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Publications (28)161.65 Total impact
Article: Transcriptional Regulators of the Trophoblast Lineage in Mammals with Hemochorial Placentation.
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ABSTRACT: Mammalian reproduction is critically dependent on the trophoblast cell lineage, which assures proper establishment of maternal-fetal interactions during pregnancy. Specification of trophoblast cell lineage begins with the development of the trophectoderm (TE) in preimplantation embryos. Subsequently, other trophoblast cell types arise with progression of pregnancy. Studies with transgenic animal models as well as trophoblast stem/progenitor cells have implicated distinct transcriptional and epigenetic regulators in trophoblast lineage development. This review focuses on our current understanding of transcriptional and epigenetic mechanisms regulating specification, determination, maintenance and differentiation of trophoblast cells.Reproduction (Cambridge, England) 09/2014; · 3.26 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Poor oocyte competence is a major factor limiting efficiency of in vitro embryo production in cattle and women. Despite decades of research, the fundamental questions of what makes an egg good or bad and how to improve egg quality in a clinical setting remain. Previous studies in our laboratory demonstrated the essential requirement of oocyte derived follistatin in early embryonic development with embryotrophic actions of follistatin in promoting early cleavage, increased rates of development to blastocyst stage and increased numbers of trophectoderm cells in bovine blastocyst stage embryos noted. Embryotrophic actions of follistatin on developmental progression of rhesus monkey embryos were also observed. Follistatin is a binding protein for specific members of the transforming growth factor β (TGFβ) superfamily and may exert its embryotrophic effects through modulation of one or more of the SMAD (SMAD2/3, SMAD1/5) or non-SMAD (e.g. AKT, JNK, ERK and P38) signaling pathways regulated by TGFβ superfamily members. The objectives of the present study were to investigate the association of AKT expression with oocyte competence/embryo developmental progression and potential effects of follistatin treatment on AKT phosphorylation in bovine embryos. In experiment 1, bovine oocytes were collected at germinal vesicle (GV) and metaphase II (MII) stages and embryos collected at pronuclear, 2-, 4-, 8- and 16-cell, morula and blastocyst stages and subjected to quantitative RT-PCR analysis of AKT mRNA abundance (n = 4 pools of 10 oocytes/embryos per pool). Results demonstrated increased transcript abundance for AKT in pronuclear through 8-cell embryos relative to GV stage oocytes. AKT transcript abundance was then decreased at 16-cell stage and further decreased in morula and blastocyst stage embryos. In experiment 2, GV stage oocytes were collected following staining with brilliant cresyl blue (BCB; a biochemical indicator of G6PDH activity and oocyte growth linked to egg quality) and differences in AKT and follistatin transcript abundance determined (n = 6 pools of 20 oocytes/group). Abundance of mRNAs for AKT and follistatin was higher in good quality oocytes that stained positive for BCB versus poorer quality oocytes negative for BCB staining. In experiment 3, the effects of exogenous follistatin treatment (0 or 10 ng/ml) of zygote stage embryos on AKT phosphorylation were determined by Western Blot analysis of samples collected at 0, 1, 2, 5 and 10 h after treatment administration (n = 20 embryos per timepoint/experiment; n = 6 replicates). Changes in levels of AKT phosphorylation (phosphorylated AKT/total AKT) in response to follistatin treatment were not detected. Collectively, results demonstrate temporal regulation of AKT mRNA abundance during bovine early embryogenesis and a positive association of AKT transcript abundance with oocyte quality (based on BCB staining) suggestive of a potential functional role in oocyte competence. However, embryotrophic actions of exogenous follistatin do not appear mediated by acute alterations in AKT phosphorylation levels in early bovine embryos. Further studies are required to elucidate the signaling pathways associated with embryotropic actions of follistatin and establish a functional role for maternally derived AKT in oocyte developmental competence and early embryonic development in cattle. (Supported by NIH grant HD072972 to GWS and JK and ARE grant JS2687 to MA)Annual meeting of the Society for the Study of Reproduction SSR, Michigan, USA; 07/2014 - [Show abstract] [Hide abstract]
ABSTRACT: Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of TGFbeta-superfamily cognate signal transduction pathway components (e.g. SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotropic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given SMAD4 is a common "SMAD" required for both SMAD2/3 and SMAD1/5/8 signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotropic actions of follistatin are SMAD4-dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via siRNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle and embryotropic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.Biology of Reproduction 07/2014; · 3.45 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: A growing body of evidence suggests oocyte developmental competence is a limiting factor in efficiency of assisted reproductive technologies and pregnancy success in livestock species and humans, but the inherent phenotypic characteristics of competent oocytes are not well understood. Oocytes gradually and sequentially acquire developmental competence (during the course of folliculogenesis) by synthesizing and accumulating transcripts and proteins critical for successful meiotic maturation, fertilization, and early embryogenesis. We have conducted fundamental studies using the bovine model to elucidate differences in oocyte transcriptome associated with poor oocyte competence and the functional and therapeutic relevance of such results. Of particular interest were studies that showed a positive association of follistatin mRNA abundance with oocyte competence in two distinct bovine models. Follistatin treatment of bovine embryos during initial stages of in vitro culture increases proportion of embryos cleaving early, proportion of embryos developing to the blastocyst stage and numbers of blastocyst trophectoderm cells. Comparative studies in the rhesus monkey model demonstrated stimulatory actions of exogenous follistatin on rates of blastocyst development and support potential clinical relevance of results in the bovine model. Complementary loss of function studies in early embryos established a functional role for follistatin in control of bovine blastocyst development and cell allocation. To increase understanding of intrinsic role of TGFβ superfamily in regulation of early embryogenesis and elucidate mechanism of action of follistatin in mediating its embryotropic effects, studies utilizing a combination of pharmacological and siRNA mediated inhibition of TGFβ superfamily signaling pathway components in the presence or absence of follistatin treatment are being conducted. Results to date suggest a functional requirement for the common SMAD (SMAD4) and SMAD2/3 signaling pathways in promoting bovine early embryonic development and indicate stimulatory effects of follistatin on blastocyst development, but not early cleavage are blocked when SMAD signaling is inhibited. Elucidation of the mechanism of action of follistatin in mediating above described embryotropic actions is critical to further understanding of the functional significance of follistatin to regulation of bovine early embryogenesis and the translational relevance of results to improvements in assisted reproductive technologies. (Supported by NIH grant HD072972 to GWS and JK).Annual meeting of the Society for the Study of Reproduction SSR, Michigan, USA; 07/2014 - [Show abstract] [Hide abstract]
ABSTRACT: Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine embryogenesis. Given JY-1 is present in oocytes throughout folliculogenesis and oocyte-derived JY-1 mRNA is temporally regulated post fertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 siRNA was microinjected into cumulus-enclosed germinal vesicle stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation, was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion and embryonic development after in vitro fertilization. Depletion of JY-1 protein during in vitro maturation effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8-16 cell stage and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion and early cleavage, but did not rescue development to 8-16 cell and blastocyst stages. However, effects of JY-1 depletion post fertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion and subsequent embryonic development.Biology of Reproduction 02/2014; · 3.45 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: In mouse blastocysts CDX2 plays a key role in silencing Oct4 and Nanog expression in the trophectoderm (TE) lineage. However, the underlying transcriptional and chromatin-based changes that are associated with CDX2-mediated repression are poorly understood. To address this a Cdx2-inducible mouse embryonic stem (ES) cell line was utilized as a model system. Induction of Cdx2 expression resulted in a decrease in Oct4/Nanog expression, an increase in TE markers, and differentiation into trophoblast-like stem (TS-like) cells within 48 to 120 hours. Consistent with the downregulation of Oct4 and Nanog transcripts, a time-dependent increase in CDX2 binding and a decrease in RNA polymerase II (RNAPII) and OCT4 binding was observed within 48 hours (P<0.05). To test whether transcriptionally active epigenetic marks were erased during differentiation, histone H3K9/14 acetylation and two of its epigenetic modifiers were evaluated. Accordingly, a significant decrease in histone H3K9/14 acetylation and loss of p300 and HDAC1 binding at the Oct4 and Nanog regulatory elements was observed by 48 hours. Accompanying these changes there was a significant increase in total histone H3 and a loss of chromatin accessibility at both the Oct4 and Nanog regulatory elements (P<0.05), indicative of chromatin remodeling. Lastly, DNA methylation analysis revealed that methylation did not occur at Oct4 and Nanog until 96 to 120 hours after induction of CDX2. In conclusion, our results show that silencing of Oct4 and Nanog is facilitated by sequential changes in transcription factor binding, histone acetylation, chromatin remodeling, and DNA methylation at core regulatory elements.Stem cells and development 09/2013; · 4.15 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: The first cell-fate decision in mammalian preimplantation embryos is the segregation of the inner cell mass (ICM) and trophectoderm (TE) cell lineages. The ICM develops into the embryo proper, whereas the TE ensures embryo implantation and is the source of the extra-embryonic trophoblast cell lineages, which contribute to the functional components of the placenta. The development of a totipotent zygote into a multi-lineage blastocyst is associated with the generation of distinct transcriptional programs. Several key transcription factors participate in the ICM and TE-specific transcriptional networks, and recent studies indicate that post-translational histone modifications as well as ATP-dependent chromatin remodeling complexes converge with these transcriptional networks to regulate ICM and TE lineage specification. This review will discuss our current understanding and future perspectives related to transcriptional and epigenetic regulatory mechanisms that are implicated in the initial mammalian lineage commitment steps, with a focus on events in mice. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.Molecular Reproduction and Development 07/2013; 81(2). · 2.68 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: During implantation the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient's placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. METHODS AND RESULTS: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. CONCLUSION: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.Biochemical and Biophysical Research Communications 06/2013; · 2.28 Impact Factor -
Article: Evidence that Transcription Factor AP-2γ Is Not Required for Oct4 Repression in Mouse Blastocysts.
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ABSTRACT: In mouse blastocysts segregation of the inner cell mass (ICM) and the trophectoderm (TE) is regulated by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2 expressed in the ICM and TE, respectively. In contrast, in other species such as bovine and human, Oct4 is not restricted to the ICM and continues to be expressed in the Cdx2-positive TE. A recent comparative study of the bovine and mouse Oct4 promoters revealed that additional mechanisms might act in conjunction with Cdx2 to downregulate Oct4 expression in the mouse TE lineage. For instance, the mouse Oct4 distal enhancer contains an AP-2γ (Tcfap2c) binding motif that is absent in the bovine and human Oct4 distal enhancer. Nonetheless, the functional relevance of Tcfap2c in Oct4 repression during mouse preimplantation development was not tested. To elucidate the role of Tcfap2c in Oct4 expression an RNA interference approach was utilized. Depletion of Tcfap2c triggered a decrease in Oct4 expression at the 8-cell and morula stage. Remarkably, at the blastocyst stage depletion of Tcfap2c and/or its family member Tcfap2a had no effect on Oct4 repression. To test whether Tcfap2c interacts with Oct4 to positively regulate Oct4 expression, chromatin immunoprecipitation and in situ co-immunoprecipitation analyses were performed. These experiments revealed Tcfap2c and Oct4 binding were enriched at the Oct4 distal enhancer in embryonic stem (ES) cells, but were rapidly lost during differentiation into trophoblast-like cells when Oct4 became repressed. Moreover, Tcfap2c and Oct4 interactions were detected at the morula stage, but were lost during blastocyst formation. In summary, these data demonstrate that Tcfap2c is not required for Oct4 silencing in mouse blastocysts, but may be necessary for the maintenance of Oct4 expression during the 8 cell-to-morula transition. These findings support the notion Cdx2 is the predominant negative regulator of Oct4 expression during blastocyst formation in mice.PLoS ONE 05/2013; 8(5):e65771. · 3.53 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: The trophectoderm epithelium is the first differentiated cell layer to arise during mammalian development. Blastocyst formation requires the proper expression and localization of tight junction, polarity, ion gradient and H(2)O channel proteins in the outer cell membranes. However, the underlying transcriptional mechanisms that control their expression are largely unknown. Here, we report that transcription factor AP-2γ (Tcfap2c) is a core regulator of blastocyst formation in mice. Bioinformatics, chromatin immunoprecipitation and transcriptional analysis revealed that Tcfap2c binds and regulates a diverse group of genes expressed during blastocyst formation. RNA interference experiments demonstrated that Tcfap2c regulates genes important for tight junctions, cell polarity and fluid accumulation. Functional and ultrastructural studies revealed that Tcfap2c is necessary for tight junction assembly and paracellular sealing in trophectoderm epithelium. Aggregation of control eight-cell embryos with Tcfap2c knockdown embryos rescued blastocyst formation via direct contribution to the trophectoderm epithelium. Finally, we found that Tcfap2c promotes cellular proliferation via direct repression of p21 transcription during the morula-to-blastocyst transition. We propose a model in which Tcfap2c acts in a hierarchy to facilitate blastocyst formation through transcriptional regulation of core genes involved in tight junction assembly, fluid accumulation and cellular proliferation.Development 11/2012; · 6.27 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.Stem cells and development 07/2011; 21(7):1134-44. · 4.15 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases.Stem cells and development 06/2011; 20(10):1669-78. · 4.15 Impact Factor - Stem Cells Dev. 01/2011;
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ABSTRACT: During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.PLoS ONE 05/2010; 5(5):e10622. · 3.53 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog transcripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were methylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic enrichment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the parameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.Cloning and Stem Cells 12/2009; 11(4):483-96. · 2.66 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog tran-scripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were meth-ylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic en-richment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the pa-rameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.11/2009; - [Show abstract] [Hide abstract]
ABSTRACT: The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells.Stem Cells 02/2009; 27(2):317-28. · 7.70 Impact Factor -
Article: Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization.
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ABSTRACT: Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca2+ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCzeta in generating changes in egg intracellular Ca2+ despite the fact that PLCzeta constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca2+ oscillations has not been examined. Reducing egg PLCbeta1 by a transgenic RNAi approach resulted in a significant decrease in Ca2+ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCbeta1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca2+ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCzeta in egg activation.Developmental Biology 01/2008; 312(1):321-30. · 3.64 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Fertilization triggers initiation of development and establishment of blocks on the egg coat and plasma membrane to prevent fertilization by multiple sperm (polyspermy). The mechanism(s) by which mammalian eggs establish the membrane block to polyspermy is largely unknown. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) appears to be the key regulator of several egg activation events (completion of meiosis, progression to embryonic interphase, recruitment of maternal mRNAs). Since sperm-induced increases in cytosolic Ca(2+) play a role in establishment of the membrane block to polyspermy in mouse eggs, we hypothesized that CaMKII was a Ca(2+)-dependent effector leading to this change in egg membrane function. To test this hypothesis, we modulated CaMKII activity in two ways: activating eggs parthenogenetically by introducing constitutively active CaMKIIalpha (CA-CaMKII) into unfertilized eggs, and inhibiting endogenous CaMKII in fertilized eggs with myristoylated autocamtide 2-related inhibitory peptide (myrAIP). We find that eggs treated with myrAIP establish a less effective membrane block to polyspermy than do control eggs, but that CA-CaMKII is not sufficient for membrane block establishment, despite the fact that CA-CaMKII-activated eggs undergo other egg activation events. This suggests that: (1) CaMKII activity contributes to the membrane block, but this not faithfully mimicked by CA-CaMKII and furthermore, other pathways, in addition to those activated by Ca(2+) and CaMKII, also participate in membrane block establishment; (2) CA-CaMKII has a range of effects as a parthenogenetic trigger of egg activation (high levels of cell cycle resumption, modest levels of cortical granule exocytosis, and no membrane block establishment).Journal of Cellular Physiology 09/2007; 212(2):275-80. · 3.87 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Fertilization in mammalian eggs is accompanied by oscillatory changes in intracellular Ca(2+) concentration, which are critical for initiating and completing egg activation events and the developmental program. Ca(2+)/Camodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is postulated to be the downstream transducer of the Ca(2+) signal in many cell types. We tested the hypothesis that CaMKII is the major integrator of Ca(2+)-induced egg activation events and embryo development by microinjecting a cRNA that encodes a constitutively active (Ca(2+)-independent) mutant form of CaMKII (CA-CaMKII) into mouse eggs. Expression of this cRNA, which does not increase intracellular Ca(2+), induced a sustained rise in CaMKII activity and triggered egg activation events, including cell cycle resumption, and degradation and recruitment of maternal mRNAs; cortical granule exocytosis, however, did not occur normally. Furthermore, when mouse eggs were injected with sperm devoid of Ca(2+)-releasing activity and activated with either CA-CaMKII cRNA or by SrCl(2), similar rates and incidence of development to the blastocyst stage were observed. These results strongly suggest that CaMKII is a major integrator of the Ca(2+) changes that occur following fertilization.Developmental Biology 09/2006; 296(2):388-95. · 3.64 Impact Factor
Publication Stats
752 | Citations | |
161.65 | Total Impact Points | |
Top Journals
Michigan State University
- Department of Animal Science
East Lansing, Michigan, United States -
EMD Serono
Rockland, Massachusetts, United States
University of Pennsylvania
- • Center for Research on Reproduction and Women's Health
- • Department of Biology
Philadelphia, Pennsylvania, United States
Hospital of the University of Pennsylvania
Philadelphia, Pennsylvania, United States
University of Massachusetts Amherst
- Department of Veterinary and Animal Sciences
Amherst Center, Massachusetts, United States


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The process of buying a new Lincoln car or truck can seem overwhelming if you don't know where to begin. Edmunds.com can get you started on the right track with a convenient directory of Lincoln car dealerships in and around Michigan City, Indiana. Compare online price quotes on the new or used car, truck, SUV, minivan, or wagon of your choice to locate the best deals. Edmunds.com makes it easy to find trusted Lincoln car dealers in Michigan City, saving car buyers time and money on what will be an important purchase.
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Thanks, I Guess: Top 10 Unwanted Christmas Gifts
- By Bossip Staff Categories: Entertainment, Multi, New, News, Originals View CommentsWal-Mart's $5 Bargain Bin Movies -
If you have TBS, TNT and FX, you'll never need to own any of these movies. What's in this bin, will always be in this bin. Forever.
Photo credit: Pennlive.com


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JAP Motorsport.net your official site of Japan parts racing for your Honda Civic and Acura Integra Models. Improve shifting response with the MFactory R&D (Synchrotech®) Carbon-Lined Synchro & Slider Kit. Recognising the extremely high cost of OEM replacement synchroniser parts, MFactory R&D (Synchrotech®) have took it upon themselves to design and develop their own range of high performance OEM replacement parts. Forged from a higher tensile steel, our Sliders are both stronger and lighter than the OEM equivalent. For the synchros, we utilize a carbon fiber lining technology which acts as braking material during gear engagement, this allows for quicker and more precise shifting and prolongs the life of your gear set. After several years of Research & Development on our championship winning race cars, we are proud to offer the most technologically advanced Synchros & Sliders on the market.
Sliders are Forged from high tensile steel
Synchros are made from high strength Japanese brass
Synchros are Carbon Fiber Lined (not just a coating!) for increased durability
100% larger surface contact area for increase performance
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* Free shipping lower 48 states. Puerto Rico, Virgin Island, Canada and International buyers contact us for shipping quotes.
JAP Motorsport.net your official site of Japan parts racing for your Honda Civic and Acura Integra Models.
Product Warranty
TERMS: Due to the nature of For Race Use Only products, Synchrotech® will provide a limited warranty for all Synchros & Sliders to be free of defects in materials and workmanship upon receipt of product and initial installation only. Parts returned must be determined by Synchrotech® to be defective before any warranty credit or replacement is issued
Synchrotech®'s obligation under warranty shall be limited to repairing or replacing, at its option, any part proven defective. The customer must prepay transportation charges
Final determination of the suitability of the products for the use contemplated by the buyer is the sole responsibility of the buyer. Synchrotech® shall not be liable for any special, direct, indirect, incidental, or consequential damages, that might be claimed as a result of the failure of any part, including claims for delay, loss of profits, or labor
Synchrotech® shall not be liable for any damage or injury to persons or property resulting from improper installation or misuse of any part subject to this warranty
There are no other warranties expressed or implied extending beyond those set forth above.


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7 results for "john hollinger"
Players like Brad Miller of Houston Rockets, DeJuan Blair of San ...
John CreganJohn Cregan discusses the possibility of using John Hollinger's PER in fantasy, and how that would affect players' fantasy ratings.
Story | Conversation | December 16, 2010 -
NBA predictions, 2010-11: John Hollinger's forecast
John HollingerOur ESPN.com NBA analyst forecasts the regular-season outcome for all 30 teams
Story | Conversation | October 22, 2010 -
John Hollinger's PER Diem: April 15, 2009
John HollingerOften overlooked during award season? Defense. We shine light on the season's top defenders.
Story | Conversation | April 15, 2009 -
John Hollinger's PER Diem: March 18, 2009
John HollingerWith reserves getting fewer minutes in the playoffs, John Hollinger examines which teams will be stronger and weaker as a result.
Story | Conversation | March 17, 2009 -
Daily Dime: Celtics ground Hawks
John HollingerIt was KG's turn to step up for the Celtics, who edged the Hawks for consecutive win No. 16, writes John Hollinger.
SpecialFeature | Conversation | December 17, 2008 -
Best and worst of the first two rounds of the playoffs
John HollingerJohn Hollinger hands out awards for the first two rounds of the playoffs.
Story | Conversation | May 16, 2008 -
From Phoenix to foul play, lots of playoff surprises so far
John HollingerJohn Hollinger takes a look at the biggest surprises in the playoffs so far.
Story | Conversation | April 28, 2008


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The Window Cleaner
A short muisc video based on the classic song "The Window Cleaner" sung by George Formby
4.39 / 5.00 6,326 ViewsThanks for the Front Page feature
Hello there Newgroundites!
Today us fine folk at the Lock Legion are proud to present to you "Taser Fun", a swell Flash movie by myself.
Watch and enjoy!
The ending was a bit weak, but the graphics were great. Another good Lock movie.
Yeah, I was at a loss for ideas towards the end, but I am glad that you liked it.
Thanks for the review!
That Made me Laugh!
That's Good Shiz YO!
Heh, thanks!


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Diet Diary: Add Makhana to your diet for that healthy edgeBy: Ishi Khosla Extracts from different parts of this water plant including its leaves, stems, roots and seeds have been reported to show several benefits, such as antioxidant, free radical scavenging, anti-inflammatory, and immuno-modulatory ... Indian Express, 18 hours ago |
6 images for ishi | ||||
Cured meats, the new favourite, aren't most healthyPreserving the entire animal after it was killed has occupied an important place in the food market today. By Ishi Khosla With westernisation of diets, cured meats are becoming increasingly popular and people travel distances to reach their ... Indian Express, 1 week ago
Diet Diary: Malnutrition in younger years marker of disorders laterBy: Ishi Khosla There is accumulating evidence to suggest a link between malnutrition early in the life cycle and risk of diabetes, obesity and other chronic degenerative diseases later in life. Recent research has suggested that risk of ... Indian Express, 2 weeks ago
Diet Diary: Decoding contradictory studies on glycemic indexBy: Ishi Khosla Scientific research is the foundation of modern medicine. Yet, when it comes to media reports, several things that have been reported one day are countered sooner than later by another study, confusing the reader. For example: ... Indian Express, 3 weeks ago
Diet Diary: Wake up to the benefits of drinking coffeeCoffee beans are cured either by air-drying or fermentation. By Ishi Khosla If you thought your favourite cup of coffee was unhealthy, think again!! Coffee's origin can be traced back to the 12th century in Ethiopia, where it is believed to ... Indian Express, 4 weeks ago
Diet Diary: Festival cheer marred by nasty hangover? Tips to avoid itSymptoms normally peak about the time alcohol level reaches zero and may continue for an entire day. By Ishi Khosla After a night of drinking and carousing, you often wake up with a hangover — pounding headache, exhausted and sick. You may ... Indian Express, 1 month ago
Buy SAIL, IDFC: Rishi KohliAccording to Rishi Kohli of Proalpha Systematic Capital, one may buy SAIL and IDFC. R ishi Kohli of Proalpha Systematic Capital told CNBC-TV18, "One may buy Steel Authority of India (SAIL) and go long in IDFC ." At 11:21 hrs Steel Authority ... Moneycontrol.com, 1 month ago
Diet diary: Switch to sugar-freeSugars can be used in moderation by most healthy active people but must be used sparingly by weight watchers. By Ishi Khosla I am often asked if consuming a certain amount of sugar is essential in our diet. During digestion in the body, all ... Indian Express, 2 months ago
Islet Holdings, Inc. Adds New Business PursuitsDENVER--(BUSINESS WIRE)-- Islet Holdings, Inc. (Pink Sheets:ISHI)(the Company) recently announced that the Company would expand its business plan to pursue additional and new medical technologies and products. Today the Company announced that it ... Pulp And Paper Online, 1 month ago
Islet Holdings Inc. Adds New Business Pursuits
PharmiWeb, 1 month ago
Islet Holdings, Inc. : Adds New Business Pursuits
4 Traders, 1 month ago
Gac launches mobile spare parts delivery vehicleGeneral Automotive Company (GAC), the official distributor for Mitsubishi Motors and the Mitsub ishi Fuso Truck and Bus Corpo ration (MFTBC) in Oman , recently introduced a dedicated parts deliv ery vehicle to its internal fleet to ensure ... 4 Traders, 6 days ago


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The Enchanted Cave 2
Delve into a strange cave with a seemingly endless supply of treasure, strategically choos
4.30 / 5.00 24,952 ViewsGhostbusters B.I.P.
COMPLETE edition of the interactive "choose next panel" comic
3.95 / 5.00 5,487 ViewsUse your super J-J-Jump powers to escape the flood in this teeny icon adventure.
Use the KEYBOARD to jump (UP/W) to avoid getting caught in the water!
That moment when its been a long time since I see anyone type in the end ''Thank you for playing'' I love that, brings back memories from the old games ;) So thank you for making this game!


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Book your room at Hotel Arco Iris
Deluxe Room
Max Occupancy: 4 guests (up to 3 children, 2 infants)Bungalow
Max Occupancy: 2 guests (up to 1 child, 1 infant)Tamarindo hotel with an outdoor pool
Situated near the airport, in Tamarindo, this hotel is close to Tamarindo Beach, Grande Beach, and Hacienda Pinilla Golf Course. Area attractions also include Las Baulas de Guanacaste National Marine Park and Avellana Beach.
Hotel Features
In addition to a restaurant, Hotel Arco Iris features an outdoor pool. Other amenities include a bar/lounge and complimentary wireless Internet access.
Room Amenities
Televisions come with premium cable channels. Guestrooms also feature complimentary wireless Internet access, refrigerators, and air conditioning.
Information missing or incorrect? Tell us!


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Add some serious attitude to your motorcycle or BMX helmet with one of Hairy R's Helmet Mohawks! Wind tested at over 200 miles per hour, these Mohawks use 4 heavy duty suction cups to stick on to your helmet, and stay there until you want to take it off. The Mohawk is made of 3 1/2 inch long strands of electric blue rubber "hair", and measures a full 12 inches long.These Helmet Mohawks make a great gift for sport bike riders, BMX riders and anyone else who wears a full face helmet. It is brand new, never used or displayed. We have a limited quantity of these, so don't miss out. Get yours now!
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Shoei J-Wing/Multitec Replacement Cheek Pads 39mmSugg. Retail: $21.50 Bobs Price: $19.99 |
Bell Rogue Helmet Muzzle BlackBobs Price: $49.95 |


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Quick View Le Capital au XXIe siècle by Thomas Piketty September 5, 2013 | Kobo Edition (eBook) | French Available for download from $31.99 See Details $31.99 was $31.99
Quick View Capital in the Twenty-First Century by Thomas Piketty June 17, 2014 | Audio Book (CD) In stock online from $32.59 What are the grand dynamics that drive the accumulation and distribution of capital? Questions about the long-term evolution of inequality, the concentration of wealth, and the prospects for economic growth lie at the heart of political economy. But satisfactory answers have been hard to find for… See Details $34.31 / $32.59 lowest price*, or used from $28.70was $51.99
Tips for store searching: Check that you entered your information correctly. If your postal code might be further than 25kms from a store, try entering a city name instead. If there are no matches in your city, try the next closest major city.


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Written on: 09/12/2009
Okay so it's not the most features packed piece of kit on the market. Neither is it the most user friendly, but it does everything I want and for £25 I think it was good value for money.
The best thing about is the fact that it switches itself off at night so that I can't be woken up by Orange's irritating - 'Sorry you didn't top up by £100 this month, no bonus for you'- texts in the middle of the night.
As rated by real users
" I got this phone more then 8 years " Read More
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" Best phone I've ever had " Read More
Written by AliceWayte
" This Nokia 9210 mobile phone can be a real boon as it can give you ... " Read More
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" I bought this nice looking phone in Nairobi-Kenya in July 2003 from... " Read More
Written by Peter Njoroge Mwangi


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Kilonewton (kN) can be converted into kilograms (kg) by first multiplying the value of kN by 1000 and then dividing it by earth's gravity, which is denoted by "g" and is equal to 9.80665 meter per second.
A kilonewton is equal to 1000 newtons. Newton, denoted by "N", is the unit of force used by the International System of Units (SI units) and is named after Sir Isaac Newton to honor his contributions in the field of classical mechanics. Kilogram is the base unit of mass used by the SI units. Since kN is a measure of force and kg is a measure of mass, they are not equivalent and can't be used interchangeably. So converting kN to kg should only be done for the sake of convenience, and either one of them can't be used as the equivalent of the other when they're part of an equation. Instead, kN can be converted into kilogram-force (kgf), which is a unit of force that is less frequently used. By definition, 1 kilogram-force is equal to the force exerted by a mass of 1 kg placed in earth's gravity. This means that 1 kilogram-force is equal to 9.80665 kilogram-meter per second per second, or 9.80665 N.
Learn MoreTo convert 161 pounds (lbs) to kilograms (kg) simply divide 161 by 2.2046, which equals 73.028. The conversion rate between pounds and kilograms is 1 kg = 2.2046 lbs.
Full Answer >If you are converting the mass of a substance from milligrams/kilograms (mg/kg) to parts per million (ppm), then the ratio is 1:1, and no conversion is necessary. Therefore, 1 mg/kg is the same as 1 ppm because 1 milligram is equal to 0.001 grams while 1 kilogram is equal to 1,000 grams. So, if you divide 0.001 by 1,000, then the result is 0.000001, which is a millionth of a gram, making it the same as 1 part per million.
Full Answer >To convert 46 kilograms to pounds, divide the number of kilograms by 0.45359237, according to Conversion of Measurement Units. Since a kilogram is equal to roughly 2.2 pounds, 46 kilograms equals approximately 101.3 pounds. For this purpose, you can round 0.4539237 to 0.4539.
Full Answer >For the greatest accuracy, an online currency converter with up-to-date exchange rate information should be used to convert British pounds to U.S. dollars. On Oct. 20, 2014, the exchange rate was 1.6138 U.S. dollars to one British pound.
Full Answer >


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From Sunday02/15/15 to Monday02/16/15
Hotels Sfântu Gheorghe
- Comprehensive hotel search for Sfântu Gheorghe online
- Find a cheap hotel in Sfântu Gheorghe!
- Book the best price!
0Booking.com $43This is the best rated 3-star hotel in Sfântu Gheorghe
Restaurant Park
3Booking.com $62Ferdinand
0Booking.com $45Bobo Panzio
0Booking.com $19Sugas
3Booking.com $46Castel
3Booking.com $31


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Demo Derby: Official PlayStation Magazine: Issue 58
Become Premium to Watch
The chaos and fun of Saints Row is fully intact in the underworld, but a new setting can only go so far.
Dimensions recaptures enough of Geometry Wars' thrills to stand as the best new dual-joystick shooter in quite a while.
The little mushroom man finally gets his name on the marquee, and the result is a charming, unique, and puzzle-heavy adventure.
Character »
A.J. Styles is a professional wrestler for TNA. He is the first-ever wrestler to have won the TNA Grand Slam Wrestling Championship
Chris Jericho, nicknamed Y2J, is professional wrestler best known for his time with WWE and he is also the lead singer for the rock band Fozzy. He has won many championships over the years, being the first Undisputed Champion, 9 time Intercontinental Champion (in which he holds the record)and more.
Edge is a retired professional wrestler of the WWE. He held over 20 titles during his career and was the first entry into the 2012 Hall of Fame Ceremony, making him the youngest inductee of all time.
Gangrel(real name David Heath) is a former wrestler, who worked for WWF in their super-popular "Attitude Era". Gangrel is best known for his gimmick, where he would portray a Vampire, who sucked other wrestler's blood and played general "mind games".
Rhino, also known as "The War Machine" or "Rhyno," is a professional wrestler most commonly identified with ECW or TNA.
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Our desire is to raise our students to engage the world they live in from the platform of Christianity and a personal faith in Jesus Christ.
It is our desire that each student learn to achieve with excellence in order to understand their purpose, their world and all things from the truth of God's word.
We believe that through a classical and Christian education our students will be equipped for the challenges that will face them as adults.
School Overview
School Membership(s)School Assoc. | Association of Christian Schools International (ACSI) Association of Classical and Christian Schools (ACCS) |
Religious Affiliation | Christian |
Grades Offered | Grades Kindergarten - 12 |
Year Founded | 1994 |
Student Body
Total Students | 57 students |
Student Body Type | Co-ed |
% Students of Color | 96% State avg.: 20% |
Students by Grade |
Academics and Faculty
Total Classroom Teachers | 9 teachers |
Student : Teacher Ratio | 7:1 National avg.: 13:1 |
% Faculty w/Advanced Degree | 50% |
Average Class Size | 6 students |
Average SAT score | 580 |
Classroom Dress Code | Formal (school uniform) |
Finances and Admission
Admission Deadline | None / Rolling |
Yearly Tuition Cost | $4,800 |
Tuition Notes | Scholarship Program available |
% on Financial Aid | 20% |
Admissions Director | Stephen Bailey |
Total Sports Offered | 2 sports |
Sports | Basketball, CheeringBasketball, Cheering, |
School Notes:
- Teaching a thoroughly Christian worldview in every academic area
- Using classical model of education as the best methodology which respects man as created in the image of God
- Teaching our students to be lovers and seekers of truth.
Nearby Schools:
The nearest high school is Portsmouth Abbey School (3.7 miles away)
The nearest elementary school is The Pennfield School (0.5 miles away)
The nearest preschool is The Pennfield School (0.5 miles away)
Show me:
- School Location Miles Students Grades
PortsmouthThe Pennfield School
110 Sandy Point Ave Litle Slocum Farm
Portsmouth, RI, 02871
(401)849-46460.5 mi | 163 students | Gr. NS-8 -
MiddletownSilveira Kindergarten & Nursery School
143 Peckham Ln
Middletown, RI, 02842
(401)846-9251Daycare / Preschool1.9 mi | 9 students | Gr. PK-KG -
MiddletownBright Ideas Preschool
1272 W Main Rd
Middletown, RI, 02842
(401)849-0055Daycare / Preschool (Non-sectarian)1.9 mi | 8 students | Gr. K -
MiddletownMiddletown KinderCare
70 Corporate Place
Middletown, RI, 02842
(401)846-1888Daycare / Preschool (Non-sectarian)2.2 mi | 8 students | Gr. NS-PK -
MiddletownAll Saints Academy
915 W Main Rd
Middletown, RI, 02842
(401)848-4300(Roman Catholic)2.2 mi | 123 students | Gr. PK-8
School Reviews
Recent Articles
Updated January 22, 2015
When making an important decision that will impact the lives of your children, you need to have all the facts in place. We compiled a list of the most important factors to consider when choosing a final private school for your child.
As you evaluate schools, take time to explore fully what the schools which you are looking at teach.
Here are some more answers and resources to help you deal with tough questions such as "What do I do when my child doesn't test well?"


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Orange Village Resort
- Suites
- Room service
- Restaurant
- Business Services
- Fitness/Health center
TripAdvisor Reviews Orange Village Resort Gangtok
Travel Blogs from Gangtok
Arranging a Tour in North Sikkim
... Traditionally fermented leaves of mustard, radish and cauliflower
- Kinema - Traditionally fermented soya bean food
- MaaChoo - Traditionally cottage cheese cooked with butter and eggs
Accompaniments: Pork with Kinema, Shyapta (Beef), Fing with Mushroom
The owner explained the dishes to us and said the set menu was big and two people could easily share. We shared and it was enough. The flavors were interesting ...
Tea time!
Darjeeling – Tea (ovbiously) and tasty food!!
The main touristy area in Darjeeling is relatively small. You could probably walk from one end to the other in half an hour or so so it didn't take us long to figure out where everything was. It was nice to have everything all close together for a change so we could walk everywhere and not have to worry about flagging down transport. We had a look in a few tea shops and book shops. It didn't actually feel as though ...
The land of tea and fog
... to cover up with and jealous of everyone else on the train who were well prepared with sheets and blankets. In the morning thankful that the night was over, I was even more thankful when Margo came to find me. I grabbed my stuff and went back with her to her and Tony's car and cabin. What a completely different environment to mine. They were the only westerner's but the young men they were sharing with were so friendly, informative and ...
One night in Gangtok makes a hard man humble
... The rivers were the color of jade, which I assume is from lime deposits or some other mineral. Along the way we continued to descend out of the mountains and down into the valley, passing monkeys and bamboo forest along the way. After some more driving, we came to the Sikkim border where we had to show our permit and get checked in. Since Sikkim is contested territory, they take extra precautions, but the check in process was simple and painless. After we crossed the border, ...
Singalila Ridge Trek
... with a lovely steady stroll, mostly downhill, which was great as the skies were still clear and you could relax and enjoy the views. After a few kilometres we went steeply downhill and into a valley to a passport checkpoint…but of course that meant we had to go back up. The following two kilometres were easily as hard as yesterdays, following the path as it wound back and forth up the side of the mountain again. Plenty of breath-stops were the ...


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- Governor Walker Supports "Jock Tax" to Help Fund New Milwaukee Arena 10:54am
- High School Sports Schedule for Tuesday, January 27th 8:41am
- High School Sports Scores: Monday, January 27th 6:11am
- Packers Sign New Punter 5:46am Jan 26
- High School Sports Schedule for Monday, January 26th 8:43am Jan 24
- Bucks Make It Rain 3's 9:01pm
Bank of America 500
Event Date: October, 12 2013
Event Times: 6:30 pm
NASCAR Sprint Cup Series -- The Bank of America 500 begins at 6:30 pm CT; WSCO broadcast starts at 5:30 pm CT.
Charlotte Motor Speedway -- Concord, NC
Felder keeps on trucking...not sure what happened to Koob this week, but no picks.
Justin Hull WHBY Radio
Marques Pfaff…
posted by Justin Hull 1 month ago
Felder is back on top after Woodsy went on skid. Watch out for Brett!!
Justin Hull WHBY Radio
Marques Pfaff…
posted by Justin Hull 1 month ago
After a few weeks similiar picks, week 13 should provide some entertainment...seperate the men from Marques and I who are…
posted by Justin Hull 1 month ago


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WalletHub: Hawaii taxpayers getting their money's worth — and then some — under ACA
- Jenna Blakely
- Reporter- Pacific Business News
- Email | Twitter
Hawaii taxpayers have among the nation's highest return when it comes to funding of the Affordable Care Act, according to a new study by WalletHub, a financial resources website that analyzed tax dollar benefit by state.
One of the report's major findings is that states that have opted to expand Medicaid coverage — funded by federal income taxes — will see a significant return on taxpayer investment.
"Taxpayers across the country are on the hook for Medicaid expansion costs no matter what, as federal income taxes are used to fund the program," WalletHub CEO Odysseas Papadimitriou said in a statement. "The only question is whether or not they get anything in return. As things currently stand, the expected benefits vary significantly based on geography, and depending on one's current health-care coverage, this could even be a reason to move."
Hawaii, which is among states expanding Medicaid coverage, ranked No. 6 on the list of states expected to see the most benefit: In Hawaii, every $1 spent in taxes will see a $2.8 dollar return. The top five states with higher return on investments than Hawaii are Vermont, West Virginia, New York, Kentucky and Oregon.
On the contrary, taxpayers in states that are not expanding Medicaid coverage still have to pay to fund health-care improvements in other states. The five states at the bottom of the list are Montana, North Carolina, Alabama, South Carolina and Mississippi, leaving $3.44 to $8.03 in federal funding on the table, respectively, that will pay for Medicaid expansion in other states.
See the full report here.
In Hawaii, Medicaid is administered through the State Department of Human Services. On Oct. 1, in compliance with the Affordable Care Act, the department launched an online system called Kolea to make it easier for people to apply and determine eligibility. Since October, the department has received more than 8,000 online applications, according to its website.
Jenna Blakely covers health care and nonprofits for Pacific Business News.
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Pronunciation: /kəˈmɪtɪd/
Translation of committed in French:
- 1 (devoted) [parent, teacher] dévoué/-e; [Christian, Socialist] fervent/-e to be committed to/to doing se consacrer à/à faire
- 2 (with commitments) pris/-e
Definition of committed in:
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Monday, December 31, 2012
The Parallel Government
Of The Entire World
All of us, every single man, woman, and child on the face of the Earth were born with the same unalienable rights; to life, liberty and the pursuit of happiness. And, if the governments of the world can't get that through their thick skulls, then, regime change will be necessary.
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""The Koran teaches fear, hatred, contempt for others, murder as a legitimate means for the dissemination and preservation of this satanic doctrine, it talks ill of women, classifies people into classes, calls for blood and ever more blood. Yet, that a camel trader sparks uproar in his tribe, that he wants to make his fellow citizens believe that he talked to the archangel Gabriel; that he boasted about being taken up into heaven and receiving a part of that indigestible book there, which can shake common sense on every page, that to gain respect for this work, he covers his country with fire and iron, that he strangles fathers, drags away daughters, that he leaves the beaten a free choice between death and his faith: now this is certainly something that no-one can excuse, unless he came as a Turk into the world, unless superstition has stifled any natural light of reason in him." --- Voltaire (1694-1788) Translation by SIMONXML from "ISLAM – Dem Untergang geweiht" by Thomas K. Luther, p.24.
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"An Islamic regime must be serious in every field," explained Ayatollah Khomeini. "There are no jokes in Islam. There is no humour in Islam. There is no fun in Islam."
"I want to be very, very clear, however: I understand and agree with the analysis of the problem. There is an imminent threat. It manifested itself on 9/11. It's real and grave. It is as serious a threat as Stalinism and National Socialism were. Let's not pretend it isn't."
"I want to be very, very clear, however: I understand and agree with the analysis of the problem. There is an imminent threat. It manifested itself on 9/11. It's real and grave. It is as serious a threat as Stalinism and National Socialism were. Let's not pretend it isn't."
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In the winter season Air Canada have begun operating both flights from Toronto and Vancouver to HKG with 777-300ERs, replacing the former combination of 777-200LRs and 777-300ERs. (1024px)
Sharklets have been installed to this A320 of this S7. SIBERIAN 545 is final approach rwy 25R from Vladivostok in the afternoon.


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.- A homosexual activist group is claiming that groups at dozens of high schools and middle schools in California, including at least one Catholic school, have signed up to show and discuss its explicit films. The films include one in which a boy "comes out" by wearing his mother's bikini.
Another film uses Native American spirituality to depict lesbian, gay, bisexual and transgender (LGBT) people as being "two-spirit" people.
Parts of the discussion materials encourage students to question whether religious and cultural celebrations such as bar mitzvahs wrongly discourage homosexual and transgender lifestyles, the Pacific Justice Institute (PJI) reports.
PJI said it was not clear whether the films are being shown predominantly during class time or in meetings of LGBT student clubs.
"They are clearly being marketed for showing in class and in school assemblies," PJI says in a press release.
The films and accompanying school materials are being distributed by Youth in Motion, a partnership between the Gay-Straight Alliance Network and the LGBT media group Frameline.
"Our goal is to give student activists and teachers new tools to educate their peers about lesbian, gay, bisexual, and transgender history and culture (and have fun doing it!). And we believe that an inclusive curriculum is one step to creating truly safe and welcoming schools," Youth in Motion says on its website.
Trying to draw on the controversy of Proposition 8 in California, Youth in Motion promotes its videos on "hot topics" such as "marriage equality."
The group claims over 250 schools have registered for the program.
The list of registered schools includes high schools throughout California and some middle and junior high schools. At least one elementary school is on the list. Many of the schools are located in the Bay Area and Southern California, but the Sacramento area and small communities are also affected.
Notre Dame High School, a Catholic girl's school in Belmont, Calif. sponsored by the Sisters of Notre Dame de Namur, is on the list of schools registered for the Youth in Motion curriculum.
"PJI will continue to investigate whether any opt-out laws are being violated by the showing of these films," said PJI president Brad Dacus. "Any parents whose opt-out requests are denied by their child's school should contact PJI immediately."
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RTC: Free agency shouldn't scare Houston
Paul KuharskyReading the coverage... Houston Texans Before he was a Pro Bowl corner for a Super Bowl team, Tramon Williams was an undrafted free agent for the Texans, says John McClain. Cameron Jordan is the pick at 11 for Houston in Lance Zierlein's first...
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Draft room tale: Remembering chaos
Paul KuharskyMike Reinfeldt's cautionary war room tale is the same as many executives who were involved with picks anywhere in close proximity to Minnesota at No. 7 in 2003. "I have one good story of chaos," said Reinfeldt, who classifies most draft rooms he's...
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Giant Haynesworth deal will have ripple effect
Paul KuharskyPosted by ESPN.com's Paul Kuharsky Haynesworth INDIANAPOLIS -- The numbers flying around the scouting combine are baffling, but we shouldn't be surprised. After Oakland signed cornerback Nnamdi A...
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Reading the coverage: Johnson shines in October
Paul KuharskyPosted by ESPN.com's Paul Kuharsky Good morning, AFC South. Let's have a look around before we put on our costumes and commence trick-or-treating. I hear you're giving away full-sized Snickers. Very generous. I may revive my Dr. Evil outfit. Houston ...
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When Jesus comes back,, HARLAN IS IN TROUBLE!!
Posted in the Harlan Forum
1 - 20 of 59 Comments
Last updated Mar 16, 2013
Mar 9, 2013
WYMT just reported this and then asked residents on the street about it:
They asked 'do you think he will turn everyone in Harlan into pillars of salt?' then they said 'or do you think he will just diarrhea on the town so much that even the flood walls will prevent his Jesus poop from pouring in to the streets of Harlan.' Everyone head for the high rise in you see liquid poop coming from the sky from a man in a white robe in sandals riding a cloud! You heard the warning here first folks. Personally, I think he will diarrhea all over the place; I mean, if you think about it, it rains all the time-and we all know that that is when he is peeing. But dude hasn't had a good poop in 2000 years!! Get a heavy duty umbrella folks and thank me later! |
Mar 9, 2013
is this your ideal of a joke this is crazy what in the hell is wrong with you
Mar 9, 2013
How can anyone lay down and sleep after writing something so awful?
Mar 9, 2013
You foul, no-good, heathen, fool how dare you spout this nonsense. I'm about rwady to find you and cast that devil spirit out of you and you need this soon! You heathens nowadays don't respect nothing or nobody, and that is a sad way to be. |
Mar 9, 2013
I must agree with you "The Apostle", preach on brother. |
Mar 9, 2013
Some believe i'm to harsh with people on here, but i'm trying to get through to some of these people. They want listen to you when you do it in a nice way, but hopefully they will see the error of their ways. |
Mar 9, 2013
holy shit. this is crazy man.
Mar 10, 2013
Christianity, is nothing more than a cult that blew-up. There isn't going to be a rapture, Jesus isn't coming back. In fact, Jesus, is never mentioned outside of the Bible. No Roman documentation of his crucifiction, nothing. No documentation of any of these mass miracles and there where historians around during the time.
Mar 10, 2013
You heathen! |
Mar 10, 2013
You nonthinking sheep. |
Mar 10, 2013
This is by far the stupidest thing I have ever heard! Who talks like that. Poop and pee, seriously, that is crazy talk!! Also sounds very nerdy and geeky. Are you high, or did you just forget to take your meds! |
Mar 10, 2013
Just what are you thinking, seriously, whacked out crazy talk and your on here defending it! Wow, I would rather not think if what I thought about was that dumb!!! |
Mar 10, 2013
Oh I agree. The original comment is crazy. Poop and pee, whatever. I'm saying religions are stupid. The Bible is so full of contradictions it defeats itself. |
Mar 10, 2013
And I challenge you to find historical documentation of Jesus, outside the Bible. Go ahead, I'll wait. |
Since: Jul 12 500 United States |
Mar 10, 2013
me to. |
Mar 10, 2013
That's right, Dogs, they can't. The only thing they really got to go on is the Pauline letters. Josephus and Tacitus, weren't even alive during the supposed life of, Jesus. They had to of written their accounts based on hearsay. The king James Bible, that is so prized here in Harlan, was written in 1611 and went through a heavy editing process that king ordered to match what the Church of England believed at the time. There was 47 people involved in this process of translating different languages. Yes, it was pieced together.
Since: Mar 13 308 Location hidden |
Mar 10, 2013
Time it's self proves it.You have a calender don't you?bc and ad;before christ and after the death.Proof enough for me! |
Mar 10, 2013
Okay, I need no proof for myself! But here's the real thing..... some people believe and some people do not! Some people pretend to be religious and some people really are! My opinion on the matter is that there is no need arguing or debating it. You know, live and let live and to each their own. And why is it that the nonbelievers are the ones that are usually trying to push their opinions on others. People shouldn't get all hung up over it! |
Mar 10, 2013
Then why did you get all hung up about it? You went on an attack and know you're retreating. |
Mar 10, 2013
The original poster seems to need psychological help and the Apostle you need to read the Bible if you truly believe. Apostle you are not supposed to call people fools! You should not be arguing with others! This gives Christianity a bad name. You should repent Apostle....read the Bible and Pray for yourself and others!!! You have to forgive others in order to be forgiven!!! You seem high minded! Pride is a sin! You have to show an example in order to be a Christian and arguing is not the example that Jesus wants. Also, I would like to apologize for all or my rude and just plain bad comments. I have come to know that the only thing that matters to me is getting saved! And that doesn't mean just saying it! That means living it as well! |
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Relevant offers
The acclaimed film director's five years of work came to fruition with the release of Two Little Boys, filmed in Invercargill and the Catlins.
1) What was your personal highlight of 2012?
It would have to be the birth of my first child, Max. But second would certainly be the New Zealand premiere of Two Little Boys in Invercargill. After five years working on the film, I could finally relax and celebrate what we'd all done with a warm and very excited crowd. It was a great night.
2) What was your biggest regret?
Not being able to attend the Berlin International Film Festival screening of Two Little Boys because Max's due date was the same as the screening date! In the end he came two weeks late so I could have gone after all.
3) What was your best purchase of the year?
A New Zealand made "Cariboo" bassinet that is suspended so when Max couldn't get to sleep we could just lean over and give him a little swing. Highly recommended to any new parent!
4) What made you angry this year?
The Government's insistence in pushing through the asset sales. Ideology over logic or economics in my opinion.
5) What made you laugh this year?
My son. Every day. I didn't realise babies could be so funny.
6) What new skill did you learn?
I learnt to juggle. Work and children that is. Got a feeling I'll be juggling for a while.
7) Most embarrassing moment of the year?
I'm not easily embarrassed these days. Worst moment of the year, however, was the film breaking down halfway through the preview screening to all the key Auckland critics. Our most influential audience had to wait in the dark for five minutes while the projectionist re-spliced it.
8) How has your life changed from January 1 to now?
This has been a year full of changes. I've had a baby, moved house, completed and released a film, started writing another. The really big changes are related to being a new parent though - new routines, no sleep, no going out - the glamorous lifestyle every parent knows well.
9) What's your goal for 2013?
Get a new project off the starting blocks and find a balance between work and parenthood that enables me to do both well.
10) Tell us a secret . . .
I can ride a unicycle and play the mandolin - but I don't do either well and I can't do them at the same time.
- The Southland Times
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"There's granddad up there, rockin' away," jokes Paul McCartney, addressing the crowd at London's O2.
Paul McCartney will marry Nancy Shevell in London tomorrow, the Mirror reported, citing a person close to the musician and former member of the Beatles that it didn't identify.
In the David H. Koch Theater at New York's Lincoln Center Thursday night, audience members craned and swiveled to catch a glimpse of Paul McCartney sitting in the first ring.
Fifty years ago this weekend, four guys got on TV before a screaming audience and drew more U.S. viewers than any show ever had. Over the next six years, they made sublime music, girls faint and a ton of money. Below, Peter Brown, right hand to Beatles manager Brian Epstein, remembers how they did it all. Republished from March of last year.
"I love branching out of fashion."
- Paul McCartney on Oct 01, 2014
NYCB's Brown on Collaborating With Paul McCartney (Audio)


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Results 1 - 5 of 5 in Kilby Correctional Facility
New sentences for men convicted of killing Prichard minister and barber 'Mr. Sam' Richardson w/Photo
... lone shooter of Richardson - sentencing him to 32 years. He is currently serving his sentence at Kilby Correctional Facility in Mt. Meigs. No one from Richardson's family, nor that of Brown or Amison, was present at the re-sentencing hearing. Asked ... Comment?
Late Kathryn Tucker Windham, who broke ground as female reporter, to be inducted into... w/Photo
... on the dead child's face in the dark to discourage predators. Later, she was assigned to visit Kilby Correctional Facility, where the children's father had been serving time, and give him the news of his daughters' deaths. Many Alabamians know ... Comment?
... and, on Dec. 29, the Tuskegee Police Department joined the search. On Dec. 30, search dogs from Kilby Correctional Facility joined VA staff and Tuskegee police in the hunt for Smith. The Alabama State Police Aviation Unit searched nearby woods from ... Comment?
2 charged, third sought in attempted murder of Tuskegee police officer w/Photo
... and Notasulga Police Departments, along with the Macon County Sheriff's Department and the Kilby Correctional Facility Team search the wooded area but did not locate the suspects. Police say they found three assault rifles and a handgun in the ... Comment?
Some Alabama corrections officers make more in overtime than regular pay, records show
From 2009 through 2013, Irvin Harris made an average of $49,351 a year in base pay working as a corrections officer at Kilby Correctional Facility - a salary that puts him close to the median household income in Alabama. Comment?
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Danny Mateo was selected by the Maui County Council to be county clerk.
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Welcome to Summer's new heart valve journal!
Hmmmm...Unfortunately, Summer has yet to post an update to this journal. That said, I'd encourage them to start posting by leaving them a note in their guestbook.
Send Summer A Note
Dr. Patrick McCarthy
Dr. McCarthy has performed over 4,000 heart valve procedures.
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- Forum: Golf Talk
- Thread: What'd You Shoot Today?
82. A pretty crappy 44 on the front 9 followed by a 38 on the back 9. Always enjoy it more when the scores get better (instead of worse) as the round progresses.
Oh, and 82 is pretty good for me!


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England game rained off
England's Twenty20 International against South Africa has been called off 24 hours in advance following heavy rain at the Chester-le-Street ground.
Authorities made the decision to call off England's only competitive Twenty20 game before their Stanford match in Antigua on November 1 after arriving at the ground to find the outfield saturated.
It means England will be without any practice in this format before they fly out to the West Indies to compete in the £10m game against a Stanford All Stars team.
Arsenal transfer news and rumours: Gabriel Paulista sent to Paris to speed up move; Luis Suarez bid planned; Gunners eye Daniele Rugani
Chelsea vs Liverpool match report: Branislav Ivanovic seals classic semi-final with extra-time header to send Blues to Wembley
Gabriel Paulista: Talented Brazilian could grow into world-class defender at Arsenal
Andy Murray vs Tomas Berydch: Murray cool over reunion with coach Dani Vallverdu at the Australian Open
Transfer news live: Gabriel Paulista to Arsenal CONFIRMED; Juan Cuadrado to Chelsea; Liverpool close in on £14m Ezequiel Lavezzi
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2013 NCAA Tournament: A Look At How No. 4 Syracuse Orange Reached the Final Four
Although the No. 4 Syracuse Orange made the Big East tournament championship game, no one gave them much credit going into the 2013 NCAA Tournament. Hence the No. 4 seed. Well, none of that matters now because Syracuse has made it all of the way to the Final Four. Let's take a look at how they got this far.
Syracuse opened up the tournament quieting many doubters with a blowout victory against No. 13 Montana. Sure, the Orange were expected to win this game, but not by 47 points.
The Orange then took on a pesky No. 12 California team and just managed to escape by six points. Syracuse still managed to win that game, despite going on a 12-minute drought without scoring a field goal. A team can pull things off like that when it has a good defense and you can definitely say that about Syracuse.
Syracuse then took on No. 1 Indiana in the Sweet 16. No way is the Orange's defense going to shutdown Indiana, let alone beat them. Wrong. The Orange held the Hoosiers to just 50 points. Coming into this game, Indiana was the third ranked offense in the nation when it came to points per game. Apparently, Syracuse could care less.
Okay, Syracuse is in the Elite Eight, but there is no way that they were going to beat another Big East opponent in the No. 3 Marquette Golden Eagles, which is the same team that already beat them this year. Well, this team looked very impressive in the Elite Eight and held Marquette to just 39 points.
Holding one team under 40 points in the NCAA Tournament is one thing, but doing it twice in the span of four games is unheard of.
Syracuse will now take on the No. 4 Michigan Wolverines in the Final Four. Although Syracuse has proved a lot to this point, there are probably still many doubters out there that think Trey Burke and the Wolverines will walk all over the Orange. That is probably just fine by Syracuse because they have been doubted a time or two this season already.
If you're a tweeter, feel free to follow me @TheTrevorLowry.
5 Must-See Big Ten Games
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Alabama vs. Kentucky: Game Preview, Prediction
The Kentucky Wildcats return home on Saturday to host the Alabama Crimson Tide in an SEC clash. Read More
5 Must-See ACC Basketball Matchups
Five of the best games remaining on the ACC conference schedule in 2015. Read More
N. Carolina vs. Louisville: Preview, Prediction
A pair of Top 25 teams meet on Saturday when the North Carolina Tar Heels visit the KFC Yum! Center to face the Louisville Cardinals. Read More
Memphis vs. Gonzaga: Preview, Prediction
Memphis will need a miracle to pull off the upset against Gonzaga on Jan. 31. Read More
Miami at Florida State: Preview, Prediction
FSU super freshman Xavier Rathan-Mayes will again light up the scoreboard against Miami. Read More
5 Bold Predictions for Duke vs. Virginia
The Virginia Cavaliers will take over the ACC with a win over the Duke Blue Devils this weekend. Read More
North Carolina at Louisville Will Be Epic Showdown
Louisville will host North Carolina in what should be a showcase game for the ACC. Read More
Oklahoma vs. Oklahoma State: Preview, Prediction
All eyes will be on Buddy Hield when the Oklahoma Sooners take on the Oklahoma State Cowboys in their second meeting of 2014-15. Read More
AAC Basketball Power Rankings For Week 12
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Stanford Backcourt Is Severely Underrated
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UCLA Saves Season With Win Over Utah
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