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In JoVE (3)

Other Publications (62)

Articles by Xiaoli Zhao in JoVE

 JoVE Biology

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

1Department of Physiology and Biophysics, Robert Wood Johnson Medical School


JoVE 2717

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

 JoVE Biology

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

1Department of Physiology and Biophysics, Confocal Microscopy and Cell Imaging Core, Robert Wood Johnson Medical School, 2Department of Physiology and Biophysics, Robert Wood Johnson Medical School, 3Muscle Biology Research Group-MUBIG Schools of Nursing & Medicine, University of Missouri-Kansas City


JoVE 3415

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

 JoVE Biology

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

1Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, 2Muscle Biology Research Group, University of Missouri-Kansas City, 3Pharmacology division, College of Pharmacy, DHLRI, Ohio State University


JoVE 4198

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Other articles by Xiaoli Zhao on PubMed

Multiecho Segmented EPI with Z-shimmed Background Gradient Compensation (MESBAC) Pulse Sequence for FMRI

A MultiEcho Segmented EPI with z-shimmed BAckground gradient Compensation (MESBAC) pulse sequence is proposed and validated for functional MRI (fMRI) study in regions suffering from severe susceptibility artifacts. This sequence provides an effective tradeoff between spatial and temporal resolution and reduces image distortion and signal dropout. The blood oxygenation level-dependent (BOLD)-weighted fMRI signal can be reliably obtained in the region of the orbitofrontal cortex (OFC). To overcome physiological motion artifacts during prolonged multisegment EPI acquisition, two sets of navigator echoes were acquired in both the readout and phase-encoding directions. Ghost artifacts generally produced by single-shot EPI acquisition were eliminated by separately placing the even and odd echoes in different k-space trajectories. Unlike most z-shim methods that focus on increasing temporal resolution for event-related functional brain mapping, the MESBAC sequence simultaneously addresses problems of image distortion and signal dropout while maintaining sufficient temporal resolution. The MESBAC sequence will be particularly useful for pharmacological and affective fMRI studies in brain regions such as the OFC, nucleus accumbens, amygdala, parahippocampus, etc.

Transient Relationships Among BOLD, CBV, and CBF Changes in Rat Brain As Detected by Functional MRI

The transient relationship between arterial cerebral blood flow (CBF(A)) and total cerebral blood volume (CBV(T)) was determined in the rat brain. Five rats anesthetized with urethane (1.2 g/kg) were examined under graded hypercapnia conditions (7.5% and 10% CO(2) ventilation). The blood oxygenation level-dependent (BOLD) contrast was determined by a gradient-echo echo-planar imaging (GE-EPI) pulse sequence, and CBV(T) changes were determined after injection of a monocrystalline iron oxide nanocolloid (MION) contrast agent using an iron dose of 12 mg/kg. The relationship between CBV(T) and CBF(A) under transient conditions is similar to the power law under steady-state conditions. In addition, the transient relationship between CBV(T) and CBF(A) is region-specific. Voxels with > or =15% BOLD signal changes from hypercapnia (7.5% CO(2) ventilation) have a larger power index (alpha = 3.26), a larger maximum possible BOLD response (M = 0.85), and shorter T(*)(2) (32 ms) caused by deoxyhemoglobin, compared to voxels with <15% BOLD signal changes (alpha = 1.82, M = 0.16, and T(*)(2) = 169 ms). It is suggested that the biophysical model of the BOLD signal can be extended under the transient state, with a caution that alpha and M values are region-specific. To avoid overestimation of the cerebral metabolic rate of oxygen changes seen using fMRI, caution should be taken to not include voxels with large veins and a large BOLD signal.

[Determination of Hypoxanthine and Xanthine in Hippocampus by HPLC Method]

A method was established for the determination of hypoxanthine and xanthine in Hippocampus by HPLC.

A Novel Double-stranded RNA-binding Protein, Disco Interacting Protein 1 (DIP1), Contributes to Cell Fate Decisions During Drosophila Development

We report the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein disconnected (disco) as a bait. DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD). Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays. DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors. In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression. Later in development DIP1 expression is decreased in dividing cells in different tissues. Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule. A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed.

Reduction of Artifacts by Optimization of the Sensitivity Map in Sensitivity-encoded Spectroscopic Imaging

Sensitivity-encoded spectroscopic imaging (SENSE-SI) reduces scanning time by using multiple coils for parallel signal acquisition. Significant artifacts could be induced by SENSE-SI, mainly due to the low-resolution nature of spectroscopic imaging. The present study introduces a novel method to reduce the artifacts. High-resolution sensitivity maps are used in low-resolution SENSE reconstruction. An intermediate unaliased image is obtained after SENSE reconstruction. Based on the intermediate image, the sensitivity maps are optimized and then the SENSE reconstruction is performed again. The final unaliased image has significantly reduced artifacts.

Uncontrolled Calcium Sparks Act As a Dystrophic Signal for Mammalian Skeletal Muscle

Most excitable cells maintain tight control of intracellular Ca(2+) through coordinated interaction between plasma membrane and endoplasmic or sarcoplasmic reticulum. Quiescent sarcoplasmic reticulum Ca(2+) release machinery is essential for the survival and normal function of skeletal muscle. Here we show that subtle membrane deformations induce Ca(2+) sparks in intact mammalian skeletal muscle. Spontaneous Ca(2+) sparks can be reversibly induced by osmotic shock, and participate in a normal physiological response to exercise. In dystrophic muscle with fragile membrane integrity, stress-induced Ca(2+) sparks are essentially irreversible. Moreover, moderate exercise in mdx muscle alters the Ca(2+) spark response. Thus, membrane-deformation-induced Ca(2+) sparks have an important role in physiological and pathophysiological regulation of Ca(2+) signalling, and uncontrolled Ca(2+) spark activity in connection with chronic activation of store-operated Ca(2+) entry may function as a dystrophic signal in mammalian skeletal muscle.

Enhanced Resistance to Fatigue and Altered Calcium Handling Properties of Sarcalumenin Knockout Mice

Sarcalumenin is a Ca2+-binding protein located in the sarcoplasmic reticulum of striated muscle cells, the physiological function of which has not been fully determined yet. Using sarcalumenin knockout (sar(-/-)) mice, we showed that sar ablation altered store-operated Ca2+ entry (SOCE) and enhanced muscle fatigue resistance. Sar(-/-) mice fatigued less with treadmill exercise, and intact isolated soleus and extensor digitorum longus muscles from sar(-/-) mice were more resistant to intermittent fatiguing stimulation than those from wild-type mice. Enhanced SOCE was observed in the sar(-/-) muscles. Biochemical analysis revealed that sar(-/-) muscles contained significantly elevated expression of mitsugumin 29 (MG29), a synaptophysin-related membrane protein located in the triad junction of skeletal muscle. Because the ablation of mg29 has been shown to cause increased fatigability and dysfunction of SOCE, the enhanced SOCE activity seen in sar(-/-) muscle may be correlated with the increased expression of MG29. Our data suggest that systemic ablation of sarcalumenin caused enhanced resistance to muscle fatigue by compensatory changes in Ca2+ regulatory proteins that effect SOCE.

Uncoupling Store-operated Ca2+ Entry and Altered Ca2+ Release from Sarcoplasmic Reticulum Through Silencing of Junctophilin Genes

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca2+ storage and altered caffeine-induced Ca2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging.

Muscle Aging is Associated with Compromised Ca2+ Spark Signaling and Segregated Intracellular Ca2+ Release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.

Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.

Differentiation of Human Embryonic Stem Cells into Smooth Muscle Cells in Adherent Monolayer Culture

Smooth muscle cell (SMC) plays critical roles in many human diseases, an in vitro system that recapitulates human SMC differentiation would be invaluable for exploring molecular mechanisms leading to the human diseases. We report a directed and highly efficient SMC differentiation system by treating the monolayer-cultivated human embryonic stem cells (hESCs) with all-trans retinoid acid (atRA). When the hESCs were cultivated in differentiation medium containing 10microM RA, more than 93% of the cells expressed SMC-marker genes along with the steadily accumulation of such SMC-specific proteins as SM alpha-actin and SM-MHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. This inducible and highly efficient in vitro human SMC system could be an important resource to study the mechanisms of SMC phenotype determination in human.

Determination of Perfluorinated Compounds in Wastewater and River Water Samples by Mixed Hemimicelle-based Solid-phase Extraction Before Liquid Chromatography-electrospray Tandem Mass Spectrometry Detection

A comparative study on the use of cetyltrimethylammonium bromide (CTAB)-coated silica and sodium dodecyl sulphate (SDS)-coated alumina mixed hemimicelles-based solid-phase extraction (SPE) for the pre-concentration of six perfluorinated compounds (PFCs) in environmental water samples was presented. The six analytes heptafluorobutyric acid (HFBA), perfluoroheptanic acid (PFHeA), perfluorooctanic acid (PFOA), perfluorooctanic sulfonic (PFOS), perfluorononanic acid (PFNA) and perfluorodecanic acid (PFDeA) were quantitatively retained on both sorbent materials. The cationic surfactant (CTAB adsorbed onto silica) was more appropriate for SPE of PFCs. The main factors affecting adsolubilization of PFCs including the amount of surfactant, pH of solution, sample loading volume and desorption were investigated and optimized. Concentration factor of 500 were achieved by SPE of 500 mL of several environmental water samples. The method detection limits obtained for HFBA, PFHeA, PFOA, PFOS, PFNA and PFDeA were 0.10, 0.28, 0.07, 0.20, 0.10 and 0.05 ng/L, respectively. The relative standard deviation of recoveries ranged from 2 to 8%, which indicated good method precision.

TRIC Channels Are Essential for Ca2+ Handling in Intracellular Stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.

Single Cell Derived Murine Embryonic Stem Cell Clones Stably Express Rex1-specific Green Fluorescent Protein and Their Differentiation Study

Embryonic stem cells (ESCs) often display high rates of apoptosis and spontaneous differentiation in routine culture, thus bring the proliferation of these cells highly inefficient. Moreover, little is known about the factors that are indispensable for sustaining self-renewal. To surmount these issues, we established transgenic mES cell lines expressing the enhanced green fluorescent protein (EGFP) under the control of the Rex1 promoter which is a key regulator of pluripotency in ES cells. In addition, we provided a simplified and improved protocol to derive transgenic mESCs from single cell. Finally, we showed that embryoid body (EB) development was faster than adherent differentiation in terms of differentiation ratio by real-time tracking of the EGFP expression. Therefore, these cell lines can be tracked and selected both in vitro and in vivo and should be invaluable for studying the factors that are indispensable for maintaining pluripotency.

Mixed Hemimicelles Solid-phase Extraction Based on Cetyltrimethylammonium Bromide-coated Nano-magnets Fe3O4 for the Determination of Chlorophenols in Environmental Water Samples Coupled with Liquid Chromatography/spectrophotometry Detection

Mixed hemimicelles solid-phase extraction (SPE) based on cetyltrimethylammonium bromide (CTAB)-coated nano-magnets Fe3O4 was investigated for the preconcentration of four chlorophenols (CPs) in environmental water samples prior to HPLC-spectrophotometry determination in this paper. By the rapid isolating (about 5 min) of Fe3O4 nanoparticles (NPs) through placing a Nd-Fe-B strong magnet on the bottom of beaker, the time-consuming preconcentration process of loading large volume sample in conversional SPE method with a column can be avoided. The unique properties of Fe3O4 NPs such as high surface area and strong magnetism were utilized adequately in the SPE process. This novel separation method produced a high preconcentration rate and factor. A comprehensive study of the adsorption conditions such as the Fe3O4 NPs zeta-potential, CTAB added amounts, pH value, standing time and maximal extraction volume was also presented. Under optimized conditions, four analytes of 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP) were quantitatively extracted. The method was then used to determine four CPs in five real environmental water samples. High concentration factors (700) were achieved for each of the analytes, with observed detection limits ranging between 0.11 and 0.15 microg L(-1). The accuracy of method was evaluated by recovery measurements on spiked samples. Good recovery results (83-98%) with satisfactory relative standard deviation (RSD) were achieved. It is important to note that satisfactory preconcentration factors and extraction recoveries for the four CPs were obtained with only a little amount of Fe3O4 NPs (0.1g) and CTAB (60 mg). To the best of our knowledge, this was the first time a mixed hemimicelles SPE method based on Fe3O4 NPs magnetic separation had been used for the pretreatment of environmental water samples.

Preparation of Silica-magnetite Nanoparticle Mixed Hemimicelle Sorbents for Extraction of Several Typical Phenolic Compounds from Environmental Water Samples

A novel type of superparamagnetic silica-coated (Fe3O4/SiO2 core/shell) magnetite nanoparticle modified by surfactants has been successfully synthesized and was applied as an effective sorbent material for the pre-concentration of several typical phenolic compounds (bisphenol A (BPA), 4-tert-octylphenol (4-OP) and 4-n-nonylphenol (4-NP)) from environmental water samples. Compared with pure magnetic particles, a thin and dense silica layer would protect the iron oxide core from leaching out in acidic conditions. In order to enhance their adsorptive tendency towards organic compounds, cetylpyridinium chloride (CPC) or cetyltrimethylammonium bromide (CTAB) were added, which adsorbed on the surface of the Fe3O4/SiO2 nanoparticles (Fe3O4/SiO2 NPs) and formed mixed hemimicelles. Main factors affecting the adsolubilization of analytes were optimized and comparative study on the use of CPC and CTAB-coated Fe3O4/SiO2 NPs mixed hemimicelles-based SPE was also carried out. CPC-coated Fe3O4/SiO2 NPs system was selected due to lower elution volume required and more effective adsorption of the target compounds. Under selected conditions, concentration factor of 1600 was achieved by using this method to extract 800 mL of different environmental water samples. The detection limits obtained for BPA, 4-OP and 4-NP with HPLC-FLD were 7, 14, and 20 ng/L, respectively.

Cetyltrimethylammonium Bromide-coated Magnetic Nanoparticles for the Preconcentration of Phenolic Compounds from Environmental Water Samples

The research presented in this paper investigates the adsorption of cation surfactants--cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC)--onto magnetic nanoparticles and the application of this mixed hemimicelles solid-phase extraction (SPE) method for the preconcentration of several typical phenolic compounds-bisphenol A (BPA), 4-tertoctylphenol (4-OP), and 4-n-nonylphenol (4-NP)--from environmental water samples. In this novel SPE method, the charged surfactants CTAB and CPC form mixed hemimicelles on Fe3O4 nanoparticles (Fe3O4 NPs), which causes retention of analytes by strong hydrophobic and electrostatic interactions. The SPE method combines the advantages of mixed hemimicelles and magnetic nanoparticles. In order to provide guidelines forthe mixed hemimicelles SPE method development, surfactant adsorption isotherms and zeta-potential isotherms were also investigated. The main factors affecting the adsolubilization of analytes, such as the amount of Fe3O4 NPs and surfactants, the type of surfactants, the solution pH,the sample loading volume, and the desorption conditions, were investigated and optimized. A concentration factor of 800 was achieved by the extraction of 800 mL of several environmental water samples using this SPE method. Under the selected conditions, detection limits obtained for BPA, 4-OP, and 4-NP were 12, 29, 34 ng/L, respectively. The accuracy of the method was evaluated by recovery measurements on spiked samples, and good recoveries (68-104%) with low relative standard deviations from 2 to 7% were achieved. The advantages of this new SPE method include high extraction yields, high breakthrough volumes, short analysis times, and easy preparation of sorbents. To the best of our knowledge, this is the first time that a mixed hemimicelles SPE method based on magnetic separation and nanoparticles has been used for the pretreatment of environmental water samples.

Compromised Store-operated Ca2+ Entry in Aged Skeletal Muscle

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca(2+) entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store-operated Ca(2+) entry (SOCE) in aged mouse skeletal muscle. Using Mn(2+) quenching of Fura-2 fluorescence and confocal-microscopic imaging of Ca(2+) movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26-27 months) as compared with those from young (2-5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin-29, a synaptophysin-related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin-29 expression and reduced SOCE may contribute to the diminished intracellular Ca(2+) homeostatic capacity generally associated with muscle aging.

Preparation of Alkanethiolate-functionalized Core/shell Fe3O4@Au Nanoparticles and Its Interaction with Several Typical Target Molecules

This paper reports the synthesis of alkanethiolate-functionalized core/shell Fe3O4/Au nanoparticles (Fe3O4@Au NPs) that combine the advantages of core/shell magnetic nanoparticles with self-assembled monolayers (SAMs). The alkanethiolates, having carboxylic acid (COOH) and methyl (CH3) terminal groups, can be easily self-assembled on the Fe3O4@Au NPs substrates. The surface composition and structure of 11-mercaptoundecanoic acid (11-MUA) and 1-dodecanethiol (11-DDT) SAMs were characterized in detail using energy-dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). To further study the surface properties of the SAMs, hydrophobic, ionic, and nonionic organic compounds were selected as probe molecules. The results indicated that the hydrophobic interaction caused strong retention of analytes, whereas the repulsive electrostatic interaction between the negatively charged 11-MUA SAMs surface and the negatively charged probe molecule would lead to the decrease of adsorption affinity.

In Vitro Effects of Nanophase Hydroxyapatite Particles on Proliferation and Osteogenic Differentiation of Bone Marrow-derived Mesenchymal Stem Cells

Coculturing scaffolds with seeded cells in vitro is an indispensable process for construction of engineered tissues. It is essential to understand effects of the constituent particles of scaffold on seeded cells. In this study, we investigated the influence of nano-sized hydroxyapatite (nHAP) particles on the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs). nHAP particles were cocultured with MSCs separated from rabbit. Cellular effects of particles were determined by cell counts, Vonkossa stains, and reverse transcription-polymerase chain reaction (RT-PCR) examinations. Results showed that nHAP particles could promote the MSCs growth when particle concentrations were lower than 20 microg/10(4) cells. This effect disappeared when the particles and the cells were cocultured in serum-free media. Higher particle concentrations could significantly inhibit the cell growth. Under the standard culture condition, the only effect of nHAP particles on osteogenic differentiation of MSCs was to enhance the expression of collagen I. Under the osteogenic-inductive culture condition, nHAP particles could inhibit mineralization of cells but promote their osteogenic differentiation. These cellular effects of particles still existed when the particles and the cells were cultured in indirect coculture system. nHAP particles could decrease calcium and phosphate concentrations of culture media, which possibly contributed to the cellular effects of nHAP particles.

Fast Thermoresponsive BAB-type HEMA/NIPAAm Triblock Copolymer Solutions for Embolization of Abnormal Blood Vessels

Thermoresponsive BAB-type HEMA/NIPAAm triblock copolymers (A = NIPAAm, B = HEMA) were prepared by atomic transfer radical polymerization (ATRP). BAB1-6 with shorter PNIPAAm blocks failed to form stable gel; while a relatively stable gel could be achieved by BAB1-8 with longer PNIPAAm blocks when copolymer aqueous solution was heated up. Introducing radiopaque agent (RA) was shown to slightly increase the transition temperature and gelation time, but the gelling ability was strengthened due to slightly weakening dehydration of copolymer in the mixture of water and RA. BAB1-8 aqueous solution about 5 wt% in the presence of RA was demonstrated to successfully occlude the cerebral rete mirabiles (RMs) and renal arteries of pigs. Within 3-month surgery, no recanalization was observed and the embolized kidney shrank considerably. Histological assay of embolized kidney demonstrated interstitial fibrosis and calcification as well as the thickening of renal small artery. This temperature sensitive copolymer with well-defined architecture holds a great potential as an embolic agent for treating arteriovenous malformations (AVMs) and renal disease due to the design flexibility of ATRP.

MG53 Nucleates Assembly of Cell Membrane Repair Machinery

Dynamic membrane repair and remodelling is an elemental process that maintains cell integrity and mediates efficient cellular function. Here we report that MG53, a muscle-specific tripartite motif family protein (TRIM72), is a component of the sarcolemmal membrane-repair machinery. MG53 interacts with phosphatidylserine to associate with intracellular vesicles that traffic to and fuse with sarcolemmal membranes. Mice null for MG53 show progressive myopathy and reduced exercise capability, associated with defective membrane-repair capacity. Injury of the sarcolemmal membrane leads to entry of the extracellular oxidative environment and MG53 oligomerization, resulting in recruitment of MG53-containing vesicles to the injury site. After vesicle translocation, entry of extracellular Ca(2+) facilitates vesicle fusion to reseal the membrane. Our data indicate that intracellular vesicle translocation and Ca(2+)-dependent membrane fusion are distinct steps involved in the repair of membrane damage and that MG53 may initiate the assembly of the membrane repair machinery in an oxidation-dependent manner.

Superparamagnetic Fe3O4 Nanoparticles As Catalysts for the Catalytic Oxidation of Phenolic and Aniline Compounds

Fe(3)O(4) magnetic nanoparticles (MNPs) with diameters about 10nm were synthesized successfully and used to remove phenol and aniline from aqueous solution. The results showed that phenol and aniline could be eliminated easily from solution under acidic and neutral conditions in the presence of MNPs and H(2)O(2). When the concentrations of Fe(3)O(4) MNPs and H(2)O(2) were 5gL(-1) and 1.2M, respectively, phenol and aniline could be removed completely after 6h of reaction at 308K, and the total organic carbon (TOC) abatement efficiency for phenol and aniline were 42.79% and 40.38%. Some intermediates such as formic acid, acetic acid, fumaric acid and hydroquinone were detected during reaction. Fe(3)O(4) MNPs exhibited good stability and reusability, also showed excellent catalysis ability to eliminate some substituted phenolic and aniline compounds from solution. Fe(3)O(4) MNPs had good superparamagnetism and was readily separated from solution by applying an external magnetic field. Finally we proposed that phenol and aniline might be degraded by the hydroxyl free radicals (.OH) released from H(2)O(2) in the presence of Fe(3)O(4) MNPs as catalysts.

[Investigation of Research Method of Fingerprint Pharmacodynamics of Traditional Chinese Medicines]

Fingerprinting techniques play a increasingly important role in the quality control standards of traditional Chinese medicines (TCM), research and establish the fingerprint about spectral-efficiency could improve the quality control of TCM. The necessity of the fingerprint pharmacodynamics research and the analysis and evaluation of the research methods in the existing literature at home and abroad were reviewed in this article, Combined with the author's laboratory research, we proposed the research methods of fingerprint pharmacodynamics of TCM and provided the basis for effectively promoting the the establishment and development of fingerprint pharmacodynamics of Chinese medicine compound preparations.

Removal of Fluoride from Aqueous Media by Fe3O4@Al(OH)3 Magnetic Nanoparticles

A novel magnetic nanosized adsorbent using hydrous aluminum oxide embedded with Fe(3)O(4) nanoparticle (Fe(3)O(4)@Al(OH)(3) NPs), was prepared and applied to remove excessive fluoride from aqueous solution. This adsorbent combines the advantages of magnetic nanoparticle and hydrous aluminum oxide floc with magnetic separability and high affinity toward fluoride, which provides distinctive merits including easy preparation, high adsorption capacity, easy isolation from sample solutions by the application of an external magnetic field. The adsorption capacity calculated by Langmuir equation was 88.48 mg g(-1) at pH 6.5. Main factors affecting the removal of fluoride, such as solution pH, temperature, adsorption time, initial fluoride concentration and co-existing anions were investigated. The adsorption capacity increased with temperature and the kinetics followed a pseudo-second-order rate equation. The enthalpy change (Delta H(0)) and entropy change (DeltaS(0)) was 6.836 kJ mol(-1) and 41.65 J mol(-1)K(-1), which substantiates the endothermic and spontaneous nature of the fluoride adsorption process. Furthermore, the residual concentration of fluoride using Fe(3)O(4)@Al(OH)(3) NPs as adsorbent could reach 0.3 mg L(-1) with an initial concentration of 20 mg L(-1), which met the standard of World Health Organization (WHO) norms for drinking water quality. All of the results suggested that the Fe(3)O(4)@Al(OH)(3) NPs with strong and specific affinity to fluoride could be excellent adsorbents for fluoride contaminated water treatment.

Highly Substituted Furo[3,4-c]azepines by Gold(I)-catalyzed Diastereoselective Tandem Double Heterocyclizations and 1,2-alkyl Migration

A Rapid and Efficient Strategy for Preparation of Super-hydrophobic Surface with Cross-linked Cyclotriphosphazene/6F-bisphenol A Copolymer Microspheres

This study reports a novel super-hydrophobic surface with fluorinated cross-linked poly[cyclotriphosphazene-co-(4,4'-(hexafluoroisopropylidene)diphenol)] microspheres prepared via a single-step precipitation polymerization and simple dip-coating route.

Anthropogenic Input of Polycyclic Aromatic Hydrocarbons into Five Lakes in Western China

The temporal distribution of polycyclic aromatic hydrocarbons (PAHs) was examined in dated sediments from five lakes in Western China. The surficial concentrations of seven carcinogenic PAHs (BaA, Chyr, BbF, BkF, BaP, DBA and INP, hereafter denoted as PAH7) were highly site-specific. The SigmaPAH7 concentrations generally increased from the deep layers to surface sediments. The temporal trends of SigmaPAH7 concentrations were clearly different from those reported in developed countries. From 1950s to 1980s, which PAHs started to decrease in developed countries, is the right period that PAHs started to increase rapidly in Western China. The fluxes and mass inventories suggest that the PAH7 input over the past three decades was much more rapidly than it was before the 1980s. Source identification indicates that the PAHs were primarily from biomass burning and domestic coal combustion, and increasing vehicular and/or industrial coal combustion emission was also found.

Isolation and Characterization of 3,5,6-trichloro-2-pyridinol-degrading Ralstonia Sp. Strain T6

A 3,5,6-trichloro-2-pyridinol (TCP)-degrading strain, T6, was isolated by continuous enrichment culture and identified as Ralstonia sp. based on morphological, physiological and biochemical tests as well as 16S rDNA sequence analysis. The bacterium metabolized 100 mg/L TCP within 12 h and 700 mg/L TCP in 80 h. A green metabolite, putatively identified as 3,6-dihydroxypyridine-2,5-dione, was detected. This is the first report of TCP-degrading isolate from the genus of Ralstonia. Strain T6 could potentially be employed in bioremediation of TCP.

Apatite-formation Ability--predictor of "bioactivity"?

The ability to trigger the formation of apatite from a supersaturated solution has been widely used to imply the bioactivity of an implant in vivo. However, the method itself may provide at best incomplete information, primarily because it is determined only by solution supersaturation, irrespective of biological processes. Bone regeneration is triggered mainly by the vitality of osteoblasts, and regulated by the expression of growth factors such as oestrogen, parathyroid hormone and bone morphogenetic proteins, while ions or other species released from an implant may affect the expression of such growth factors, and so bone resorption or formation. The misinterpretation of the outcome of such tests must result in misunderstanding of the true effects and behaviour of materials intended for use in embedded biological contexts. Thus, the underlying and motivating hypothesis needs to be carefully reconsidered, along with the results of all work founded on the concept. It would seem that it is only viable to test using osteoblasts, whether in vivo or in vitro.

Diastereo- and Enantioselective Gold(I)-catalyzed Intermolecular Tandem Cyclization/[3+3]cycloadditions of 2-(1-alkynyl)-2-alken-1-ones with Nitrones

Increased Store-operated Ca2+ Entry in Skeletal Muscle with Reduced Calsequestrin-1 Expression

Store-operated Ca(2+) entry (SOCE) contributes to Ca(2+) handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn(2+) fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca(2+) store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39 degrees C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca(2+) permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca(2+) as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca(2+) entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca(2+) signaling in MH.

Benzotriazole Removal from Water by Zn-Al-O Binary Metal Oxide Adsorbent: Behavior, Kinetics and Mechanism

In this study, a novel Zn-Al-O binary metal oxide adsorbent was prepared and used to remove the emerging polar contaminant benzotriazole from water. The adsorption behavior, kinetics and mechanism were systemically studied. Results showed that benzotriazole was rapidly and effectively adsorbed by the adsorbent. Instantaneous adsorption was observed under each studied condition, and the adsorption reached equilibrium within 30 min. High initial benzotriazole concentration enhanced the adsorption. The amount of absorbed benzotriazole increased with increasing adsorbent dosage, but decreased with increasing ionic strength. Solution pH had little effect on benzotriazole adsorption. The adsorption isotherm was consistent with S-type. Langmuir isotherm model fitted the equilibrium data better than Freundlich, Dubinin-Radushkevich and Temkin isotherm models. The maximum monolayer adsorptive capacity of benzotriazole with and without electrolytes was 7.30 mg g(-1) and 9.51 mg g(-1), respectively. Elovich and pseudo-second-order models were most suitable for describing the adsorption kinetics. Interactions between the surface sites of the adsorbent and benzotriazole may be a combination of electrostatic interaction, ion exchange and hydrogen bond.

China Embarking on Development of Its Own National Water Quality Criteria System

Ca2+ Overload and Sarcoplasmic Reticulum Instability in Tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K(+), Cl(-), and H(+) channels may facilitate charge neutralization during Ca(2+) release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca(2+) release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a(-/-) skeletal muscle showed Ca(2+) overload inside the SR with frequent formation of Ca(2+) deposits compared with the wild type muscle. This elevated SR Ca(2+) pool in the tric-a(-/-) muscle could be released by caffeine, whereas the elemental Ca(2+) release events, e.g. osmotic stress-induced Ca(2+) spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of "alternan" behavior with isolated tric-a(-/-) skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca(2+ ATPase) function could lead to aggravation of the stress-induced alternans in the tric-a(-/-) muscle. Our data suggests that absence of TRIC-A may lead to Ca(2+) overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca(2+) movement across the SR membrane. The observed alternan behavior with the tric-a(-/-) muscle may reflect a skeletal muscle version of store overload-induced Ca(2+) release that has been reported in the cardiac muscle under stress conditions.

[Simultaneous Determination of Chlorogenic Acid, Cryptochlorogenic Acid, Caffeic Acid, Naringin, Hesperidin and Linarin in Xiao'erjinning Oral Liquid by an HPLC Method]

To develop a HPLC method for the simultaneous determination of chlorogenic acid, cryptochlorogenic acid, caffeic acid, naringin, hesperidin and linarin in xiao' erjinning oral liquid.

Novel Preparation of Polyphosphazene-coated Carbon Nanotubes As a Pt Catalyst Support

This study reports a noncovalent functionalization of MWCNTs with a fluorinated cross-linked polymer coating of poly[cyclotriphosphazene-co-(4,4'-(hexafluoroisopropylidene)diphenol)] and their application as the support of Pt for electrocatalytic oxidation of methanol.

A Loop-controlled RrnB P1 Promoter for High-level Expression of Heterologous Proteins in Escherichia Coli

An effective protein expression system was constructed in Escherichia coli utilizing the rRNA rrnB P1 promoter and the regulatory element of the lac operon (lacO). To regulate the transcriptional activity of the rrnB P1 promoter, we designed two lacO sites with an intervening loop structure; expression was verified by measuring the levels of the β-1,4-glucanase gene, cel5G. Basal expression from the looped promoter construct was reduced by 92% when compared to expression from the T7 promoter. We also found that the host cell type had a significant effect on the regulation of the rrnB P1 promoter: E. coli DH5α and DH10B had high expression levels, whereas the expression in BL21(DE3) was more stringent.

Production of Rabbit Monoclonal Antibodies Against Mouse Embryonic Stem Cells and Identification of Pluripotency-associated Surface Antigens

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of the blastocyst. ES cell surface molecules are important for the identification, labeling, sorting, quality control and functional studies of ES cells. Currently, knowledge of ES surface molecules is limited. To identify new surface molecules, we generated a panel of rabbit monoclonal antibodies (rMabs) against mouse ES (mES) cells. We identified three monoclonal antibodies that interact with molecules on the mES cell surface and found that the expression of their respective antigens decreased upon mES cell differentiation. The antigen of the rMab ZjuESrMab29 was identified as granulocyte macrophage colony-stimulating factor receptor α (GM-CSFR α) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study demonstrated that rabbit monoclonal antibody production via whole-cell immunization could be a practical method for the discovery of stem cell surface antigens.

A Versatile Single-plasmid System for Tissue-specific and Inducible Control of Gene Expression in Transgenic Mice

We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.

Solvent-induced Crystallization of Poly(3-dodecylthiophene): Morphology and Kinetics

We report on the self-assembly of poly(3-dodecylthiophene) (P3DDT) into nanowhiskers for the first time via addition of the marginal solvent anisole into its well-dissolved solution. By controlling the solvent composition and aging time, we observed a morphology evolution from nanowhiskers to two-dimensional nanoribbons and foliated aggregates, which was ascribed to diverse driving forces for self-assembly in the process of crystallization. UV-vis absorption spectroscopy and dynamic lighting scattering (DLS) measurements were employed to in situ monitor crystallization kinetics of P3DDT induced by mixed solvents. It has been shown that conformational transition serves as a critical factor for P3AT to perform π-π stacking to form nanowhiskers. From a thermodynamic point of view, P3AT dispersion dissolved in mixed solvents is actually not a thermodynamic equilibrium system, but a multicomponent and multiphase case whose phase composition and properties evolve with time. The understanding in morphology transition mechanisms and crystallization kinetics of P3DDT can provide guidelines for optimization of processing parameters and enhance performance of photovoltaic devices.

Microstructures and Mechanical Properties of Metastable Ti-30Zr-(Cr, Mo) Alloys with Changeable Young's Modulus for Spinal Fixation Applications

In order to develop a novel alloy with a changeable Young's modulus for spinal fixation applications, we investigated the microstructures, Young's moduli, and tensile properties of metastable Ti-30Zr-(Cr, Mo) alloys subjected to solution treatment (ST) and cold rolling (CR). All the alloys comprise a β phase and small athermal ω phase, and they exhibit low Young's moduli after ST. During CR, deformation-induced phase transformation occurs in all the alloys. The change in Young's modulus after CR is highly dependent on the type of deformation-induced phase. The increase in Young's modulus after CR is attributed to the deformation-induced ω phase on {3 3 2} mechanical twinning. Ti-30Zr-3Cr-3Mo (3Cr3Mo), which exhibits excellent tensile properties and a changeable Young's modulus, shows a smaller springback than Ti-29Nb-13Ta-4.6Zr, a β-type titanium alloy expected to be useful in spinal fixation applications. Thus, 3Cr3Mo is a potential candidate for spinal fixation applications.

Store-operated Ca(2+) Entry (SOCE) Contributes to Normal Skeletal Muscle Contractility in Young but Not in Aged Skeletal Muscle

Muscle atrophy alone is insufficient to explain the significant decline in contractile force of skeletal muscle during normal aging. One contributing factor to decreased contractile force in aging skeletal muscle could be compromised excitation-contraction (E-C) coupling, without sufficient available Ca(2+) to allow for repetitive muscle contractility, skeletal muscles naturally become weaker. Using biophysical approaches, we previously showed that store-operated Ca(2+) entry (SOCE) is compromised in aged skeletal muscle but not in young ones. While important, a missing component from previous studies is whether or not SOCE function correlates with contractile function during aging. Here we test the contribution of extracellular Ca(2+) to contractile function of skeletal muscle during aging. First, we demonstrate graded coupling between SR Ca(2+) release channel-mediated Ca(2+) release and activation of SOCE. Inhibition of SOCE produced significant reduction of contractile force in young skeletal muscle, particularly at high frequency stimulation, and such effects were completely absent in aged skeletal muscle. Our data indicate that SOCE contributes to the normal physiological contractile response of young healthy skeletal muscle and that defective extracellular Ca(2+) entry through SOCE contributes to the reduced contractile force characteristic of aged skeletal muscle.

Chronic Helicobacter Pylori Infection and Ischemic Stroke Subtypes

Chronic infection by Helicobacter pylori is regarded as an etiological factor for vascular diseases. However, there are conflicting results on the relevance of chronic infection by Helicobacter pylori as a risk factor for ischemic stroke. The aim of our study was to investigate the association between Helicobacter pylori infection and ischemic stroke subtypes in Chinese.

Derivation of Multipotent Nestin(+)/CD271 (-)/STRO-1 (-) Mesenchymal-like Precursors from Human Embryonic Stem Cells in Chemically Defined Conditions

The successful establishment of stem cell-based therapies requires multipotent, immunocompatible stem cells, highly efficient strategies for direct differentiation, and most importantly, optimal culture conditions for large-scale expansion of such cell populations. Other than adult tissues, human embryonic stem cells (hESCs) represent another infinitely expansible source for mesenchymal stem cell (MSC) derivation. Here, we reproducibly derived a population of Nestin(+)/CD271(-)/STRO-1(-) mesenchymal-like precursors from hESCs (hESC-MPs) in chemically defined conditions, without requiring any serum or serum replacement of animal origin, based on a Y-27632-assisted monolayer culture system. These cells showed slim fibroblastic morphology, and satisfied the criteria of MSCs including self-renewal, the expression of multiple MSC-specific markers and the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Compared with previously reported hESC-derived MSCs, our hESC-MPs were more multipotent, and could differentiate into representative derivatives of all three embryonic germ layers including mature smooth muscle cells, cardiomyocytes, functional hepatocytes and neural cells expressing various neurotransmitter phenotypes, making them an attractive cell source for regenerative medicine.

Molecular Architecture of Ca2+ Signaling Control in Muscle and Heart Cells

Ca2+ signaling in skeletal and cardiac muscles is a bi-directional process that involves cross-talk between signaling molecules in the sarcolemmal membrane and Ca2+ release machinery in the intracellular organelles. Maintenance of a junctional membrane structure between the sarcolemmal membrane and the sarcoplasmic reticulum (SR) provides a framework for the conversion of action potential arrived at the sarcolemma into release of Ca2+ from the SR, leading to activation of a variety of physiological processes. Activity-dependent changes in Ca2+ storage inside the SR provides a retrograde signal for the activation of store-operated Ca2+ channel (SOC) on the sarcolemmal membrane, which plays important roles in the maintenance of Ca2+ homeostasis in physiology and pathophysiology. Research progress during the last 30 years had advanced our understanding of the cellular and molecular mechanisms for the control of Ca2+ signaling in muscle and cardiovascular physiology. Here we summarize the functions of three key molecules that are located in the junctional membrane complex of skeletal and cardiac muscle cells: junctophilin as a "glue" that physiologically links the SR membrane to the sarcolemmal membrane for formation of the junctional membrane framework, mitsugumin29 as a muscle-specific synaptophysin family protein that contributes to maintain the coordinated Ca2+ signaling in skeletal muscle, and TRIC as a novel cation-selective channel located on the SR membrane that provides counter-ion current during the rapid process of Ca2+ release from the SR.

Effect of Dual-specificity Protein Phosphatase 5 on Pluripotency Maintenance and Differentiation of Mouse Embryonic Stem Cells

The MAPK/Erk signaling pathway is considered as a key regulator of the pluripotency and differentiation of embryonic stem (ES) cells, while dual-specificity protein phosphatases (DUSPs) are negative regulators of MAPK. Although DUSPs are potential embryogenesis regulators, their functions in the regulation of ES cell differentiation have not been demonstrated. The present study revealed that Dusp5 was expressed in mouse ES (mES) cells and that its expression was correlated with the undifferentiated state of these cells. Exogenous Dusp5 expression enhanced mES cell clonogenicity and suppressed mES cell differentiation by maintaining Nanog expression via the inhibition of the Erk pathway. Following Dusp5 knockdown, Nanog and Oct4 expression was significantly attenuated and the Erk signaling pathway was activated. Additionally, EBs derived from Dusp5 knockdown mES cells (KDEBs) exhibited a weak adherence capability, very little outgrowth, and a reduction in the number of epithelial-like cells. The expression of Gata6 (an endodermal marker) and Flk1 and Twist1 (mesodermal markers) was inhibited in KDEBs, which indicated that Dusp5 influenced the differentiation of these germ layers during EB development. Collectively, this study suggested that Dusp5 plays an important role in the maintenance of pluripotency in mES cells, and that Dusp5 may be required for EB development.

[Effects of Glycyrrhiza Extract on Pharmacokinetics Property of Daphnetin in Rats]

To research the influence of glycyrrhiza extract on the pharmacokinetics characteristic parameters of daphnetin, which was aimed to explore the rationality of concert application of drugs.

N-acetyl-L-cysteine Enhances the Osteogenic Differentiation and Inhibits the Adipogenic Differentiation Through Up Regulation of Wnt 5a and Down Regulation of PPARG in Bone Marrow Stromal Cells

Nowadays, the treatment of osteoporosis is still a great challenge in the medical field. The combination of enhancement of osteogenesis and the inhibition of adipogenesis of bone marrow stromal cells (BMSCs) is considered an efficient therapeutic strategy for the treatment of osteoporosis. In the present study, we investigated the effects of N-acetyl-L-cysteine (NAC) on the proliferation, osteogenesis and adipogenesis of BMSCs. NAC treatment enhanced the alkaline phosphatase activity, mineral deposition and mRNA expression levels of osteogenesis markers collagen I, osteopontin, and signal pathway related protein Wingless-type family member 5a in addition to Wingless-type family member 3a during osteogenic induction, and inhibited the accumulation of lipid droplets and the expression levels of lipoprotein lipase, fatty acid binding protein 4 and peroxisome proliferator-activated receptor gamma mRNA during adipogenic induction. Meanwhile, NAC had the same effects as enhancing mineral deposition in regular culture condition. In addition, cell proliferation was also promoted by NAC treatment in regular culture condition. These results suggested that NAC may enhance osteogenic differentiation and inhibit adipogenic differentiation of BMSCs, which is at least partially mediated by up regulating Wnt 5a and down regulating PPARG. Taking into account the extensive protective effects of NAC and that the maintenance of BMSCs number is an important factor in osteoporosis prevention and treatment, these observations suggested that NAC is a promising potential drug for the prevention and treatment of osteoporosis and its associated diseases.

Relationship Between (210)Pb(ex) Activity and Sedimentary Organic Carbon in Sediments of 3 Chinese Lakes

This report demonstrates that organic matter was an important factor in lake sediment (210)Pb(ex) dating. Sediment cores from lakes in central and western China with different-trophic levels were collected, and the (210)Pb(ex) activity and total organic carbon (TOC) were measured. The Rock-Eval pyrolysis technique was used to deconvolute TOC into free hydrocarbons (S1), thermally less-stable macromolecular organic matter (S2a), kerogen (S2b), and residual carbon (RC). The results show significant correlations between TOC and (210)Pb(ex), particularly between S2a and (210)Pb(ex), in all the sediment cores. This indicated that the algal-derived organic component S2a may play the most important role in controlling the distribution of (210)Pb(ex). Scavenging by algal-derived organic matter may be the main mechanism. As chronology is the key to the understanding of pollution reconstruction and early diagenesis in sediments, more attention should be paid to the influence of organic matter on (210)Pb(ex).

Store-operated Calcium Entry is Present in HL-1 Cardiomyocytes and Contributes to Resting Calcium

Store-operated Ca(2+) entry (SOCE) has recently been shown to be of physiological and pathological importance in the heart, particularly during cardiac hypertrophy. However, measuring changes in intracellular Ca(2+) during SOCE is very difficult to study in adult primary cardiomyocytes. As a result there is a need for a stable and reliable in vitro model of SOCE which can be used to test cardiac drugs and investigate the role of SOCE in cardiac pathology. HL-1 cells are the only immortal cardiomyocyte cell line available that continuously divides and spontaneously contracts while maintaining phenotypic characteristics of the adult cardiomyocyte. To date the role of SOCE has not yet been investigated in the HL-1 cardiac cell line. We report for the first time that these cells expressed stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel Orai1, which are essential components of the SOCE machinery. In addition, SOCE was tightly coupled to sarcoplasmic reticulum (SR)-Ca(2+) release in HL-1 cells, and such response was not impaired in the presence of voltage dependent Ca(2+) channels (L-type and T-type channels) or reverse mode Na(+)/Ca(2+) exchanger (NCX) inhibitors. We were able to abolish the SOCE response with known SOCE inhibitors (BTP-2 and SKF-96365) and by targeted knockdown of Orai1 with RNAi. In addition, knockdown of Orai1 resulted in lower baseline Ca(2+) and an attenuated response to thapsigargin (TG) and caffeine, indicating that SOCE may play a role in Ca(2+) homeostasis during unstressed conditions in cardiomyocytes. Currently, there is little knowledge about SOCE in cardiomyocytes, and the present results suggest that HL-1 cells will be of great utility in investigating the role of SOCE in the heart.

Relationship Between Various Deformation-induced Products and Mechanical Properties in Metastable Ti-30Zr-Mo Alloys for Biomedical Applications

Nowadays, there is a significant research focus on the development of bio-implant materials that have not only a low Young's modulus but also other unique characteristics such as a changeable Young's modulus and the ability to prevent calcium phosphate formation. Taking advantage of deformation-induced phases is an effective way to obtain the changeable Young's modulus. This study investigated the relationship between the various deformation-induced products and the mechanical properties-including Young's modulus, microstructure, and tensile properties-of Ti-30Zr-(5,6,7)mass%Mo alloys subjected to solution treatment (ST) and cold-rolling (CR). After ST, each alloy is composed of a β phase and a small amount of athermally formed ω phase, and exhibits a low Young's modulus. During CR, deformation-induced phase transformation occurs in all the alloys. The change in Young's modulus due to CR is highly dependent on the types of deformation-induced products. The decrease in Young's modulus due to CR is related to the deformation-induced α' phase transformation accompanying with the disappearance of athermal ω phase, and the increase in Young's modulus is attributed to the deformation-induced ω phase, which mainly exists in {332}β mechanical twins.

Sedimentary Record of Polycyclic Aromatic Hydrocarbons in Lake Erhai, Southwest China

The temporal distribution of polycyclic aromatic hydrocarbons (PAHs) was investigated in a sediment core from Lake Erhai in Southwest China using gas-chromatography/mass spectrometry (GC/MS) method. The total organic carbon (TOC) normalized total PAHs concentrations (sum of US Environmental Protection Agency proposed 16 priority PAHs) ranged from 31.9 to 269 microg/g dry weight (dw), and were characterized by a slowly increasing stage in the deeper sediments and a sharp increasing stage in the upper sediments. The PAHs in the sediments were dominated by low molecular weight (LMW) PAHs, suggesting that the primary source of PAHs was low- and moderate temperature combustion processes. However, both the significant increase in high molecular weight (HMW) PAHs in the upper sediments and the vertical profile of diagnostic ratios pointed out a change in the sources of PAHs from low-temperature combustion to high-temperature combustion. The ecotoxicological assessment based on consensus-based sediment quality guidelines implied that potential adverse biological impacts were possible for benzo(ghi)perylenelene and most LMW PAHs. In addition, the total BaP equivalent quotient of seven carcinogenic polycyclic aromatic hydrocarbons (BaA, CHr, BbF, BkF, BaP, DBA and INP) was 106.1 ng/g, according to the toxic equivalency factors. Although there was no great biological impact associated with the HMW PAHs, great attention should be paid to these PAH components based on their rapid increase in the upper sediments.

Picogram Per Liter Level Determination of Hydroxylated Polybrominated Diphenyl Ethers in Water by Liquid Chromatography-electrospray Tandem Mass Spectrometry

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) have obtained increasing attention; however, few analytical methods are available for sensitive identification of these compounds in water. In this paper, we developed a highly sensitive method for simultaneous determination of nine OH-PBDEs in water by using hydrophilic-lipophilic balanced cartridge extraction, silica cartridge purification, and the combination of derivatization with liquid chromatography-electrospray tandem mass spectrometry. The effective sample pretreatment process and greatly increased instrumental sensitivity by derivatization allow for the quantification of nine OH-PBDEs at a method detection limit of 0.04-3.5pg/L with 1-L wastewater treatment plant effluent or river water. This method was applied to wastewater effluent and river water samples collected in Beijing, China, where two OH-tetraPBDEs were detected at concentrations ranging from 0.63 to 1.0pg/L (5-OH-BDE-47) and from 0.92 to 1.3pg/L (6-OH-BDE-47).

Polybrominated Diphenyl Ethers and Decabromodiphenylethane in Sediments from Twelve Lakes in China

Sediment cores from 12 Chinese lakes were analyzed to investigate the historical inputs of polybrominated diphenyl ethers (PBDEs) and decabromodiphenylethane (DBDPE) during the past few decades. Concentrations of ΣPBDE(1) (sum of tri- to hepta-BDEs), ΣPBDE(2) (sum of nona- to deca-BDEs) and DBDPE in the surface sediments were 0.02-0.29 ng g(-1), 0.46-46.6 ng g(-1) and 1.02-3.64 ng g(-1), respectively. The temporal trends of PBDEs and DBDPE followed a general increase from the bottom to the surface. The calculated fluxes for ΣPBDE(1), ΣPBDE(2) and DBDPE were 0.001-0.09, 0.03-4.24, and 0.05-0.31 ng cm(-2) yr(-1), and the inventories were 0.09-7.86, 0.91-461, and 3.83-24.6 ng cm(-2), respectively. The urbanization and industrialization are highly related to the contamination of PBDEs and DBDPE in sediments. The DBDPE input in recent years was still lower than ΣPBDE(2) but the temporal trends indicated that the contamination would increase with the increasing usage of DBDPE in the future.

Improved Motion Correction Capabilities for Fast Spin Echo T(1) FLAIR Propeller Using Non-Cartesian External Calibration Data Driven Parallel Imaging

Patient motion is a common challenge in the clinical setting and fast spin echo longitudinal relaxation time fluid attenuating inversion recovery imaging method with motion correction would be highly desirable. The motion correction provided by transverse relaxation time- and diffusion-weighted periodically rotated overlapping parallel lines with enhanced reconstruction methods has seen significant clinical adoption. However, periodically rotated overlapping parallel lines with enhanced reconstruction with fast spin echo longitudinal relaxation time fluid attenuating inversion recovery-weighting has proved challenging since motion correction requires wide blades that are difficult to acquire while also maintaining short echo train lengths that are optimal for longitudinal relaxation time fluid attenuating inversion recovery-weighting. Parallel imaging provides an opportunity to increase the effective blade width for a given echo train lengths. Coil-by-coil data-driven autocalibrated parallel imaging methods provide greater robustness in the event of motion compared to techniques relying on accurate coil sensitivity maps. However, conventional internally calibrated data-driven parallel imaging methods limit the effective acceleration possible for each blade. We present a method to share a single calibration dataset over all imaging blades on a slice by slice basis using the APPEAR non-Cartesian parallel imaging method providing an effective blade width increase of 2.45×, enabling robust motion correction. Results comparing the proposed technique to conventional Cartesian and periodically rotated overlapping parallel lines with enhanced reconstruction methods demonstrate a significant improvement during subject motion and maintaining high image quality when no motion is present in normal and clinical volunteers. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.

Taurine Postponed the Replicative Senescence of Rat Bone Marrow-derived Multipotent Stromal Cells in Vitro

The aging of many mammalian tissues is associated with replicative decline in somatic stem cells. Postponing this decline is a direct way of anti-aging. Bone marrow-derived multipotent stromal cells (BMSCs) hold promise for an increasing list of therapeutic uses due to their multilineage potential. Clinical application of BMSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often facing the replicative senescence problem. We demonstrated that taurine exhibited anti-replicative senescence effect on rat BMSCs by promoting colony forming unit-fibroblast formation and cell proliferation, shortening cell population doubling time, enormously inhibiting senescence-associated beta-galactosidase activity and slowing the loss of differentiation potential, while having no significant effect on the maximum passage number and total culture time, and slight influences on the cell surface CD molecules expressions. Taurine is a quite safe antioxidant and nutrient extensively used in food addition and clinical treatment. These suggested that taurine is a promising anti-replicative senescence additive for ex vivo expansion of BMSCs in experimental and clinical cell therapies.

Parathyroid Hormone Induces Differentiation of Mesenchymal Stromal/stem Cells by Enhancing Bone Morphogenetic Protein Signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1(+) CD45(-) CD11b(-) MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of (125) I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1(+) CD45(-) CD11b(-) MSCs into the bone marrow cavity of Rag2(-/-) immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling.

Decreased Expression of Survivin, Estrogen and Progesterone Receptors in Endometrial Tissues After Radiofrequency Treatment of Dysfunctional Uterine Bleeding

ABSTRACT: BACKGROUND: The purpose of the research is to study the histopathology and expression of survivin, estrogen and progesterone receptors (ER/PR) in the endometrium of patients with dysfunctional uterine bleeding (DUB) treated with adiofrequency endometrial ablation (REA). METHODS: A total of 98 DUB patients were enrolled in this case-control study. Among them, 66 underwent REA treatment and 32 optioned for hormone therapy as the control group. Immunohistochemical analysis for survivin, ER and PR expression was carried out on endometrial tissue samples collected before and 6 to 7 months after treatment for both groups. RESULTS: Both hormone and REA treatment ameliorated menstrual bleeding of DUB patients, with the latter showing a significantly higher effective rate. Endometrial surface tissue was replaced by fibrosis tissue in the REA treatment group. REA treatment also significantly reduced the expression of survivin, ER, and PR. Endometrial surface tissues collected from the hormonetreated control group neither showed any apparent morphological alteration nor in the expression of those receptors. CONCLUSIONS: REA treatment changed endometrial surface tissue type from gland rich to gland poor, and significantly decreased the expression of survivin, ER, and PR. This may be an important contributing mechanism for the long-term curative effect and prevention of DUB recurrence.

Recombinant MG53 Protein Modulates Therapeutic Cell Membrane Repair in Treatment of Muscular Dystrophy

Mitsugumin 53 (MG53), a muscle-specific TRIM family protein, is an essential component of the cell membrane repair machinery. Here, we examined the translational value of targeting MG53 function in tissue repair and regenerative medicine. Although native MG53 protein is principally restricted to skeletal and cardiac muscle tissues, beneficial effects that protect against cellular injuries are present in nonmuscle cells with overexpression of MG53. In addition to the intracellular action of MG53, injury to the cell membrane exposes a signal that can be detected by MG53, allowing recombinant MG53 protein to repair membrane damage when provided in the extracellular space. Recombinant human MG53 (rhMG53) protein purified from Escherichia coli fermentation provided dose-dependent protection against chemical, mechanical, or ultraviolet-induced damage to both muscle and nonmuscle cells. Injection of rhMG53 through multiple routes decreased muscle pathology in the mdx dystrophic mouse model. Our data support the concept of targeted cell membrane repair in regenerative medicine, and present MG53 protein as an attractive biological reagent for restoration of membrane repair defects in human diseases.

The Detection of HTERC Amplification Using Fluorescence in Situ Hybridization in the Diagnosis and Prognosis of Cervical Intraepithelial Neoplasia: a Case Control Study

ABSTRACT: BACKGROUND: Currently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN) and cervical cancer are Thinprep cytology test (TCT) and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients' prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC) in the diagnosis and prognosis of CIN. METHODS: The study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n = 11) or radical surgery (n = 14). hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women. RESULTS: The percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2% [PLUS-MINUS SIGN] 4.6% and 44.4% (12/27) respectively in patients with CIN I; 16.0% [PLUS-MINUS SIGN] 14.4% and 85.1% (46/54) in patients with CIN II/III; 19.7% [PLUS-MINUS SIGN] 13.3% and 88.3% (15 /17) in patients with carcinoma in situ; 47.0% [PLUS-MINUS SIGN] 25.2% and 100% (28/28)in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P <0.01), and between the patients with various diseases within the study group (P <0.05). CONCLUSION: The detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.

Ultrastrong Fibers Assembled from Giant Graphene Oxide Sheets

Continuous, ultrastrong graphene fibers are achieved by wet-spinning of giant graphene oxide liquid crystals, followed by wet-drawing and ion-cross-linking. The giant size and regular alignment of graphene sheets render the fibers with high mechanical strength and good conductivity. Such graphene fibers promise wide applications in functional textiles, flexible and wearable sensors, and supercapacitor devices.

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