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Background mutations in parental cells account for most of the genetic heterogeneity of Induced Pluripotent Stem Cells

Young, Margaret A.; Larson, David E.; Sun, Chiao-Wang; George, Daniel R.; Ding, Li; Miller, Christopher A.; Lin, Ling; Pawlik, Kevin M.; Chen, Ken; Fan, Xian; Schmidt, Heather; Kalicki-Veizer, Joelle; Cook, Lisa L.; Swift, Gary W.; Demeter, Ryan T.; Wendl
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
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To assess the genetic consequences of induced Pluripotent Stem Cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. This data suggests that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which “captures” their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.

Temporal Dissection of K-rasG12D Mutant In Vitro and In Vivo Using a Regulatable K-rasG12D Mouse Allele

Wang, Zuoyun; Feng, Yan; Bardessy, Nabeel; Wong, Kwok-Kin; Liu, Xin-Yuan; Ji, Hongbin
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 11/05/2012 Português
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Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-rasG12D (ER-K-rasG12D) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 µM works optimally for activation of ER-K-rasG12D independent of the gender status. Furthermore, tamoxifen-inducible activation of K-rasG12D promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-rasG12D in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.

Multi-Patterned Dynamics of Mitochondrial Fission and Fusion in a Living Cell

Wang, Shiqi; Xiao, Weiming; Shan, Sicong; Jiang, Chunsun; Chen, Ming; Zhang, Yan; Lü, Shouqin; Chen, Juan; Zhang, Chuanmao; Chen, Quan; Long, Mian
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 23/05/2012 Português
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Mitochondria are highly-dynamic organelles, but it is challenging to monitor quantitatively their dynamics in a living cell. Here we developed a novel approach to determine the global occurrence of mitochondrial fission and fusion events in living human epithelial cells (Hela) and mouse embryonic fibroblast cells (MEF). Distinct patterns of sequential events including fusion followed by fission (Fu-Fi), the so-called “kiss and run” model previously described, fission followed by fusion (Fi-Fu), fusion followed by fusion (Fu-Fu), and fission followed by fission (Fi-Fi) were observed concurrently. The paired events appeared in high frequencies with short lifetimes and large sizes of individual mitochondria, as compared to those for unpaired events. The high frequencies of paired events were found to be biologically significant. The presence of membrane uncoupler CCCP enhanced the frequency of paired events (from both Fu-Fi and Fi-Fu patterns) with a reduced mitochondrial size. Knock-out of mitofusin protein Mfn1 increased the frequency of fission with increased lifetime of unpaired events whereas deletion of both Mfn1 and Mfn2 resulted in an instable dynamics. These results indicated that the paired events were dominant but unpaired events were not negligible...

Embryonic Fibroblasts Enable the Culture of Primary Ovarian Follicles Within Alginate Hydrogels

Tagler, David; Tu, Tao; Smith, Rachel M.; Anderson, Nicholas R.; Tingen, Candace M.; Woodruff, Teresa K.; Shea, Lonnie D.
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
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Hydrogel-encapsulating culture systems support the consistent growth of ovarian follicles from various species, such as mouse, non-human primate, and human; however, further innovations are required for the efficient production of quality oocytes from early-stage follicles. In this report, we investigated the coculture of mouse ovarian follicles with mouse embryonic fibroblasts (MEFs), commonly used as feeder cells to promote the undifferentiated growth of embryonic stem (ES) cells, as a means to provide the critical paracrine factors necessary for follicle survival and growth. Follicles were encapsulated within alginate hydrogels and cocultured with MEFs for 14 days. Coculture enabled the survival and growth of early secondary (average diameter of 90–100 μm) and primary (average diameter of 70–80 μm) follicles, which developed antral cavities and increased in diameter to 251–347 μm. After 14 days, follicle survival ranged from 70% for 100-μm follicles to 23% for 70-μm follicles. Without MEF coculture, all follicles degenerated within 6–10 days. Furthermore, 72%–80% of the oocytes from surviving follicles underwent germinal vesicle breakdown (GVBD), and the percentage of metaphase II (MII) eggs was 41%–69%. Medium conditioned by MEFs had similar effects on survival...

Damage Associated Molecular Pattern Molecule-Induced microRNAs (DAMPmiRs) in Human Peripheral Blood Mononuclear Cells

Unlu, Sebnem; Tang, Siuwah; Wang, E. na; Martinez, Ivan; Tang, Daolin; Bianchi, Marco E.; Zeh, Herbert J.; Lotze, Michael T.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 22/06/2012 Português
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Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10−4 and p<3.7×10−3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1+/+) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1−/− MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic “PAMPmiR”. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate...

Physiochemical, Optical and Biological Activity of Chitosan-Chromone Derivative for Biomedical Applications

Kumar, Santosh; Koh, Joonseok
Fonte: Molecular Diversity Preservation International (MDPI) Publicador: Molecular Diversity Preservation International (MDPI)
Tipo: Artigo de Revista Científica
Publicado em 18/05/2012 Português
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This paper describes the physiochemical, optical and biological activity of chitosan-chromone derivative. The chitosan-chromone derivative gels were prepared by reacting chitosan with chromone-3-carbaldehyde, followed by solvent exchange, filtration and drying by evaporation. The identity of Schiff base was confirmed by UV-Vis absorption spectroscopy and Fourier-transform infrared (FTIR) spectroscopy. The chitosan-chromone derivative was evaluated by X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), photoluminescence (PL) and circular dichroism (CD). The CD spectrum showed the chitosan-chromone derivative had a secondary helical structure. Microbiological screening results demonstrated the chitosan-chromone derivative had antimicrobial activity against Escherichia coli bacteria. The chitosan-chromone derivative did not have any adverse effect on the cellular proliferation of mouse embryonic fibroblasts (MEF) and did not lead to cellular toxicity in MEFs. These results suggest that the chitosan-chromone derivative gels may open a new perspective in biomedical applications.

Differential Roles of Proteasome and Immunoproteasome Regulators Pa28αβ, Pa28γ and Pa200 in the Degradation of Oxidized Proteins

Pickering, Andrew. M.; Davies, Kelvin. J. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The response and functions of proteasome regulators Pa28αβ (or 11S), Pa28γ, and Pa200 in oxidative-stress adaptation (also called hormesis) was studied in murine embryonic fibroblasts (MEF), using a well-characterized model of cellular adaptation to low concentrations (1.0 to 10.0μM) of hydrogen peroxide (H2O2), which alter gene expression profiles, increasing resistance to higher levels of oxidative-stress. Pa28αβ bound to 20S proteasomes immediately upon H2O2-treatment, whereas 26S proteasomes were disassembled at the same time. Over the next 24 hours, the levels of Pa28αβ, Pa28γ, and Pa200 proteasome regulators increased during H2O2-adaptation, whereas the 19S regulator was unchanged. Purified Pa28αβ, and to a lesser extent Pa28γ, significantly increased the ability of purified 20S proteasome to selectively degrade oxidized proteins; Pa28αβ also increased the capacity of purified immunoproteasome to selectively degrade oxidized proteins but Pa28γ did not. Pa200 regulator actually decreased 20S proteasome and immunoproteasome’s ability to degrade oxidized proteins but Pa200 and poly-ADP ribose polymerase may cooperate in enabling initiation of DNA repair. Our results indicate that cytoplasmic Pa28αβ and nuclear Pa28γ may both be important regulators of proteasome’s ability to degrade oxidatively-damaged proteins...

The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome

Fusco, Carmela; Micale, Lucia; Egorov, Mikhail; Monti, Maria; D’Addetta, Ester Valentina; Augello, Bartolomeo; Cozzolino, Flora; Calcagnì, Alessia; Fontana, Andrea; Polishchuk, Roman S.; Didelot, Gerard; Reymond, Alexandre; Pucci, Piero; Merla, Giusepp
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 09/07/2012 Português
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In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.

Thrombospondin-1 Interacts with Trypanosoma cruzi Surface Calreticulin to Enhance Cellular Infection

Johnson, Candice A.; Kleshchenko, Yulia Y.; Ikejiani, Adaeze O.; Udoko, Aniekanabasi N.; Cardenas, Tatiana C.; Pratap, Siddharth; Duquette, Mark A.; Lima, Maria F.; Lawler, Jack; Villalta, Fernando; Nde, Pius N.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 11/07/2012 Português
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Trypanosoma cruzi causes Chagas disease, which is a neglected tropical disease that produces severe pathology and mortality. The mechanisms by which the parasite invades cells are not well elucidated. We recently reported that T. cruzi up-regulates the expression of thrombospondin-1 (TSP-1) to enhance the process of cellular invasion. Here we characterize a novel TSP-1 interaction with T. cruzi that enhances cellular infection. We show that labeled TSP-1 interacts specifically with the surface of T. cruzi trypomastigotes. We used TSP-1 to pull down interacting parasite surface proteins that were identified by mass spectrometry. We also show that full length TSP-1 and the N-terminal domain of TSP-1 (NTSP) interact with T. cruzi surface calreticulin (TcCRT) and other surface proteins. Pre-exposure of recombinant NTSP or TSP-1 to T. cruzi significantly enhances cellular infection of wild type mouse embryo fibroblasts (MEF) compared to the C-terminal domain of TSP-1, E3T3C1. In addition, blocking TcCRT with antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Taken together, our findings indicate that TSP-1 interacts with TcCRT on the surface of T. cruzi through the NTSP domain and that this interaction enhances cellular infection. Thus surface TcCRT is a virulent factor that enhances the pathogenesis of T. cruzi infection through TSP-1...

Efficient Derivation and Genetic Modifications of Human Pluripotent Stem Cells on Engineered Human Feeder Cell Lines

Zou, Chunlin; Chou, Bin-Kuan; Dowey, Sarah N.; Tsang, Kitman; Huang, Xiaosong; Liu, Cyndi F.; Smith, Cory; Yen, Jonathan; Mali, Prashant; Zhang, Yu Alex; Cheng, Linzhao; Ye, Zhaohui
Fonte: Mary Ann Liebert, Inc. Publicador: Mary Ann Liebert, Inc.
Tipo: Artigo de Revista Científica
Português
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Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs...

Family perspectives in lynch syndrome becoming a family at risk, patterns of communication and influence on relations

Bartuma, Katarina; Nilbert, Mef; Carlsson, Christina
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em 25/05/2012 Português
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Loss of manganese superoxide dismutase leads to abnormal growth and signal transduction in mouse embryonic fibroblasts

Zhang, Yiqiang; Zhang, Hong-Mei; Shi, Yun; Lustgarten, Michael; Li, Yan; Qi, Wenbo; Zhang, Bin-Xian; Van Remmen, Holly
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Manganese superoxide dismutase (MnSOD) in the mitochondria plays an important role in cellular defense against oxidative damage. Homozygous MnSOD knockout (Sod2−/−) mice are neonatal lethal, indicating the essential role of MnSOD in early development. To investigate the potential cellular abnormalities underlying the aborted development of Sod2−/− mice, we examined the growth of isolated mouse embryonic fibroblasts (MEF) from Sod2−/− mice. We found that the proliferation of Sod2−/− MEFs was significantly decreased when compared with wild type MEFs despite the absence of morphological differences. The Sod2−/− MEFs produced less cellular ATP, had lower O2 consumption, generated more superoxide, and expressed less Prdx3 protein. Furthermore, the loss of MnSOD dramatically altered several markers involved in cell proliferation and growth, including decreased growth stimulatory function of mTOR signaling and enhanced growth inhibitory function of GSK-3β signaling. Interestingly, the G protein coupled receptor-mediated intracellular Ca2+ ([Ca2+]i) signal transduction was also severely suppressed in Sod2−/− MEFs. Finally, the ratio of LC3-II/LC3-I, an index of autophagic activity, was increased in Sod2−/− MEFs...

Discovery of NSAID and anticancer drugs enhancing reprogramming and iPS cell generation

Yang, Chao-Shun; Lopez, Claudia G.; Rana, Tariq M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/2011 Português
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Recent breakthroughs in creating induced pluripotent stem cells (iPSCs) provide alternative means to obtain ES-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/ Lin28) into somatic cells. iPSCs are versatile tools for investigating early developmental processes and could become sources of tissues or cells for regenerative therapies. Here, for the first time, we describe a strategy to analyze genomics datasets of mouse embryonic fibroblasts (MEFs) and embryonic stem (ES) cells to identify genes constituting barriers to iPSC reprogramming. We further show that computational chemical biology combined with genomics analysis can be used to identify small molecules regulating reprogramming. Specific down-regulation by small interfering RNAs (siRNAs) of several key MEF-specific genes encoding proteins with catalytic or regulatory functions, including WISP1, PRRX1, HMGA2, NFIX, PRKG2, COX2, and TGFβ3, greatly increased reprogramming efficiency. Based on this rationale, we screened only 17 small molecules in reprogramming assays and discovered that the NSAID Nabumetone and the anti-cancer drug OHTM can generate iPS cells without Sox2. Nabumetone could also produce iPS cells in the absence of c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. In summary...

GRP78/BiP is a Novel Downstream Target of IGF-1 Receptor Mediated Signaling

PFAFFENBACH, KYLE T.; PONG, MICHELLE; MORGAN, TODD E.; WANG, HONGJUN; OTT, KATE; ZHOU, BEIYUN; LONGO, VALTER D.; LEE, AMY S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/2012 Português
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Glucose regulated protein 78/immunoglobulin binding protein (GRP78/BiP) is an ER chaperone protein and master regulator of the unfolded protein response (UPR). The response of GRP78 to overt pharmacologically induced ER stress is well established, whereas the modulation of GRP78 to physiologic changes is less characterized. In this study, we examined the regulation of GRP78 in response to reduced IGF-1 growth factor signaling, a common consequence of calorie restriction (CR). ER chaperone protein expression was quantified in cell lysates prepared from the livers of calorie restricted (CR) and ad libitum fed mice, as well as MEFs grown in normal medium or serum starved. The requirement of IGF-1 signaling on GRP78 expression was studied using MEFs with IGF-1 receptor overexpression (R+) or deletion (R−), and the regulatory mechanism was examined using mTORC1 and PI3K inhibitors, as well as R− cells with knockdown of transcription factor FOXO1 compared to shRNA control. We observed a 40% reduction in GRP78 protein expression in CR mice and in serum-starved MEF cells. R− cells had drastically reduced AKT phosphorylation and exhibited lower levels of ER chaperones, in particular 80% less GRP78. Despite an 80% reduction in GRP78 expression...

Doxorubicin Induces Cytotoxicity through Upregulation of pERK–Dependent ATF3

Park, Eun-Jung; Kwon, Hyuk-Kwon; Choi, Yong-Min; Shin, Hyeon-Jun; Choi, Sangdun
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 13/09/2012 Português
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Although doxorubicin is commonly used in the treatment of many cancer types, its use in chemotherapy has been limited, largely because of its severe side effects, including cardiotoxicity and nephrotoxicity. In this study, we aimed to identify the mechanism of doxorubicin-induced cytotoxicity by using the human kidney proximal tubule cell line HK-2. Furthermore, we investigated the role of activating transcription factor 3 (ATF3) as a mediator of doxorubicin-induced cytotoxicity by using wild-type mouse embryonic fibroblasts (MEF) cells and ATF3 knockout (KO) cells. In HK-2 cells, doxorubicin decreased cell viability in a dose-dependent manner and induced an increase in cells in the sub G1 and G2/M phases at all doses. Doxorubicin treatment showed the following dose-dependent effects: increase in the secretion of tumor necrosis factor alpha; decrease in the expression of phosphorylated protein kinase A and Bcl-2; and increase in the expression of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal-regulated kinase (ERK), and ATF3. Based on these results, we suggest that doxorubicin induces cytotoxicity through an ERK-dependent pathway, and ATF3 plays a pivotal role as a transcriptional regulator in this process.

Metal-enhanced intrinsic fluorescence of nucleic acids using platinum nanostructured substrates

Akbay, Nuriye; Mahdavi, Farhad; Lakowicz, Joseph R.; Ray, Krishanu
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/2012 Português
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We investigated the feasibility of using platinum nanostructures to accomplish the metal-enhanced fluorescence (MEF) in the UV spectral region. We examine the possibility for detection of the intrinsic fluorescence from nucleotides and G-quadruplex DNA on platinum nanoparticles. Guanosine monophosphate (GMP) showed significant increases (~20-fold) in fluorescence intensities in the presence of platinum nanostructures when compared to quartz controls. G-quadruplex DNA demonstrated ~5-fold increase in fluorescence intensity and higher photostability in the presence of Pt nanostructures. We performed finite element method simulations to explore how Pt nanoparticles interact with plane waves and conformed that the Pt nanostructures are promising for enhancing the fluorescence emission in the UV region.

Toward xeno-free culture of human embryonic stem cells

Mallon, Barbara S.; Park, Kye-Yoon; Chen, Kevin G.; Hamilton, Rebecca S.; McKay, Ronald D.G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.

Gadd45a inhibits cell migration and invasion by altering the global RNA expression

Shan, Zhanhai; Li, Guiyuan; Zhan, Qimin; Li, Dan
Fonte: Landes Bioscience Publicador: Landes Bioscience
Tipo: Artigo de Revista Científica
Publicado em 01/09/2012 Português
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Gadd45a, the first well-defined p53 downstream gene, can be induced by multiple DNA-damaging agents, which plays important roles in the control of cell cycle checkpoint, DNA repair process and signaling transduction. Our previous findings suggested that Gadd45a maintains cell-cell adhesion and cell contact inhibition. However, little is known about how Gadd45a participates in the suppression of malignancy in human cancer cells. To examine the functions of Gadd45a in cell invasion and metastasis, we performed the adhesion, wound-healing and transwell assays in Gadd45a+/+ and Gadd45a−/− MEF cell lines. We found the adhesion, migration and invasive abilities were much higher in Gadd45a deficient cells. We furthermore applied high-throughput cDNA microarray analysis and bioinformatics analysis to analyze the mechanisms of Gadd45a gene in invasion and metastasis. Compared with the Gadd45a wild type cells, the Gadd45a deficient cells showed a wide range of transcripts alterations. The altered gene pathways were predicted by the MAS software, which indicated focal adhesion,cell communication,ECM-receptor interaction as the three main pathways. Real-time PCR was employed to validate the differentially expressed genes. Interestingly, we figured out that the deregulations of these genes are caused neither by genomic aberrations nor methylation status. These findings provided a novel insight that Gadd45a may involve in tumor progression by regulating related genes expressions.

Induction of Lung GSH and Glutamate Cysteine Ligase by 1,4-phenylenebis(methylene)selenocyanate and its Glutathione Conjugate: Role of Nuclear factor-erythroid 2-Related Factor 2

Emmert, Sans W.; El-Bayoumy, Karam; Das, Arunangshu; Sun, Yuan-Wan; Amin, Shantu; Desai, Dhimant; Aliaga, Cesar; Richie, John P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The synthetic organoselenium agent 1,4- phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG), are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamate cysteine ligase (GCL), through activation of antioxidant response elements (ARE) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular related kinase (ERK) (220%) and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), while the naturally-occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE reporter cells up to 3-fold for p-XSC and ≥5-fold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2 (siNrf2). The dependence of GCL induction on Nrf2 was confirmed in Nrf2 deficient mouse embryonic fibroblasts (MEF) where p-XSeSG induced GCL subunits in wildtype...

MicroRNA-450a-3p Represses Cell Proliferation and Regulates Embryo Development by Regulating Bub1 Expression in Mouse

Luo, Min; Weng, Yaguang; Tang, Jian; Hu, Min; Liu, Qiang; Jiang, Fengbing; Yang, Dandan; Liu, Chen; Zhan, Xiaoqin; Song, Peipei; Bai, Huili; Li, Baolin; Shi, Qiong
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 22/10/2012 Português
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Bub1 is a critical component of the spindle assembly checkpoint (SAC) and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3′-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF) cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.