter ions. However, quantification of reporter ions results in distorted ratio measurements due to contaminating peptides that are co-selected in the same precursor isolation window. Here, we show that quantitation of isobaric peptide fragment isotopologues in tandem mass spectra reduces precursor interference. The method is based on the relative quantitation of isobaric isotopologues of dimethylated peptide fragments in tandem mass spectra following higher energy collisional dissociation (HCD). The approach enables precise quantification of a proteome down to single spectra per protein and quantifies >90% of proteins in a MudPIT experiment and accurately measures proteins in a model cell line for cystic fibrosis.
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Effects of annealing conditions on the dielectric properties of solution-processed Al2O3 layers for indium-zinc-tin-oxide thin-film transistors.
J Nanosci Nanotechnol
PUBLISHED: 11-20-2013
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In this paper, the effects of annealing conditions on the dielectric properties of solution-processed aluminum oxide (Al2O3) layers for indium-zinc-tin-oxide (IZTO) thin-film transistors (TFTs) have been investigated. The dielectric properties of Al2O3 layers such as leakage current density and dielectric strength were largely affected by their annealing conditions. In particular, oxygen partial pressure in rapid thermal annealing, and the temperature profile of hot plate annealing had profound effects on the dielectric properties. From a refractive index analysis, the enhanced dielectric properties of Al2O3 gate dielectrics can be attributed to higher film density depending on the annealing conditions. With the low-temperature-annealed Al2O3 gate dielectric at 350 degrees C, solution-processed IZTO TFTs with a field-effect mobility of approximately 2.2 cm2/Vs were successfully fabricated.
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Enhanced bias stability of solution-processed zinc-tin-oxide thin film transistors using self-assembled monolayer as a selective channel passivation.
J Nanosci Nanotechnol
PUBLISHED: 11-20-2013
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The enhanced positive bias stability of amorphous zinc-tin-oxide thin-film transistors (a-ZTO TFTs) were obtained by applying self-assembled monolayer (SAM) as a selective passivation layer on the metal-oxide back channel area. The a-ZTO TFTs with passivation layers such as poly(methyl methacylate) (PMMA), SAM, and SAM/PMMA were fabricated by simple solution methods. After deposition of the passivation layers, the electrical characteristics of a-ZTO TFTs have not been changed and the threshold voltage shift (deltaV(th)) under gate-bias stress for around 10(4) seconds was improved. The deltaV(th) of the devices with PMMA, SAM, and SAM/PMMA dual layer were 3.79 V, 3.2 V, and 2.17 V, respectively.
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Morphological evolution of carbon nanofibers encapsulating SnCo alloys and its effect on growth of the solid electrolyte interphase layer.
ACS Nano
PUBLISHED: 07-22-2013
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Two distinctive one-dimensional (1-D) carbon nanofibers (CNFs) encapsulating irregularly and homogeneously segregated SnCo nanoparticles were synthesized via electrospinning of polyvinylpyrrolidone (PVP) and polyacrylonitrile (PAN) polymers containing Sn-Co acetate precursors and subsequent calcination in reducing atmosphere. CNFs synthesized with PVP, which undergoes structural degradation of the polymer during carbonization processes, exhibited irregular segregation of heterogeneous alloy particles composed of SnCo, Co3Sn2, and SnO with a size distribution of 30-100 nm. Large and exposed multiphase SnCo particles in PVP-driven amorphous CNFs (SnCo/PVP-CNFs) kept decomposing liquid electrolyte and were partly detached from CNFs during cycling, leading to a capacity fading at the earlier cycles. The closer study of solid electrolyte interphase (SEI) layers formed on the CNFs reveals that the gradual growth of fiber radius due to continuous increment of SEI layer thickness led to capacity fading. In contrast, SnCo particles in PAN-driven CNFs (SnCo/PAN-CNFs) showed dramatically reduced crystallite sizes (<10 nm) of single phase SnCo nanoparticles which were entirely embedded in dense, semicrystalline, and highly conducting 1-D carbon matrix. The growth of SEI layer was limited and saturated during cycling. As a result, SnCo/PAN-CNFs showed much improved cyclability (97.9% capacity retention) and lower SEI layer thickness (86 nm) after 100 cycles compared to SnCo/PVP-CNFs (capacity retention, 71.9%; SEI layer thickness, 593 nm). This work verifies that the thermal behavior of carbon precursor is highly responsible for the growth mechanism of SEI layer accompanied with particles detachment and cyclability of alloy particle embedded CNFs.
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Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast.
J. Proteome Res.
PUBLISHED: 04-09-2013
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A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near-complete yeast proteome from a whole cell tryptic digest. This modified online two-dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient. A total of 4269 protein identifications were made from 4189 distinguishable protein families from yeast during log phase growth. The "Micro" MudPIT separation performed as well as a standard MudPIT separation in 40% less gradient time. The majority of the yeast proteome can now be routinely covered in less than a days time with high reproducibility and sensitivity. The newly devised separation method was used to detect changes in protein expression during cellular quiescence in yeast. An enrichment in the GO annotations "oxidation reduction", "catabolic processing" and "cellular response to oxidative stress" was seen in the quiescent cellular fraction, consistent with their long-lived stress resistant phenotypes. Heterogeneity was observed in the stationary phase fraction with a less dense cell population showing reductions in KEGG pathway categories of "Ribosome" and "Proteasome", further defining the complex nature of yeast populations present during stationary phase growth. In total, 4488 distinguishable protein families were identified in all cellular conditions tested.
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Identification of long-lived proteins reveals exceptional stability of essential cellular structures.
Cell
PUBLISHED: 03-14-2013
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Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here, we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cells life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. PAPERCLIP:
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The Li-ion rechargeable battery: a perspective.
J. Am. Chem. Soc.
PUBLISHED: 01-18-2013
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Each cell of a battery stores electrical energy as chemical energy in two electrodes, a reductant (anode) and an oxidant (cathode), separated by an electrolyte that transfers the ionic component of the chemical reaction inside the cell and forces the electronic component outside the battery. The output on discharge is an external electronic current I at a voltage V for a time ?t. The chemical reaction of a rechargeable battery must be reversible on the application of a charging I and V. Critical parameters of a rechargeable battery are safety, density of energy that can be stored at a specific power input and retrieved at a specific power output, cycle and shelf life, storage efficiency, and cost of fabrication. Conventional ambient-temperature rechargeable batteries have solid electrodes and a liquid electrolyte. The positive electrode (cathode) consists of a host framework into which the mobile (working) cation is inserted reversibly over a finite solid-solution range. The solid-solution range, which is reduced at higher current by the rate of transfer of the working ion across electrode/electrolyte interfaces and within a host, limits the amount of charge per electrode formula unit that can be transferred over the time ?t = ?t(I). Moreover, the difference between energies of the LUMO and the HOMO of the electrolyte, i.e., electrolyte window, determines the maximum voltage for a long shelf and cycle life. The maximum stable voltage with an aqueous electrolyte is 1.5 V; the Li-ion rechargeable battery uses an organic electrolyte with a larger window, which increase the density of stored energy for a given ?t. Anode or cathode electrochemical potentials outside the electrolyte window can increase V, but they require formation of a passivating surface layer that must be permeable to Li(+) and capable of adapting rapidly to the changing electrode surface area as the electrode changes volume during cycling. A passivating surface layer adds to the impedance of the Li(+) transfer across the electrode/electrolyte interface and lowers the cycle life of a battery cell. Moreover, formation of a passivation layer on the anode robs Li from the cathode irreversibly on an initial charge, further lowering the reversible ?t. These problems plus the cost of quality control of manufacturing plague development of Li-ion rechargeable batteries that can compete with the internal combustion engine for powering electric cars and that can provide the needed low-cost storage of electrical energy generated by renewable wind and/or solar energy. Chemists are contributing to incremental improvements of the conventional strategy by investigating and controlling electrode passivation layers, improving the rate of Li(+) transfer across electrode/electrolyte interfaces, identifying electrolytes with larger windows while retaining a Li(+) conductivity ?(Li) > 10(-3) S cm(-1), synthesizing electrode morphologies that reduce the size of the active particles while pinning them on current collectors of large surface area accessible by the electrolyte, lowering the cost of cell fabrication, designing displacement-reaction anodes of higher capacity that allow a safe, fast charge, and designing alternative cathode hosts. However, new strategies are needed for batteries that go beyond powering hand-held devices, such as using electrode hosts with two-electron redox centers; replacing the cathode hosts by materials that undergo displacement reactions (e.g. sulfur) by liquid cathodes that may contain flow-through redox molecules, or by catalysts for air cathodes; and developing a Li(+) solid electrolyte separator membrane that allows an organic and aqueous liquid electrolyte on the anode and cathode sides, respectively. Opportunities exist for the chemist to bring together oxide and polymer or graphene chemistry in imaginative morphologies.
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Does Li4Ti5O12 need carbon in lithium ion batteries? Carbon-free electrode with exceptionally high electrode capacity.
Chem. Commun. (Camb.)
PUBLISHED: 11-29-2011
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A carbon-free Li(4)Ti(5)O(12) electrode has shown excellent electrochemical performance without any effort to enhance the electrical conductivity. Partial reduction of Ti(4+) and a metallic Li(7)Ti(5)O(12) phase are suggested to be possible origins of the exceptional behavior.
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Low-dose combinations of LBH589 and TRAIL can overcome TRAIL-resistance in colon cancer cell lines.
Anticancer Res.
PUBLISHED: 10-04-2011
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Despite the considerable advances in the treatment of colorectal cancer, substantial changes in treatment strategies are required to overcome the problems of drug resistance and toxicity.
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Controlled deposition of a high-performance small-molecule organic single-crystal transistor array by direct ink-jet printing.
Adv. Mater. Weinheim
PUBLISHED: 08-08-2011
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Ink-jet printed small-molecule organic single-crystal transistors are realized by using selective surface energy modification, precise control of volume density of ink droplets on spatially patterned areas, and a co-solvent system to control solvent evaporation properties. The single-crystal formation in bottom-contact-structured transistors via direct printing is expected to permit high-density array fabrication in large-area electronics.
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Modulation of retinal wound healing by systemically administered bone marrow-derived mesenchymal stem cells.
Korean J Ophthalmol
PUBLISHED: 07-22-2011
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To evaluate whether systemically injected bone marrow-derived mesenchymal stem cells (MSCs) can be incorporated into neuroretinal tissues and play an important role in retinal wound healing in the laser-induced retinal trauma model.
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Esculetin inhibits cell proliferation through the Ras/ERK1/2 pathway in human colon cancer cells.
Oncol. Rep.
PUBLISHED: 04-07-2011
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Esculetin, a phenolic compound, has been shown to inhibit the growth of colon tumors in animal studies. However, the roles of signaling pathways and cell cycle regulation in the esculetin-induced inhibition of cancer cell growth, remain to be elucidated. The present study suggests a novel mechanism for the Ras/ERK1/2 pathway in esculetin-treated human colon cancer HCT116 cells. The treatment of cells with esculetin resulted in significant growth inhibition and G1 phase cell cycle arrest, which led to the down-regulation of cyclin and cyclin-dependent kinase (CDK) expressions. This G1 phase cell cycle arrest was associated with the up-regulation of p27KIP expression. In addition, ERK1/2 was activated by esculetin. The pre-treatment of cells with the MEK1/2-specific inhibitor, PD98059, blocked the p27KIP expression induced by esculetin. Blockage of the ERK1/2 function consistently prevented the inhibition of cell proliferation and decreased G1 phase cell cycle protein levels. Furthermore, Ras activation was increased by the esculetin treatment. Transient transfection of the dominant negative Ras (RasN17) mutant gene abolished both the ERK1/2 activity and p27KIP expression induced by esculetin. Finally, the overexpression of RasN17 suppressed the esculetin-induced reduction in cell proliferation and cell cycle proteins. In conclusion, these results indicate that the Ras/ERK1/2 pathway is mediated by the p27KIP1 induction, leading to a reduction in cyclin/CDK complexes in the esculetin-induced inhibition of colon cancer cell growth. Overall, these findings indicate that the molecular action of esculetin has therapeutic potential for the treatment of colon malignancies.
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Differential proteomic analysis of mammalian tissues using SILAM.
PLoS ONE
PUBLISHED: 01-20-2011
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Differential expression of proteins between tissues underlies organ-specific functions. Under certain pathological conditions, this may also lead to tissue vulnerability. Furthermore, post-translational modifications exist between different cell types and pathological conditions. We employed SILAM (Stable Isotope Labeling in Mammals) combined with mass spectrometry to quantify the proteome between mammalian tissues. Using (15)N labeled rat tissue, we quantified 3742 phosphorylated peptides in nuclear extracts from liver and brain tissue. Analysis of the phosphorylation sites revealed tissue specific kinase motifs. Although these tissues are quite different in their composition and function, more than 500 protein identifications were common to both tissues. Specifically, we identified an up-regulation in the brain of the phosphoprotein, ZFHX1B, in which a genetic deletion causes the neurological disorder Mowat-Wilson syndrome. Finally, pathway analysis revealed distinct nuclear pathways enriched in each tissue. Our findings provide a valuable resource as a starting point for further understanding of tissue specific gene regulation and demonstrate SILAM as a useful strategy for the differential proteomic analysis of mammalian tissues.
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[Clinical characteristics and ALB gene mutation analysis of Korean patients with bisalbuminemia].
Korean J Lab Med
PUBLISHED: 07-07-2010
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Bisalbuminemia is a hereditary or an acquired condition characterized by the presence of 2 albumin variants with different mobilities on serum protein electrophoresis (SPE). The clinical significance of bisalbuminemia has not been clearly established. However, some regions of the albumin variant may affect the biochemical analysis of biomolecules such as steroid or thyroid hormones by altering their albumin-binding affinities. In this study, we analyzed the clinical manifestations, genetic variations, and the albumin-binding characteristics in Korean patients with bisalbuminemia.
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Suppression of O2 evolution from oxide cathode for lithium-ion batteries: VO(x)-impregnated 0.5Li2MnO3-0.5LiNi(0.4)Co(0.2)Mn(0.4)O2 cathode.
Chem. Commun. (Camb.)
PUBLISHED: 05-10-2010
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A VO(x)-impregnated oxide cathode for lithium ion batteries exhibits a substantial drop in oxygen evolution during high voltage operation. An electrolyte was found to catalyze the gas evolution, and the VO(x) layer could protect the cathode oxide surface from the electrolyte and stabilize the surface oxide ions during their electrochemical oxidation.
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Quantitative proteomics approach for identifying protein-drug interactions in complex mixtures using protein stability measurements.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-03-2010
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Knowledge about the protein targets of therapeutic agents is critical for understanding drug mode of action. Described here is a mass spectrometry-based proteomics method for identifying the protein target(s) of drug molecules that is potentially applicable to any drug compound. The method, which involves making thermodynamic measurements of protein-folding reactions in complex biological mixtures to detect protein-drug interactions, is demonstrated in an experiment to identify yeast protein targets of the immunosuppressive drug, cyclosporin A (CsA). Two of the ten protein targets identified in this proof of principle work were cyclophilin A and UDP-glucose-4-epimerase, both of which are known to interact with CsA, the former through a direct binding event (K(d) approximately 70 nM) and the latter through an indirect binding event. These two previously known protein targets validate the methodology and its ability to detect both the on- and off-target effects of protein-drug interactions. The other eight protein targets discovered here, which include several proteins involved in glucose metabolism, create a new framework in which to investigate the molecular basis of CsA side effects in humans.
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Tailoring the electrochemical properties of composite electrodes by introducing surface redox-active oxide film: VO(x)-impregnated LiFePO4 electrode.
Chem. Commun. (Camb.)
PUBLISHED: 03-12-2010
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When the electrode is specifically designed by impregnation with an electronic and ionic mixed-conductor, improvement in the electrochemical properties was observed. The presence of an active redox center may contribute to the enhancement of the surface electronic/ionic transport properties by enhancing electrical connection and isotropic Li(+) ion transport.
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Census for proteome quantification.
Curr Protoc Bioinformatics
PUBLISHED: 03-06-2010
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Quantitative analysis has become increasingly important in the proteomics field; however, the large amount of mass spectrometric data and the different types of quantitative strategies make data analysis ever challenging. Here we describe a quantitative software tool called Census to analyze high-throughput mass spectrometry data from shotgun proteomics experiments in an efficient way. Census is capable of analyzing various stable isotope labeling experiments (using, e.g., (15)N, (18)O, SILAC, iTRAQ, TMT) in addition to labeling-free experiments. With high-resolution data, Census increases the quantitative accuracy by minimizing the contributions of interfering peaks and chemical noise with a small accuracy tolerance for each isotope peak. Census provides various scoring algorithms including least-squares correlation, weight average, singleton peptide detection with discriminant analysis, and probability score for each peptide. Furthermore, Census has built-in multiple statistical filters to maintain robust quality control on quantitative results.
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Identification of novel substrates for the serine protease HTRA1 in the human RPE secretome.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 03-05-2010
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PURPOSE. To define the role of the serine protease HTRA1 in age-related macular degeneration (AMD) by examining its expression level and identifying its potential substrates in the context of primary RPE cell extracellular milieu. METHODS. Primary RPE cell cultures were established from human donor eyes and screened for CFH, ARMS2, and HTRA1 risk genotypes by using an allele-discrimination assay. HTRA1 expression in genotyped RPE cells was determined by using real-time PCR and quantitative proteomics. Potential HTRA1 substrates were identified by incubating RPE-conditioned medium with or without human recombinant HTRA1. Selectively cleaved proteins were quantified by using the differential stable isotope labeling by amino acids in cell culture (SILAC) strategy. RESULTS. HTRA1 mRNA levels were threefold higher in primary RPE cells homozygous for the HTRA1 promoter risk allele than in RPE cells with the wild-type allele, which translated into a twofold increase in HTRA1 secretion by RPE cells with the risk genotype. A total of 196 extracellular proteins were identified in the RPE secretome, and only 8 were found to be selectively cleaved by the human recombinant HTRA1. These include fibromodulin with 90% cleavage, clusterin (50%), ADAM9 (54%), vitronectin (54%), and alpha2-macroglobulin (55%), as well as some cell surface proteins including talin-1 (21%), fascin (40%), and chloride intracellular channel protein 1 (51%). CONCLUSIONS. Recombinant HTRA1 cleaves RPE-secreted proteins involved in regulation of the complement pathway (clusterin, vitronectin, and fibromodulin) and of amyloid deposition (clusterin, alpha2-macroglobulin, and ADAM9). These findings suggest a link between HTRA1, complement regulation, and amyloid deposition in AMD pathogenesis.
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Long-term survival in a patient with acute promyelocytic leukemia with isolated meningeal relapse.
Korean J Hematol
PUBLISHED: 03-01-2010
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The meningeal involvement is rare in acute promyelocytic leukemia. We experienced a 39-year-old woman who achieved complete remission with all-trans retinoic acid, idarubicin and cytarabine therapy. Several months later, she complained of non-specific headache. Her complete blood cell count was normal, but magnetic resonance image of brain revealed focal meningeal enhancement and cerebrospinal fluid showed leukemic promyelocytes and PML/RARA rearrangement. Bone marrow study showed hematologic, cytogenetic and molecular remission. She was treated with intrathecal and systemic chemotherapy and whole brain radiotherapy. The patient has survived for 68 months since the last systemic chemotherapy.
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Role of PI3K on the regulation of BMP2-induced beta-Catenin activation in human bone marrow stem cells.
Bone
PUBLISHED: 01-08-2010
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Bone morphogenetic protein 2 (BMP2), a very potent bone-inducing agent, promotes the differentiation of bone marrow stem cells (BMSCs) to osteoblasts. However, the potency of BMP2 action is variable and its perturbed dynamic signaling pathways in human BMSCs has not been fully elucidated. In this study, we used a combination of stable isotope labeling by amino acids during cell culture (SILAC) and liquid-chromatography electrospray ionization mass spectrometry (LC-ESI-MS/MS) technology to reveal the BMP2 action in BMSC. In this quantitative proteomic analysis, 414 of 449 proteins were successfully quantified with 79.2% peptide quantification efficiency. Interestingly, beta-Catenin was identified in BMP2-stimulated heavy isotope-labeled cells, and further analysis confirmed that BMP2 increased beta-Catenin mRNA and protein levels. The increment effects of BMP2 on the beta-Catenin expression levels and its translocation to nucleus were diminished by blocking the PI3K signal pathway. In addition, BMP2-induced beta-Catenin activity and ALP activity were blocked by PI3K inhibition. Thus, our quantitative proteomics analysis and further biochemical investigations showed that BMP2 modulates beta-Catenin signaling via PI3K pathway and that this pathway plays roles in BMP2-induced osteoblast differentiation of hBMSCs.
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Inhibitory effects of the ethanol extract of Gleditsia sinensis thorns on human colon cancer HCT116 cells in vitro and in vivo.
Oncol. Rep.
PUBLISHED: 11-04-2009
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The thorns of Gleditsia sinensis have traditionally been used in the treatment of several diseases, which includes their use as anti-tumor agents, but there has been no scientific evidence of this anti-tumor effect. However, the present study has identified a novel mechanism for the anti-tumor effect of Gleditsia sinensis thorns in the treatment of colon cancer. Treatment with the ethanol extract of Gleditsia sinensis thorns (EEGS) resulted in significant growth inhibition together with G2/M-phase cell cycle arrest at a dose of 600 microg/ml (IC50) in HCT116 cells. In addition, treatment with EEGS induced p27 expression and down-regulated expression of cyclins and cyclin-dependent kinases. Moreover, EEGS treatment induced phosphorylation of extracellular signal-regulated kinases (ERK), p38 MAP kinase and JNK (c-Jun N-terminal kinases). Among the pathways examined, only PD98059 (ERK-specific inhibitor) abolished EEGS-dependent p27 expression. Similarly, suppression of ERK function reversed EEGS-mediated cell proliferation inhibition and decreased cell cycle proteins. In addition, tumor necrosis factor-alpha (TNF-alpha)-induced matrix metalloproteinase-9 (MMP-9) expression was inhibited by EEGS treatment via decreased transcriptional activity of both activator protein-1 (AP-1) and nuclear factor-kappaB. Finally, EEGS treatment significantly reduced tumor sizes in HCT116 cell-xenografted tumor tissues, which was associated with the changed levels of ERK phosphorylation, p27 and MMP-9 expression. Overall, these results have identified a novel molecular mechanism for EEGS in the treatment of colon cancer and might provide a theoretical basis for the potential therapeutic use of EEGS in the treatment of malignancies.
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Shotgun protein identification and quantification by mass spectrometry in neuroproteomics.
Methods Mol. Biol.
PUBLISHED: 08-18-2009
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Shotgun proteomics is based on identification and quantification of peptides from digested proteins using tandem mass spectrometry. In this chapter, we discuss computational methods to analyze tandem mass spectra of peptides, including database searching, de novo peptide sequencing, hybrid approaches, library searching, and unrestricted modification search. A special focus is given to database searching programs, since they are the most widely used. The process of inferring proteins from identified peptides is then discussed. We also provide description of key steps in the quantitative analysis of mass spectrometry proteomics data. These methods are valuable tools for discovery and hypothesis-driven analyses in neuroproteomics.
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Two-stage double-technique hybrid (TSDTH) identification strategy for the analysis of BMP2-induced transdifferentiation of premyoblast C2C12 cells to osteoblast.
J. Proteome Res.
PUBLISHED: 08-07-2009
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Transdifferentiation offers new opportunities in the area of cell replacement therapy; however, the molecular mechanism by which transdifferentiation occurs is not fully understood. Our understanding about the sophisticated regulations of transdifferentiation is limited yet since their comprehensive proteome regulations have not been fully elucidated. Studies on bone morphogenic protein-2 (BMP2)-induced transdifferentiation of murine C2C12 cells, a myogenic lineage committed premyoblast, to osteogenic cells can provide a full picture of the dynamic events that occur at the level of protein activity and/or expression. Here, we investigated the overall dynamic regulatory proteome associated with BMP2-induced osteoblast transdifferentiation in premyoblast C2C12 cells using a novel Two-Stage Double-Technique Hybrid (TSDTH) strategy for proteomic analysis. Here, we took the approach of a TSDTH involving phosphoproteomic analysis after a short-term treatment (stage one, 30 min) and a long-term treatment (stage two, 3 days); SILAC (Stable isotope labeling with amino acids in cell culture)-proteomics was used to map the proteins. In these experiments, a total of 1321 potential phosphoproteins were identified in stage one analysis and 433 proteins were quantified in stage two analysis. Among them, 374 BMP2-specific phosphoproteins and 54 up- or down-regulated proteins were selected. In first stage analysis, several deubiquitination enzymes including Uch-l3 as well as ubiquitination related proteins were newly identified, and its inhibitor reduced the stability of phosphorylated Smad1, and the BMP2-induced ALP levels of C2C12 cells were detected. In second stage analysis, Thrombospondin1 was identified as the highest up-regulated protein by BMP2-long time stimulation and this was confirmed with immunoblot analysis. Furthermore, pathway enrichment and network analyses revealed that insulin-like growth factor (IGF) and calcium signaling pathways as well as TGFbeta/BMP signaling proteins are found to be potentially involved in the early and long-term actions of BMP2. Collectively, our TSDTH is a useful simple strategy to obtain comprehensive molecular mechanism of cellular processes such as transdifferentiation.
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Nature of insulating-phase transition and degradation of structure and electrochemical reactivity in an olivine-structured material, LiFePO4.
Inorg Chem
PUBLISHED: 08-07-2009
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Synthesis time using microwave irradiation was varied to elucidate the electrochemical degradation mechanism of LiFePO(4) related to the evolution of Fe(2)P. When the amount of Fe(2)P was above a critical level, LiFePO(4) tended to change into an insulating phase, Li(4)P(2)O(7). The correlation between structural analysis and electrochemical analysis attributed the initial degradation of LiFePO(4) to the low electronic conductivity of Li(4)P(2)O(7), whereas the deficiency of P and O evolved by Li(4)P(2)O(7) resulted in the cyclic degradation of LiFePO(4). This kind of correlation between structure and electrochemical performance in intercalation materials will significantly contribute to an explanation of their degradation mechanism for their application.
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Shotgun protein identification and quantification by mass spectrometry.
Methods Mol. Biol.
PUBLISHED: 06-23-2009
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Shotgun proteomics is based on identification and quantification of peptides from digested proteins using tandem mass spectrometry. In this chapter, we discuss computational methods to analyze tandem mass spectra of peptides, including database searching, de novo peptide sequencing, hybrid approaches, library searching, and unrestricted modification search. A special focus is given to database searching programs since they are most widely used. The process of inferring proteins from identified peptides is then discussed. We also provide description of key steps in the quantitative analysis of mass spectrometry proteomics data.
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Inhibition of proliferation and migration by piceatannol in vascular smooth muscle cells.
Toxicol In Vitro
PUBLISHED: 04-16-2009
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Piceatannol (3,5,3,4-tetrahydroxy- trans-stilbene), a resveratrol analogue, is a polyphenol present in the skins of grapes and in wine and other foods. The present study aimed to investigate for the first time the cardioprotective effects of piceatannol on vascular smooth muscle cells (VSMC). The treatment of cells with piceatannol inhibited cell proliferation by reducing extracellular signal-regulated kinase (ERK) 1/2 and JNK activity in cultured VSMC in the presence of tumor necrosis factor-alpha (TNF-alpha). These inhibitory effects were also associated with G1 cell cycle arrest, and resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins. Piceatannol treatment strongly induced the expression of p21WAF1 via independence of p27KIP and p53 expression. The effect of piceatannol was not restricted to cell proliferation, as TNF-alpha-induced invasion and migration was also suppressed in VSMC. Moreover, piceatannol treatment strongly decreased matrix metalloproteinase-9 (MMP-9) expression and promoter activity in a dose-dependent manner in response to TNF-alpha. It was further demonstrated that piceatannol abrogated the transcriptional activity of nuclear factor kappa B (NF-kappaB), an important nuclear transcription factor involved in MMP-9 expression. Overall, these results demonstrate that piceatannol inhibits proliferation and migration of VSMC treated with TNF-alpha. Therefore, piceatannol may be an effective therapeutic approach to treat atherosclerosis.
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Preliminary quantitative profile of differential protein expression between rat L6 myoblasts and myotubes by stable isotope labeling with amino acids in cell culture.
Proteomics
PUBLISHED: 03-03-2009
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Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high-accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracellular matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In summary, our results provide new clues for understanding the mechanism of myogenesis.
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A phase II study of gemcitabine in combination with oxaliplatin as first-line chemotherapy in patients with inoperable biliary tract cancer.
Cancer Chemother. Pharmacol.
PUBLISHED: 01-14-2009
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The aim of this study is to investigate the efficacy and safety of gemcitabine and oxaliplatin combination chemotherapy as first-line therapy in patients with inoperable biliary tract cancer (BTC).
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A hypereosinophilic syndrome presenting as eosinophilic colitis.
Clin Endosc
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HYPEREOSINOPHILIC SYNDROME (HES) HAS THREE DEFINING FEATURES: marked hypereosinophilia for at least 6 months, no confirmed etiology for the eosinophilia, and eosinophilia-related symptoms or organ dysfunction. However, a shorter period of hypereosinophilia with symptoms requiring eosinophil-lowering therapy is also acceptable. We report a case of HES presenting as eosinophilic colitis. Although hypereosinophilia was present for 3 months, this patient needed to be treated with eosionphil-lowering therapy for severe hematochezia. After systemic corticosteroid therapy, symptoms caused by organ involvement were dramatically improved.
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Digestion and depletion of abundant proteins improves proteomic coverage.
Nat. Methods
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Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases and proteomic pipelines.
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Fludarabine, cytarabine, and attenuated-dose idarubicin (m-FLAI) combination therapy for elderly acute myeloid leukemia patients.
Am. J. Hematol.
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We performed a phase II trial to evaluate the efficacy and safety of the modified fludarabine, cytarabine, and attenuated-dose idarubicin (m-FLAI) regimen in elderly acute myeloid leukemia (AML) patients. Elderly (?60 years) AML patients who had not previously received chemotherapy were enrolled in the study. Patients received two consecutive cycles of m-FLAI chemotherapy as an induction. The m-FLAI regimen comprised fludarabine (25 mg/m(2) , days 1-4), cytarabine (1,000 mg/m(2) , days 1-4), and attenuated-dose idarubicin (5 mg/m(2) , days 1-3). The primary end point was complete remission (CR) rate. Secondary end points were overall survival (OS), event-free survival (EFS), and treatment-related mortality (TRM). There were 108 patients (median age 68.4 years, M:F = 64:44) enrolled in the study. CR was achieved in 56.5% of patients, and the TRM rate was 21.3%. Median OS and median EFS were 10.2 and 6.6 months, respectively. The mortality at 30 and 60 days was 15 and 21%, respectively. Performance status and comorbidity did not have prognostic value in this patient cohort. Bone marrow expression of CD117 was associated with increased EFS and OS. m-FLAI is an effective induction regimen for previously untreated AML in elderly patients. In addition, bone-marrow CD117 expression is an independent favorable prognostic factor in elderly AML patients. (ClinicalTrials.gov number, NCT01247493).
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Flexible metal-oxide devices made by room-temperature photochemical activation of sol-gel films.
Nature
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Amorphous metal-oxide semiconductors have emerged as potential replacements for organic and silicon materials in thin-film electronics. The high carrier mobility in the amorphous state, and excellent large-area uniformity, have extended their applications to active-matrix electronics, including displays, sensor arrays and X-ray detectors. Moreover, their solution processability and optical transparency have opened new horizons for low-cost printable and transparent electronics on plastic substrates. But metal-oxide formation by the sol-gel route requires an annealing step at relatively high temperature, which has prevented the incorporation of these materials with the polymer substrates used in high-performance flexible electronics. Here we report a general method for forming high-performance and operationally stable metal-oxide semiconductors at room temperature, by deep-ultraviolet photochemical activation of sol-gel films. Deep-ultraviolet irradiation induces efficient condensation and densification of oxide semiconducting films by photochemical activation at low temperature. This photochemical activation is applicable to numerous metal-oxide semiconductors, and the performance (in terms of transistor mobility and operational stability) of thin-film transistors fabricated by this route compares favourably with that of thin-film transistors based on thermally annealed materials. The field-effect mobilities of the photo-activated metal-oxide semiconductors are as high as 14 and 7?cm(2)?V(-1)?s(-1) (with an Al(2)O(3) gate insulator) on glass and polymer substrates, respectively; and seven-stage ring oscillators fabricated on polymer substrates operate with an oscillation frequency of more than 340?kHz, corresponding to a propagation delay of less than 210?nanoseconds per stage.
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Characterization of Breast Cancer Interstitial Fluids by TmT Labeling, LTQ-Orbitrap Velos Mass Spectrometry, and Pathway Analysis.
J. Proteome Res.
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Cancer is currently considered as the end point of numerous genomic and epigenomic mutations and as the result of the interaction of transformed cells within the stromal microenvironment. The present work focuses on breast cancer, one of the most common malignancies affecting the female population in industrialized countries. In this study, we perform a proteomic analysis of bioptic samples from human breast cancer, namely, interstitial fluids and primary cells, normal vs disease tissues, using tandem mass tags (TmT) quantitative mass spectrometry combined with the MudPIT technique. To the best of our knowledge, this work, with over 1700 proteins identified, represents the most comprehensive characterization of the breast cancer interstitial fluid proteome to date. Network analysis was used to identify functionally active networks in the breast cancer associated samples. From the list of differentially expressed genes, we have retrieved the associated functional interaction networks. Many different signaling pathways were found activated, strongly linked to invasion, metastasis development, proliferation, and with a significant cross-talking rate. This pilot study presents evidence that the proposed quantitative proteomic approach can be applied to discriminate between normal and tumoral samples and for the discovery of yet unknown carcinogenesis mechanisms and therapeutic strategies.
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Single-step inline hydroxyapatite enrichment facilitates identification and quantitation of phosphopeptides from mass-limited proteomes with MudPIT.
J. Proteome Res.
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Herein we report the characterization and optimization of single-step inline enrichment of phosphopeptides directly from small amounts of whole cell and tissue lysates (100-500 ?g) using a hydroxyapatite (HAP) microcolumn and Multidimensional Protein Identification Technology (MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide enrichment study, ?80% of the phosphopeptides identified using HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation motifs between the two enrichment methods illustrates the complementarity of calcium- and iron-based enrichment methods and the higher sensitivity and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how the identification of more multiply phosphorylated peptides from HAP-MudPIT can be used to quantify phosphorylation cooperativity. Through optimization of HAP-MudPIT on a whole cell lysate we routinely achieved identification and quantification of ca. 1000 phosphopeptides from a ?1 h enrichment and 12 h MudPIT analysis on small quantities of material. Finally, we applied this optimized method to identify phosphorylation sites from a mass-limited mouse brain region, the amygdala (200-500 ?g), identifying up to 4000 phosphopeptides per run.
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Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity.
Cell Metab.
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Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here, we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary hepatocytes and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis.
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Dynamics of subcellular proteomes during brain development.
J. Proteome Res.
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Many neurological disorders are caused by perturbations during brain development, but these perturbations cannot be readily identified until there is comprehensive description of the development process. In this study, we performed mass spectrometry analysis of the synaptosomal and mitochondrial fractions from three rat brain regions at four postnatal time points. To quantitate our analysis, we employed (15)N labeled rat brains using a technique called SILAM (stable isotope labeling in mammals). We quantified 167429 peptides and identified over 5000 statistically significant changes during development including known disease-associated proteins. Global analysis revealed distinct trends between the synaptic and nonsynaptic mitochondrial proteomes and common protein networks between regions each consisting of a unique array of expression patterns. Finally, we identified novel regulators of neurodevelopment that possess the identical temporal pattern of known regulators of neurodevelopment. Overall, this study is the most comprehensive quantitative analysis of the developing brain proteome to date, providing an important resource for neurobiologists.
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Determination of the esculetin contents of medicinal plants by liquid chromatography-tandem mass spectrometry.
Biomed. Chromatogr.
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We developed a LC-MS/MS method for the determination of esculetin contents in medicinal plants. The analysis was performed using multiple reaction monitoring in negative mode, and an XBridge™ C(18) column (2.1?×?100?mm, 3.5?µm) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r(2) ?>?0.9993). The limits of detection and quantitation were 0.02 and 0.07?ng/mL, respectively. The intra-day and inter-day precisions were 1.5-6.8 and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini Cortex showed the highest content of esculetin (761-5475?mg/kg). In Mori Folium and Artemisiae Capillaris Herba, 5.2-21.5 and 7.0-17.6?mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10?mg/kg. The analysis method appears to be simple, sensitive and reproducible. Contrary to expectations based on traditional medical knowledge, although Artemisiae Capillaris Herba contains a large amount of esculetin, it appears from this study that Fraxini Cortex contains a greater amount. The pharmacological effects of esculetin isolated from medicinal plants should be investigated as part of new medicines development.
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The effects of Mo doping on 0.3Li[Li0.33Mn0.67]O2·0.7Li[Ni0.5Co0.2Mn0.3]O2 cathode material.
Dalton Trans
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Mo doped Li excess transition metal oxides formulated as 0.3Li[Li(0.33)Mn(0.67)]O(2)·0.7Li[Ni(0.5-x)Co(0.2)Mn(0.3-x)Mo(2x)]O(2) were synthesized using the co-precipitation process. The effects of the substitution of Ni and Mn with Mo were investigated for the density of the states, the structure, cycling stability, rate performance and thermal stability by tools such as first principle calculations, synchrotron X-ray diffraction, field-emission SEM, solid state (7)Li MAS nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), elemental mapping by scanning TEM (STEM), inductively coupled plasma atomic emission spectrometry (ICP-AES) and a differential scanning calorimeter (DSC). It was confirmed that high valence Mo(6+) doping of the Li-excess manganese-nickel-cobalt layered oxide in the transition metal enhanced the structural stability and electrochemical performance. This increase was due to strong Mo-O hybridization inducing weak Ni-O hybridization, which may reduce O(2) evolution, and metallic behavior resulting in a diminishing cell resistance.
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Assurance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cascade.
EMBO Rep.
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Sqstm1/p62 functions in the non-canonical activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, its physiological relevance is not certain. Here, we show that p62(-/-) mice exhibited an accelerated presentation of ageing phenotypes, and tissues from these mice created a pro-oxidative environment owing to compromised mitochondrial electron transport. Accordingly, mitochondrial function rapidly declined with age in p62(-/-) mice. In addition, p62 enhanced basal Nrf2 activity, conferring a higher steady-state expression of NAD(P)H dehydrogenase, quinone 1 (Nqo1) to maintain mitochondrial membrane potential and, thereby, restrict excess oxidant generation. Together, the p62-Nrf2-Nqo1 cascade functions to assure mammalian longevity by stabilizing mitochondrial integrity.
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