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Find video protocols related to scientific articles indexed in Pubmed.
Enhancement of 1.53???m emission in erbium/cerium-doped germanosilicate glass pumped by common 808??nm laser diode.
Appl Opt
PUBLISHED: 10-17-2014
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Erbium-doped germanosilicate glasses with various cerium ions contents have been prepared. Optical absorption and 1.53 ?m emission spectra were measured to characterize the spectroscopic performances of prepared samples. A detailed study of 1.53 ?m spectroscopic properties was carried out when pumped by an 808 nm laser diode. Moreover, an energy level diagram and an energy transfer mechanism between Er3+ and Ce3+ were proposed to elucidate the enhanced 1.53 ?m fluorescence. It is found that the prepared samples have optimal spectroscopic properties when the Ce3+ concentration is fixed to 0.5 mol. %. High spontaneous radiative transition probability (172.66??s-1), large effective emission bandwidth (74 nm), and emission cross section (9.49×10-21??cm2 indicate that 808 nm pumped Er3+/Ce3+ codoped germanosilicate glass might be a suitable material for a broadband optical amplifier.
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A Gain-of-Function Mutation in Tnni2 Impeded Bone Development through Increasing Hif3a Expression in DA2B Mice.
PLoS Genet.
PUBLISHED: 10-01-2014
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Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.
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Mid-infrared fluorescence, energy transfer process and rate equation analysis in Er(3+) doped germanate glass.
Sci Rep
PUBLISHED: 05-30-2014
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Er(3+) doped Y2O3 and Nb2O5 modified germanate glasses with different Er(3+) concentrations were prepared. J-O intensity parameters were computed to estimate the structural changes due to the additions of Y2O3 and Nb2O5. The main mid-infrared spectroscopic features were investigated. To shed light on the observed mid-infrared radiative behavior, 975?nm and 1.53??m emission spectra along with their decay lifetimes were also discussed. Moreover, the energy transfer processes of (4)I11/2 and (4)I13/2 level were quantitatively analyzed. In view of the experimental lifetimes, the simplified rate equation was utilized to calculate the energy transfer upconversion processes of upper and lower laser level of 2.7??m emission. The theoretical calculations are in good agreement with the observed 2.7??m fluorescence phenomena. Finally, the stimulated emission and gain cross sections were calculated and the results indicate that Er(3+) doped germanate glasses have great potential for mid-infrared application.
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Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome.
Nature
PUBLISHED: 05-07-2014
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Cytosolic inflammasome complexes mediated by a pattern recognition receptor (PRR) defend against pathogen infection by activating caspase 1. Pyrin, a candidate PRR, can bind to the inflammasome adaptor ASC to form a caspase 1-activating complex. Mutations in the Pyrin-encoding gene, MEFV, cause a human autoinflammatory disease known as familial Mediterranean fever. Despite important roles in immunity and disease, the physiological function of Pyrin remains unknown. Here we show that Pyrin mediates caspase 1 inflammasome activation in response to Rho-glucosylation activity of cytotoxin TcdB, a major virulence factor of Clostridium difficile, which causes most cases of nosocomial diarrhoea. The glucosyltransferase-inactive TcdB mutant loses the inflammasome-stimulating activity. Other Rho-inactivating toxins, including FIC-domain adenylyltransferases (Vibrio parahaemolyticus VopS and Histophilus somni IbpA) and Clostridium botulinum ADP-ribosylating C3 toxin, can also biochemically activate the Pyrin inflammasome in their enzymatic activity-dependent manner. These toxins all target the Rho subfamily and modify a switch-I residue. We further demonstrate that Burkholderia cenocepacia inactivates RHOA by deamidating Asn?41, also in the switch-I region, and thereby triggers Pyrin inflammasome activation, both of which require the bacterial type VI secretion system (T6SS). Loss of the Pyrin inflammasome causes elevated intra-macrophage growth of B. cenocepacia and diminished lung inflammation in mice. Thus, Pyrin functions to sense pathogen modification and inactivation of Rho GTPases, representing a new paradigm in mammalian innate immunity.
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Tunable mid-infrared luminescence from Er(3+) -doped germanate glass.
Luminescence
PUBLISHED: 02-13-2014
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Er(3+) -doped germanate glasses with superior thermal stability were prepared. Judd-Ofelt intensity parameters and important spectroscopic properties were discussed in detail. Upon 800 nm and 980 nm LD pumping, 2.7 µm fluorescence characteristics were investigated and it was found that the effective 2.7 µm emission bandwidth can reach to 101.79 nm in prepared glasses. The tunability of the 2.7 µm emission band can be realized by adjusting the Er(3+) content. Moreover, a high-emission cross-section (11.09 ×10(-21) cm(2) ), large gain bandwidth (772.30 ×10(-28) cm(3) ) and gain coefficient (6.72 cm(-1) ) were obtained in the prepared sample. Hence, Er(3+) -doped germanate glass might be a promising mid-infrared material for tunable amplifiers or lasers. Copyright © 2014 John Wiley & Sons, Ltd.
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Self-adaptive strain-relaxation optimization for high-energy lithium storage material through crumpling of graphene.
Nat Commun
PUBLISHED: 01-23-2014
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High-energy lithium battery materials based on conversion/alloying reactions have tremendous potential applications in new generation energy storage devices. However, these applications are limited by inherent large volume variations and sluggish kinetics. Here we report a self-adaptive strain-relaxed electrode through crumpling of graphene to serve as high-stretchy protective shells on metal framework, to overcome these limitations. The graphene sheets are self-assembled and deeply crumpled into pinecone-like structure through a contraction-strain-driven crumpling method. The as-prepared electrode exhibits high specific capacity (2,165?mAh?g(-1)), fast charge-discharge rate (20?A?g(-1)) with no capacity fading in 1,000 cycles. This kind of crumpled graphene has self-adaptive behaviour of spontaneous unfolding-folding synchronized with cyclic expansion-contraction volumetric variation of core materials, which can release strain and maintain good electric contact simultaneously. It is expected that such findings will facilitate the applications of crumpled graphene and the self-adaptive materials.
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Analysis of structure origin and luminescence properties of Yb(3+)-Er(3+) co-doped fluorophosphate glass.
Spectrochim Acta A Mol Biomol Spectrosc
PUBLISHED: 01-17-2014
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The near infrared luminescence properties of Yb(3+)-Er(3+) co-doped fluorophosphate glasses have been investigated. The various effects on structure and 1.53 ?m emission were analyzed as a function of Yb(3+) concentration. The energy transfer mechanism was proposed. High measured lifetime (10.75 ms), large effective full widths at half maximum (73.71 nm) and large gain per unit length (62.8 × 10(-)(24)cm(2)s) have been achieved in prepared glass. The present glass co-doped with 6mol% YbF3 and 2 mol% ErF3 showed magnificent luminescence properties for telecommunication application.
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Reduced syncytin-1 expression in choriocarcinoma BeWo cells activates the calpain1-AIF-mediated apoptosis, implication for preeclampsia.
Cell. Mol. Life Sci.
PUBLISHED: 01-12-2014
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Placentas associated with preeclampsia are characterized by extensive apoptosis in trophoblast lineages. Syncytin-1 (HERVWE1) mediates the fusion of cytotrophoblasts to form syncytiotrophoblasts, which assume the placental barrier, fetal-maternal exchange and endocrine functions. While decreased syncytin-1 expression has been observed in preeclamptic placentas, it is not clear if this alteration is involved in trophoblast apoptosis. In the current study, we found that siRNA-mediated knockdown of syncytin-1 led to apoptosis in choriocarcinoma BeWo, a cell line of trophoblastic origin. Characterization of the apoptotic pathways indicated that this effect does not rely on the activation of caspases. Rather, decreased syncytin-1 levels activated the apoptosis inducing factor (AIF) apoptotic pathway by inducing the expression, cleavage, and nuclear translocation of AIF. Moreover, calpain1, the cysteine protease capable of cleaving AIF, was upregulated by syncytin-1 knockdown. Furthermore, treatment with calpain1 inhibitor MDL28170 effectively reversed AIF cleavage, AIF nuclear translocation, and cell apoptosis triggered by syncytin-1 downregulation, verifying the specific action of calpain1-AIF pathway in trophoblast apoptosis. We confirmed that preeclamptic placentas express lower levels of syncytin-1 than normal placentas, and observed an inverse correlation between syncytin-1 and AIF/calpain1 mRNA levels, a result consistent with the in vitro findings. Immunohistochemistry analyses indicated decreased syncytin-1 and increased AIF and calpain1 protein levels in apoptotic cells of preeclamptic placentas. These findings have for the first time revealed that decreased levels of syncytin-1 can trigger the AIF-mediated apoptosis pathway in BeWo cells. This novel mechanism may contribute to the structural and functional deficiencies of syncytium frequently observed in preeclamptic placentas.
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Integral proteomic analysis of blastocysts reveals key molecular machinery governing embryonic diapause and reactivation for implantation in mice.
Biol. Reprod.
PUBLISHED: 01-01-2014
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Among nearly 100 mammalian species, implantation can be suspended at blastocyst stage for a certain time and reactivated under favorable conditions, a phenomenon known as embryonic diapause. Until now, the underlying molecular mechanism governing embryonic diapause and reactivation for implantation remained largely unknown. Here we conducted the first integral proteomic analysis of blastocysts from diapause to reactivation by using a physiologically relevant mouse delayed implantation model. More than 6000 dormant and reactivated blastocysts were used for the proteomic analysis. A total of 2255 proteins were detected. Various cellular and molecular processes, including protein translation, aerobic glycolysis, pentose phosphate pathway, purine nucleotide biosynthesis, glutathione metabolism, and chromatin organization were identified as differentially regulated. In particular, we demonstrated a remarkable activation of mitochondria in blastocysts upon reactivation from dormancy, highlighting their essential physiological significance. Moreover, the activities of the endosome-lysosome system were prominently enhanced in the mural trophectoderm of reactivated blastocysts, accompanied by active phagocytosis at the fetal-maternal interface, suggesting a critical role in promoting trophoblast invasion. Collectively, we provided an integral proteomic view upon the regulatory network of blastocyst reactivation from diapause, which will help to better interpret the nature of embryonic diapause and reactivation in wild animals and to identify molecular indicators for selecting blastocysts with high implantation competency.
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Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.
PLoS ONE
PUBLISHED: 01-01-2014
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Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.
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Nanowire templated semihollow bicontinuous graphene scrolls: designed construction, mechanism, and enhanced energy storage performance.
J. Am. Chem. Soc.
PUBLISHED: 11-21-2013
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Graphene scrolls have been widely investigated for applications in electronics, sensors, energy storage, etc. However, graphene scrolls with tens of micrometers in length and with other materials in their cavities have not been obtained. Here nanowire templated semihollow bicontinuous graphene scroll architecture is designed and constructed through "oriented assembly" and "self-scroll" strategy. These obtained nanowire templated graphene scrolls can achieve over 30 ?m in length with interior cavities between the nanowire and scroll. It is demonstrated through experiments and molecular dynamic simulations that the semihollow bicontinuous structure construction processes depend on the systemic energy, the curvature of nanowires, and the reaction time. Lithium batteries based on V3O7 nanowire templated graphene scrolls (VGSs) exhibit an optimal performance with specific capacity of 321 mAh/g at 100 mA/g and 87.3% capacity retention after 400 cycles at 2000 mA/g. The VGS also shows a high conductivity of 1056 S/m and high capacity of 162 mAh/g at a large density of 3000 mA/g with only 5 wt % graphene added which are 27 and 4.5 times as high as those of V3O7 nanowires, respectively. A supercapacitor made of MnO2 nanowire templated graphene scrolls (MGSs) also shows a high capacity of 317 F/g at 1A/g, which is over 1.5 times than that of MnO2 nanowires without graphene scrolls. These excellent energy storage capacities and cycling performance are attributed to the unique structure of the nanowire templated graphene scroll, which provides continuous electron and ion transfer channels and space for free volume expansion of nanowires during cycling. This strategy and understanding can be used to synthesize other nanowire templated graphene scroll architectures, which can be extended to other fabrication processes and fields.
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P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium.
Arch. Biochem. Biophys.
PUBLISHED: 10-15-2013
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Cells respond differently to DNA damaging agents, which may related to cell context and differentiation status. The aim of present study was to observe the cellular and molecular responses of cells in different differentiation status to ionizing irradiation (IR).
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A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 08-08-2013
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Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxons rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
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Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay.
Gastroenterology
PUBLISHED: 04-10-2013
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Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.
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Brief report: CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells.
Stem Cells
PUBLISHED: 02-24-2013
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Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive "active" stem cells as well as HOPX positive "facultative" stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 (CD24-/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease.
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Syncytin-1 modulates placental trophoblast cell proliferation by promoting G1/S transition.
Cell. Signal.
PUBLISHED: 01-08-2013
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Placental syncytiotrophoblasts formed by the fusion of cytotrophoblasts constitute the interface between maternal and fetal circulations. The syncytium, composed of a continuous layer of syncytiotrophoblasts, assumes the fetal-maternal nutrient exchange, placental barrier, and endocrine functions important for the maintenance of normal pregnancy. Syncytin-1, an endogenous retroviral gene product, mediates the fusion of cytotrophoblasts. While the fusogenic function of syncytin-1 has been well established, little is known regarding its nonfusogenic activities. This study investigates the role of syncytin-1 in trophoblast proliferation. We found that syncytin-1 knockdown significantly inhibited BeWo cell growth and DNA synthesis. Moreover, time course studies on key cell cycle regulators demonstrated an upregulation of p15 and downregulation of CDK4, E2F1, PCNA, and c-Myc, which consequently led to a reduced level of CDK1. These results, together with those from flow cytometry analysis, indicated that syncytin-1 knockdown blocked the G1/S transition phase of the cell cycle. Moreover, syncytin-1 overexpression promoted CHO cell proliferation and led to changes opposite to those observed in syncytin-1 knockdown experiments, confirming the critical role of syncytin-1 for G1/S transition. Thus, syncytin-1, through both nonfusogenic and fusogenic, functions, may co-regulate the input (proliferation) and output (fusion) of the cytotrophoblast "pool". Such co-regulation could be an efficient way to achieve the balance between these two opposing processes, which is required for syncytium homeostasis. Since decreased syncytin-1 expression has been shown to be associated with preeclamptic and hypoxic condition, insufficient replenishing of the cytotrophoblast "pool" may contribute to syncytium deficiency, a critical pathological change frequently found in preeclamptic placentas.
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Wnt6 is essential for stromal cell proliferation during decidualization in mice.
Biol. Reprod.
PUBLISHED: 01-01-2013
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Postimplantation uterine development involves extensive stromal cell proliferation and decidual transformation with polyploidization, which is essential for normal pregnancy establishment. However, it remains largely unknown how stromal proliferation versus decidual polyploidization is differentially regulated during decidualization. Utilizing Wnt6-mutant mice, we show here that Wnt6 deficiency impairs stromal cell proliferation without much adverse effects on decidual polyploidization. Applying a primary stromal cell culture model, we further reveal that loss of Wnt6 prolongs the cell cycle length via downregulating cyclin B1 expression, thus attenuating stromal cell proliferation. Our study provides the first genetic evidence that Wnt6 is critical for normal stromal cell proliferation in mice, highlighting the concept that there are differential machineries governing the process of stromal cell proliferation versus decidual transformation during early pregnancy. This finding has high clinical relevance because Wnt signaling is known to be important for human implantation and endometrial function.
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Topical application of hPDGF-A-modified porcine BMSC and keratinocytes loaded on acellular HAM promotes the healing of combined radiation-wound skin injury in minipigs.
Int. J. Radiat. Biol.
PUBLISHED: 06-02-2011
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To evaluate the efficacy of cultured cutaneous substitute (CCS) in accelerating the healing of combined radiation-skin wound injury (CRWI) in minipigs.
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Schwann cells promote neurite outgrowth of dorsal root ganglion neurons through secretion of nerve growth factor.
Indian J. Exp. Biol.
PUBLISHED: 04-02-2011
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The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.
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Active deformation of apoptotic intestinal epithelial cells with adhesion-restricted polarity contributes to apoptotic clearance.
Lab. Invest.
PUBLISHED: 11-01-2010
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Dying epithelial cells are thought to be squeezed out of the epithelium by the contraction of an actomyosin ring formed in live neighboring cells, which simultaneously closes any potential gap, thereby maintaining the integrity of the epithelial layer. The shrinkage and contraction of apoptotic cells contribute little to the extrusion process. In contrast, the clearance of dying intestinal columnar epithelial cells in vivo usually leaves a transient gap via an unknown mechanism. By using freshly isolated small intestinal villus units with or without basal lamina, we found that the nucleus of apoptotic enterocytes moved apically until they budded off, leaving the cytoplasmic residue in the transient gap. Apical polarity of nucleus movement was restricted unless the basal lamina was artificially removed. F-actin mainly accumulated in apoptotic cells rather than neighboring live cells, even after the addition of resistance force against extrusion. The actin accumulation in apoptotic cells does not depend on the living state of neighboring cells. Apoptotic cells can complete the shedding process when neighboring a goblet cell, as the majority of space is occupied by mucin granules and the cytoplasm consists of intermediate filaments and microtubules, but lacks F-actin. We found that the elongation and deformation of apoptotic cells depend on the stretching force generated inside the cell, rather than the force generated by neighboring cells extending. Our findings clearly demonstrate that intestinal epithelial shedding does not depend on the formation and contraction of an actomyosin ring in live neighboring cells. Apoptotic epithelial cells may undergo an active process of cell deformation with adhesion-restricted polarity, which may contribute to maintaining barrier function during a high rate of cellular turnover.
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Novel importin-alpha family member Kpna7 is required for normal fertility and fecundity in the mouse.
J. Biol. Chem.
PUBLISHED: 08-10-2010
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Nuclear importing system and nuclear factors play important roles in mediating nuclear reprogramming and zygotic gene activation. However, the components and mechanisms that mediate nuclearly specific targeting of the nuclear proteins during nuclear reprogramming and zygotic gene activation remain largely unknown. Here, we identified a novel member of the importin-? family, AW146299(KPNA7), which is predominantly expressed in mouse oocytes and zygotes and localizes to the nucleus or spindle. Mutation of Kpna7 gene caused reproductivity reduction and sex imbalance by inducing preferential fetal lethality in females. Parthenogenesis analysis showed that the cell cycle of activated one-cell embryos is loss of control and ahead of schedule but finally failed to develop into blastocyst stage. Further RT-PCR and epigenetic modification analysis showed that knocking out of Kpna7 induced abnormalities of gene expression (dppa2, dppa4, and piwil2) and epigenetic modifications (down-regulation of histone H3K27me3). Biochemical analysis showed that KPNA7 interacts with KPNB1 (importin-?1). In summary, we identified a novel Kpna7 gene that is required for normal fertility and fecundity.
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A synthetic cantharidin analog for the enhancement of doxorubicin suppression of stem cell-derived aggressive sarcoma.
Biomaterials
PUBLISHED: 07-02-2010
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Failure to cure many cancers once they are disseminated has been attributed to the presence of resistant cancer stem cells. Cantharidin, a natural compound isolated from the beetles and other insects has been traditionally used as anticancer agent, but limited by its significant toxicity. It has shown that cantharidin can force cancer cells prematurely into cell cycle and subsequently induce apoptotic cell death through the inhibition of protein phosphatase 2A (PP2A). In this study, we showed that a synthesized analog of cantharidin, LB1, with significant PP2A inhibition activity but without apparent toxicity, greatly enhanced the effectiveness of the standard anti-sarcoma chemotherapeutic agent, doxorubicin (DOX), in the xenograft growth inhibition and lung metastases prevention of an aggressive sarcoma derived from transformed mesenchymal stem cells in syngeneic rats. We report here on the possibility of, pharmacologic inhibition of PP2A with low toxicity cantharidin derivatives may be a useful strategy to enhance the effectiveness of DNA-damaged chemotherapeutic drugs against stem cell-derived cancer.
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Differentiation of embryonic stem cells in adult bone marrow.
J Genet Genomics
PUBLISHED: 05-06-2010
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Embryonic stem cells (ESCs) are a potential source of generating transplantable hematopoietic stem and progenitor cells, which in turn can serve as "seed" cells for hematopoietic regeneration. In this study, we aimed to gauge the ability of mouse ESCs directly differentiating into hematopoietic cells in adult bone marrow (BM). To this end, we first derived a new mouse ESC line that constitutively expressed the green fluorescent protein (GFP) and then injected the ESCs into syngeneic BM via intra-tibia. The progeny of the transplanted ESCs were then analyzed at different time points after transplantation. Notably, however, most injected ESCs differentiated into non-hematopoietic cells in the BM whereas only a minority of the cells acquired hematopoietic cell surface markers. This study provides a strategy for evaluating the differentiation potential of ESCs in the BM micro-environment, thereby having important implications for the physiological maintenance and potential therapeutic applications of ESCs.
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Bone marrow derivation of interstitial cells of cajal in small intestine following intestinal injury.
J. Biomed. Biotechnol.
PUBLISHED: 01-27-2010
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Interstitial cells of Cajal (ICCs) in gastrointestinal tract are specialized cells serving as pacemaker cells. The origin of ICCs is currently not fully characterized. In this work, we aimed to study whether bone marrow-derived cells (BMDCs) could contribute to the origin of ICCs in the muscular plexus of small intestine using GFP-C57BL/6 chimeric mice.Engraftment of BMDCs in the intestine was investigated for GFP expression. GFP positive bone marrow mononuclear cells reached a proportion of 95.65% +/- 3.72% at different times in chimerism. Donor-derived cells distributed widely in all the layers of the gastrointestinal tract. There were GFP positive BMDCs in the myenteric plexus, which resembled characteristics of ICCs, including myenteric location, c-Kit positive staining, and ramified morphology. Donor-derived ICCs in the myenteric plexus contributed to a percentage ranging 9.25% +/- 4.9% of all the ICCs in the myenteric plexus. In conclusion, here we described that donor-derived BMDCs might differentiate into gastrointestinal ICCs after radiation injury, which provided an alternative source for the origin of the ICCs in the muscular plexus of adult intestine. These results further identified the plasticity of BMDCs and indicated therapeutic implications of BMDCs for the gastrointestinal dysmotility caused by ICCs disorders.
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Gonadotropin-regulated expressions of lanosterol 14alpha-demethylase, sterol Delta14-reductase and C-4 sterol methyl oxidase contribute to the accumulation of meiosis-activating sterol in rabbit gonads.
Prostaglandins Other Lipid Mediat.
PUBLISHED: 01-23-2010
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Meiosis-activating sterol (MAS), the intermediate of cholesterol biosynthesis, is an important lipophilic molecule mediating gonadotropins action in inducing oocyte meiotic resumptions in various mammalian species. With respect to MASs physiological relevance during oocyte maturation in the rabbit, early study has demonstrated that luteinizing hormone (LH), but not follicle stimulating hormone (FSH) can induce FF-MAS accumulation facilitating oocyte maturation in rabbits. However, the potential underlying mechanism for the MAS accumulation in the rabbit gonad remained unclear. We hypothesized that differential expression of MAS synthetic and metabolic enzymes would contribute to the timely MAS accumulation in the rabbit gonad. To address this issue, in the present investigation, we first cloned the cDNAs encoding there pre- and post-MAS enzymes, lanosterol 14alpha-demethylase (CYP51), sterol Delta14-reductase (14-SR) and C-4 sterol methyl oxidase (C4MO), respectively, using rapid amplification of cDNA ends (RACE) cloning, and then performed northern hybridization experiments to explore their expression profiles in the rabbit ovary, testis, and various other tissues. We observed that CYP51 expression was significantly upregulated only by LH/hCG in the antral follicle exhibiting its peak levels in preovulatory follicles; whereas both FSH and LH significantly downregulated 14-SR expression with the progression of antral follicular development. These findings provided here novel evidence that an inverse upregulation of CYP51 and downregulation of 14-SR expression under FSH/LH stimulation functions as the machinery for FF-MAS accumulation in preovulatory follicles prior to ovulation in the rabbit.
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More insight into mesenchymal stem cells and their effects inside the body.
Expert Opin Biol Ther
PUBLISHED: 01-22-2010
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The pan-tissue existence and multipotency of differentiation make mesenchymal stem cells (MSCs) an attractive source of cells as tissue repair cells, seeds of engineered tissue, vehicles for gene therapy or in combination to promote tissue regeneration in wound healing and disease recovery.
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Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine.
Anal. Biochem.
PUBLISHED: 09-24-2009
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Gene expression studies in intestinal epithelial and stromal cells are a common tool for investigating the mechanisms by which the homeostasis of the small intestine is regulated under normal and pathological conditions. Quantitative real-time PCR (qPCR) is a sensitive and highly reproducible method of gene expression analysis, with expression levels quantified by normalization against reference genes in most cases. However, the lack of suitable reference genes for epithelial cells with different differentiation states and nonepithelial tissue cells has limited the application of qPCR in gene expression studies of small intestinal samples. In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP, and TUBA1, were analyzed to determine their applicability for isolated crypt cells, villus cells, deepithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expressions of the commonly used reference genes GAPDH, B2M, and ACTB, and ribosomal protein genes RPL13A, RPS29, and RPLP0 were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine.
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Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro.
Differentiation
PUBLISHED: 07-30-2009
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A murine embryonic mesenchymal cell line C3H/10T1/2 possesses the potential to differentiate into multiple cell phenotypes and has been recognized as multipotent mesenchymal stem cells, but no in vitro model of its endothelial differentiation has been established and the effect of angiogenic factors on the differentiation is unknown. The aim of the present study was to evaluate the role of angiogenic factors in inducing endothelial differentiation of C3H/10T1/2 cells in vitro. C3H/10T1/2 cells were treated with angiogenic factors, VEGF (10 ng/mL) and bFGF (5 ng/mL). At specified time points, cells were subjected to morphological study, immunofluorescence staining, RT-PCR, LDL-uptake tests and 3-D culture for the examination of the structural and functional characteristics of endothelial cells. Classic cobblestone-like growth pattern appeared at 6 day of the induced differentiation. Immunofluorescence staining and RT-PCR analyses revealed that the induced cells exhibited endothelial cell-specific markers such as CD31, von Willebrand factor, Flk1, Flt1, VE-cadherin, Tie2, EphrinB2 and Vezf1 at 9 day. The induced C3H/10T1/2 cells exhibited functional characteristics of the mature endothelial phenotype, such as uptake of acetylated low-density lipoproteins (Ac-LDL) and formation of capillary-like structures in three-dimensional culture. At 9 day, Weibel-Palade bodies were observed under a transmission electron microscope. This study demonstrates, for the first time, endothelial differentiation of C3H/10T1/2 cells induced by angiogenic factors, VEGF and bFGF, and confirms the multipotential differentiation ability. This in vitro model is useful for investigating the molecular events in endothelial differentiation of mesenchymal stem cells.
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Defective chromatin structure in somatic cell cloned mouse embryos.
J. Biol. Chem.
PUBLISHED: 07-14-2009
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Epigenetic reprogramming plays a central role in the development of cloned embryos generated by somatic cell nuclear transfer, and it is believed that aberrant reprogramming leads to the abnormal development of most cloned embryos. Recent studies show that trimethylation of H3K27 (H3K27me3) contributes to the maintenance of embryonic stem cell pluripotency because the differentiation genes are always occupied by nucleosomes trimethylated at H3K27, which represses gene expression. Here, we provide evidence that differential H3K27me3 modification exists between normal fertilization-produced blastocysts and somatic cell nuclear transfer cloned blastocysts; H3K27me3 was specifically found in cells of the inner cell mass (ICM) of normal blastocysts, whereas there was no modification of H3K27me3 in the ICM of cloned blastocysts. Subsequently, we demonstrated that the differentiation-related genes, which are marked by H3K27me3 in embryonic stem cells, were expressed at significantly higher levels in cloned embryos than in normal embryos. The polycomb repressive complex 2 (PRC2) component genes (Eed, Ezh2, and Suz12), which are responsible for the generation of H3K27me3, were expressed at lower levels in the cloned embryos. Our results suggest that reduced expression of PRC2 component genes in cloned embryos results in defective modification of H3K27me3 to the differentiation-related genes in pluripotent ICM cells. This results in premature expression of developmental genes and death of somatic cloned embryos shortly after implantation. Taken together, these studies suggest that H3K27me3 might be an important epigenetic marker with which to evaluate the developmental potential of cloned embryos.
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Differential methylation status of imprinted genes in nuclear transfer derived ES (NT-ES) cells.
Genomics
PUBLISHED: 03-06-2009
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Compared with fertilized embryo derived ES (F-ES) cells, somatic cell nuclear transfer (SCNT) produced ES (NT-ES) cells were proposed appropriate for cell transplantation based therapies. Although previous studies indicated that NT-ES cells and F-ES cells were transcriptionally and functionally indistinguishable, characterization of DNA methylation patterns of imprinted genes in NT-ES cells is lacking. Here, we show that DNA methylation patterns in the differentially methylated region (DMR) of paternally imprinted gene, H19, displayed distinct abnormalities in certain NT-ES and F-ES cell lines after long-term culture in vitro. DNA methylation profiles of H19 appeared very dynamic in most ES cell lines examined, either hypermethylation or hypomethylation could be observed in specific ES cell lines. In contrast to H19, maternally imprinted genes, Mest and Peg3, showed relatively stable methylation patterns in ES cells, especially Peg3, which displayed better capability in enduring long-term culture in vitro. Our results indicate that abnormal methylation profiles of certain imprinted genes could be observed in both NT-ES and F-ES cell lines after long-term culture in vitro although these cell lines were proved to be pluripotent with germline transmission competent. Stringent screening of epigenetically normal NT-ES cells might be potentially necessary for further therapeutic application of these cells.
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Isolation and characterization of novel cellulase genes from uncultured microorganisms in different environmental niches.
Microbiol. Res.
PUBLISHED: 02-20-2009
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Four different environmental DNA libraries were prepared from microbial consortium collected from forest soil, dung of elephant, cow rumen and rotted tree. Seven independent clones specifying cellulase activities (five endo-beta-1,4-glucanases and two beta-glucosidases) were isolated and identified. Sequence analysis of the retrieved genes revealed that the encoded products of these cellulase genes shared less than 50% identities and 70% similarities to cellulases in the databases. Domain analysis predicted that four endo-beta-1,4-glucanases conform to glycolsyl hydrolase family 5 (GHF 5) and one endo-beta-1,4-glucanase to glycolsyl hydrolase family 9 (GHF 9), while both beta-glucosidases belong to glycolsyl hydrolase family 3 (GHF 3). Further sequence analysis indicated that although a solid affiliation could be made for the two endo-beta-1,4-glucanases to the typical ruminal microbe Prevotella ruminicola, the rest formed deep-branched lineages with no close relatives. The revelation of the phylogenetic novelty provided a snapshot on the great diversity of cellulases in these natural environments.
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Long-term repopulation effects of donor BMDCs on intestinal epithelium.
Dig. Dis. Sci.
PUBLISHED: 02-05-2009
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Bone marrow-derived cells (BMDCs) have the ability to differentiate into intestinal epithelial cells after transplantation and participate in the regeneration process of damaged epithelium.
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Increased frequencies of Th17 and Th22 cells in the peripheral blood of patients with secondary syphilis.
FEMS Immunol. Med. Microbiol.
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T-helper (Th) 17 and the more recently identified Th22 cells are of great importance in host defense against pathogens, but can also be responsible for chronic inflammatory disorders. However, the roles of the two cell subsets in syphilis remain elusive. In this study, we show that the frequencies of Th17 and Th22 cells are significantly increased in the peripheral blood of patients with secondary syphilis (SS). A significant positive correlation is observed between Th17 and Th22 cells, whereas a negative correlation exists between Th17 and Th1 cells. Moreover, the frequency of Th17 cells has a significant positive correlation with the plasma interleukin 6 (IL-6) or IL-1? levels, and the frequency of Th22 cells is positively correlated with the IL-6 or IL-23 levels. Finally, the elevated frequencies of Th17 and Th22 cells are positively associated with plasma C-reactive protein levels. Our results suggest that Th17 and Th22 cells may be implicated in the pathogenesis of the SS.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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