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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Adenoviral Gene Delivery Can Reprogram Human Fibroblasts to Induced Pluripotent Stem Cells†‡§
Version of Record online: 20 AUG 2009
DOI: 10.1002/stem.201
Copyright © 2009 AlphaMed Press
Volume 27, Issue 11, pages 2667–2674, November 2009
Additional Information
How to Cite
Zhou, W. and Freed, C. R. (2009), Adenoviral Gene Delivery Can Reprogram Human Fibroblasts to Induced Pluripotent Stem Cells. STEM CELLS, 27: 2667–2674. doi: 10.1002/stem.201
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Author contributions: W.Z.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; C.R.F.: conception and design, data analysis and interpretation, manuscript writing.
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Disclosure of potential conflicts of interest is found at the end of this article.
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First published online in STEM CELLS EXPRESS 2009.
Publication History
- Issue online: 6 NOV 2009
- Version of Record online: 20 AUG 2009
- Accepted manuscript online: 20 AUG 2009 12:00AM EST
- Manuscript Accepted: 8 AUG 2009
- Manuscript Received: 18 MAY 2009
Funded by
- Anna Mitchell Family Foundation
- Human iPS cells;
- Adenoviral vector;
- DNA integration;
- Human ES cells;
- Parkinson's disease;
- Dopamine neurons
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Mouse and human fibroblasts have been transformed into induced pluripotent stem (iPS) cells by retroviral transduction or plasmid transfection with four genes. Unfortunately, viral and plasmid DNA incorporation into chromosomes can lead to disruption of gene transcription and malignant transformation. Tumor formation has been found in offspring of mice generated from blastocysts made mosaic with iPS cells. To proceed with iPS cells for human therapy, reprogramming should be done with transient gene expression. Recently, adenoviral vectors have been used to produce mouse iPS cells without viral integration. Here, we report the successful creation of human iPS cells from embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. After screening 12 colonies, three stable iPS cell lines were established. Each cell line showed human embryonic stem cell morphology and surface markers. Southern blots and polymerase chain reaction demonstrated that there was no viral DNA integration into iPS cells. Fingerprinting and karyotype analysis confirmed that these iPS cell lines are derived from the parent human fibroblasts. The three human iPS cell lines can differentiate to all three germ layers in vitro, including dopaminergic neurons. After s.c. injection into nonobese diabetic–severe combined immunodeficient mice, each human iPS line produced teratomas within 5 weeks postimplantation. We conclude that adenoviral vectors can reprogram human fibroblasts to pluripotent stem cells for use in individualized cell therapy without the risk for viral or oncogene incorporation. STEM CELLS 2009;27:2667–2674
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Stem cell research offers great promise for understanding basic mechanisms of human development and differentiation, as well as the hope for new treatments for diseases such as diabetes, Parkinson's disease, and myocardial infarction. However, human embryonic stem (ES) cell research also raises ethical and political controversies. The groundbreaking work of Shinya Yamanaka has shown that adult mouse and human fibroblasts can be transformed into induced pluripotent stem (iPS) cells by retroviral transduction with only four genes encoding the transcription factors c-Myc, Klf4, Oct4, and Sox2 [1, 2]. This work was confirmed by others in murine and human systems [3–5]. The reprogramming of somatic cells to iPS cells avoids the ethical problems specific to ES cell research. Reprogramming fibroblasts to stem cells makes it possible to create unique pluripotent stem cells to treat individual patients and study human disease.
A shortcoming of the initial reprogramming method was the use of a retrovirus or lentivirus to transduce fibroblasts. These viruses are integrated into host chromosomes where they can cause insertional mutagenesis, interfere with gene transcription, and induce malignant transformation [6]. The average retroviral infection leads to 10-20 retroviral integration sites in human iPS cell lines [2, 7]. In the first report of germline-competent mouse iPS cells, 20% of chimeric mice developed tumors that were attributable to the reactivation of the c-Myc proviral transgene that had integrated into the host cell genome [8]. This result was corroborated by a report from another group describing cancer-related deaths in 18 of 36 iPS chimeric mice [9]. Higher rates of malignancies are not usually observed in chimeric mice derived from ES cells; therefore, the tumorigenicity of iPS cells constitutes a major safety concern.
To proceed with iPS cells for human therapy, reprogramming should be done with nonintegrating strategies. Several groups have sought alternatives to retroviral gene integration. Yamanaka's group transfected plasmids without using virus to generate mouse iPS cells [10]. Two recent papers have shown that mouse and human iPS cells can be produced by piggyBac transposition with four genes in a single plasmid, thus significantly improving induction efficiency [11, 12]. Another method has used Cre-recombinase-excisable viruses to reprogram mouse cells [13, 14]. Thomson's laboratory has described production of human iPS cells using nonintegrating episomal vectors [15]. After removal of the episome, iPS cells were free of vector and transgene sequences. Ding's laboratory reported the generation of mouse iPS cells by the insertion of transducing proteins into the cytoplasm of fibroblasts in the presence of valproic acid [16].
Replication-defective adenoviral vectors have proven useful for gene insertion into cells without integration into chromosomal DNA [17, 18]. Gene expression can persist for days, which should provide sufficient time to reprogram fibroblasts to pluripotent cells. Stadtfeld and colleagues have used adenoviral vectors to generate mouse iPS cells from liver cells and fibroblasts without viral integration [19].
In this report, we successfully produced iPS cells from human embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. The human iPS cell lines express ES cell-specific markers and can be differentiated into all three germ layers in vitro and in vivo. We demonstrate that human iPS cells generated with adenovirus are free of any viral or transgene integration. Because our laboratory has a long interest in cell therapy for Parkinson's disease, we show that the human iPS cells can be differentiated into dopamine neurons.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Construction of Adenovirus
We used the Ad-Easy system (The Johns Hopkins Oncology Center, Baltimore, MD, http://www.coloncancer.org/adeasy.htm) to create adenoviruses expressing human Oct4, Sox2, Klf4 and c-Myc. Four expressed sequence tag clones containing full coding sequences were obtained from Open Biosystems (Huntsville, AL, http://www.openbiosystems.com). A 1.4-kb fragment of human Oct4 cDNA was cloned into NotI-XbaI sites of pShuttle-cytomegalovirus (CMV) vector [18, 20]. Similarly, cDNA fragments for human Sox2 (1.5 kb), Klf4 (2.0 kb), and c-Myc (1.5 kb) were cloned into XhoI-HindIII sites of pShuttle-CMV vector. Sequences were verified by restriction digestion. Homologous recombinations were performed in bacterial AdEasy-1 cells. The adenoviruses were produced in HEK293 cells and purified to high titer, as we and others have described [18, 20-22]. Viral titer was determined by plaque assays and is expressed as plaque-forming units per μl (pfu/μl).
Culture of Human Embryonic Fibroblast IMR90 Cells
Human embryonic fibroblast IMR90 cells were purchased from the American Type Culture Collection (Manassas, VA, http://www.atcc.org; Catalog No. CCL-186). IMR90 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin–streptomycin–L-glutamine (Invitrogen). IMR90 cells were passaged using 0.05% trypsin-EDTA (Invitrogen) and replated at 1:5 dilutions.
Adenovirus Transduction of IMR90 Cells
IMR90 cells were plated at a density of 5 × 104 to 5 × 106 cells per 100-mm tissue culture plate. The next day (day 2), IMR90 cells were about 40% confluent. The cells were incubated with medium containing four adenoviruses (Ad-cMyc, Ad-Klf4, Ad-Oct4, and Ad-Sox2) plus Ad-green fluorescent protein (GFP), each at a multiplicity of infection of 50-500 pfu/cell. Twenty-four hours later, adenoviruses were removed and replaced with normal IMR90 culture medium. On day 4, IMR90 cells had grown to near confluence. The cells were transduced with the same five adenoviruses at the same concentrations as on day 2. Twenty-four hours later, adenoviruses were removed and replaced with normal IMR90 culture medium. On day 7, IMR90 cells were dissociated using 0.05% trypsin-EDTA and plated onto mouse embryonic fibroblast feeder layers (PMEF) (Millipore, Billerica, MA, http://www.millipore. com) at a concentration of 1 × 106 cells per 100-mm plate. From day 8 onward, the culture medium was changed to human ES cell medium consisting of DMEM/F12, 20% Knockout Serum Replacement, nonessential amino acids, sodium pyruvate, 1% penicillin–streptomycin–L-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng/ml basic fibroblast growth factor (bFGF). The medium was changed every other day.
iPS Cell Generation
By days 25-30, a total of 26 colonies showing ES cell-like morphology emerged under various conditions (Table 1). Twelve colonies with promising morphology were selected with a P-20 pipette tip. These colonies were briefly dissociated in 0.05% trypsin-EDTA and plated on mouse embryonic fibroblasts in individual wells of a 96-well plate in human ES cell medium. The colonies were further expanded in 24-well plate and six-well plate cultures. Finally, three iPS clones were established. The doubling time of iPS cells was about 36 hours.
Immunofluorescent Staining for ES Cell Markers and Three Germ Layers
Human iPS cell colonies and differentiated cells were fixed in 4% paraformaldehyde for 20 minutes. The cells were washed three times with phosphate-buffered saline (PBS) and incubated in blocking buffer (PBS, 5% normal goat serum, 0.1% Triton X-100) for 30 minutes. Cells were incubated overnight at 4°C in the following primary antibody solutions (from Chemicon, Temecula, CA, http://www.chemicon.com, unless otherwise noted): rabbit anti-Oct4 (1:200), rabbit anti-Nanog (1:200), rat anti-stage-specific embryonic antigen (SSEA)-3 (1:100), mouse anti-SSEA-4 (1:100), mouse anti-TRA-1-60 (1:100), mouse anti-TRA-1-81 (1:100), mouse anti-smooth muscle actin (SMA) (1:200), mouse anti-α-fetoprotein (AFP) (1:200), rabbit anti-Nestin (1:200), mouse anti-microtubule-associated protein (MAP)2 (1:200), mouse anti-βIII-tubulin (TuJ1; 1:200; Promega, Madison, WI, http://www.promega.com), rabbit anti-tyrosine hydroxylase (TH) (1:150, Pel-Freez, Rogers, AK, http://www.invitrogen.com). After three washes with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibodies for 1 hour. Fluorescent staining was examined with a Nikon Diaphot fluorescent microscope (Nikon Instruments, Inc., Melville, NY, http://www.nikoninstruments.com), and images were taken by SPOT digital camera (SPOT Imaging Solutions, a division of Diagnostic Instruments, Inc., Sterling Heights, MI, http://www.diaginc.com). For alkaline phosphatase (ALP) staining, iPS cells were fixed with 4% paraformaldehyde for 2 minutes, followed by a 15-minute incubation with staining solution (Alkaline Phosphatase Detection Kit; Millipore).
Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was purified from human iPS cells using the RNeasy Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Reverse transcription-polymerase chain reaction (RT-PCR) was performed according to established methods [23] using an RT-PCR kit (Invitrogen) with the primers listed in supplemental online Table 1. Primers overlapping the coding region and 3′-untranslated region were used for endogenous mRNA. Primers covering only the coding region were used for adenovirus-mediated mRNA. Total RNA from human ES cells (BG01V) was used as a positive control. For adenovirus-mediated transcription, total RNA from HEK293 cells infected with each adenovirus was used as a positive control.
Bisulfite Sequencing
Genomic DNA was purified from human iPS cells using the DNeasy Kit (Qiagen). The conversion of unmethylated cytosines to uracil was carried out as described in the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, http://www.zymoresearch.com). One microgram genomic DNA was treated in each reaction, and 2 μl of elution was used for each PCR reaction. The primers GAG GCT GGA GCA GAA GGA TTG CTT TGG and CCC CCC TGG CCC ATC ACC TCC ACC ACC TGG were used to amplify a genomic DNA fragment (460 bp) in the human Oct4 promoter using the following conditions (Herculase II Fusion DNA Polymerase system; Stratagene, La Jolla, CA, http://www.stratagene.com): 1× Herculase buffer, 2 mM MgCl2, 0.2 mM deoxynucleotide triphosphate (dNTP), 0.25 μM primers, 100 ng genomic DNA, and 1 μl Herculase DNA polymerase. The cycling conditions were 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 59°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 minutes. The resultant PCR products were cloned into pCR2.1 vector (Invitrogen) and sequenced.
Karyotyping and DNA Fingerprinting
The human iPS cells were cultured in six-well plates as described above. Cells at 80%–90% confluence were mitotic arrested with colcemid (0.05% μg/ml) for 3 hours and harvested using 0.05% trypsin-EDTA. Hypotonization was performed with 0.075 M KCl, and a 3:1 mixture of methanol and glacial acidic acid was used for cell fixation. Slides were dropped and aged for 8 hours at 57°C. Giemsa/trypsin/Leishman banding was performed following a standard protocol, with 2.5-minute incubations in 0.6% trypsin and Leishman's stain. Imaging and karyotyping were performed using BandView software (Applied Spectral Imaging, Vista, CA, http://www.spectral-imaging.com). To confirm that human iPS clones were derived from IMR90, short tandem repeat (STR) analysis was performed in the DNA Sequencing Core at the University of Colorado – Denver.
PCR Analysis for Viral Gene Integration
Human iPS cells were cultured in six-well plates as described above. Cells were dissociated using 0.05% trypsin-EDTA. After centrifugation at 200g for 5 minutes, cell pellets were suspended in 200 μl PBS with 20 μl proteinase K (10 mg/ml) and 4 μl RNaseA (100 mg/ml). Total genomic DNA was purified using the DNeasy Blood and Tissue Kit according to provided protocols (Qiagen). The DNA concentration was measured by absorbance at 260 nm. For genomic DNA PCR, we used the Herculase II Fusion DNA Polymerase system (Stratagene) with the following setup: 1× Herculase buffer, 2 mM MgCl2, 0.2 mM dNTP, 0.25 μM primers, 100 ng genomic DNA, and 1 μl Herculase DNA polymerase. The cycling conditions were 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 minutes. The primers are listed in supplemental online Table 2. We used 1 pg of adenoviral vector as a positive control. We analyzed 15 μl PCR products in 1% agarose gel.
Southern Blotting
The probes for Southern blots were generated by random-primed DNA labeling with digoxigenin (DIG)-2′-deoxyuridine 5′-triphosphate (Roche, Basel, Switzerland, http://www.roche-applied-science.com). DNA fragments for Oct4, Klf4, c-Myc, and Sox2 (1.5-2.0 kb) were purified from pCMV-Shuttle vector after digestion with NotI-XbaI or XhoI-HindIII. The pAd-Klf4 adenoviral plasmid DNA was digested with BamHI-HindIII, which in theory would generate 9 DNA fragments (2, 2.8, 2.9, 3.2, 3.5, 4.5, 4.7, 5.3, and 6.0 kb). All adenoviral fragments were purified and used for producing probes for whole adenoviral vector blots.
Genomic DNA was purified from iPS cells, IMR90cells, and HEK293 cells with the DNeasy kit (Qiagen). Purified genomic DNA (5 μg) was digested with BamHI overnight. As a positive control for whole vector blotting, pAd-Klf4 adenoviral plasmid DNA was digested with BamHI-HindIII. As a positive control for cDNA blots, pAd-Oct4 adenoviral plasmid DNA was digested with NotI-XbaI, whereas pAd-Klf4, pAd-cMyc, and pAd-Sox2 adenoviral plasmid DNA was digested with XhoI-HindIII.
Digested DNA fragments were separated on 0.8% agarose gels and transferred to a nylon membrane (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com). The membrane was incubated with DIG-labeled DNA probes in DIG Easy Hyb buffer (Roche) at 42°C overnight with constant agitation. After washing, ALP-conjugated anti-DIG antibody (1:10,000; Roche) was added to the membrane. Chemiluminescent signals were detected using x-ray film after incubation with CDP-Star (Roche).
In Vitro Differentiation
Human iPS cells were dissociated using 0.05% trypsin-EDTA and were replated on noncoated Petri dishes at 1 × 105 cells per 35-mm dish. The medium was the same as human ES cell medium, but without bFGF. The medium was changed every 2 days. After 7 days in floating culture, embryoid bodies formed. We then transferred embryoid bodies to 0.1% gelatin-coated tissue culture dishes (12-well) using the same medium. The medium was changed every other day once embryoid bodies were attached to the dish. Differentiated cells were fixed after 8 days in adherent culture.
Dopamine Neuron Differentiation
Mouse PA6 stromal cells were cultured in DMEM with 10% fetal calf serum and 1% penicillin–streptomycin–L-glutamine. The day before iPS cell differentiation, PA6 cells were dissociated using 0.05% trypsin-EDTA and seeded on 0.1% gelatin-coated 12-well plates at 1 × 105 cells/well [24–26]. Human iPS cells were dissociated using 0.05% trypsin-EDTA and seeded on the PA6 cell layer at 1,000 iPS cells/well. The medium was ES cell medium without bFGF and was changed every 2 days. The iPS cells were differentiated on PA6 cells for 14-16 days. After differentiation, cells were fixed and stained with antibodies for βIII-tubulin (TuJ1), MAP2, and TH [23].
Teratoma Formation
To examine whether human iPS cells can form teratomas in vivo, we s.c. injected 2 × 106 iPS cells in 0.1 ml PBS to 8-week-old nonobese diabetic severe combined immunodeficient (NOD/SCID) (National Cancer Institute, Bethesda, MD, http://www.cancer.gov) mice. Two mice were injected for each iPS clone. For a control, IMR90 cells (2 × 106 cells in 0.1 ml PBS) were also injected into two mice. After 5-6 weeks, tumors were dissected and fixed in 4% paraformaldehyde. Samples were embedded in paraffin and processed with hematoxylin and eosin staining in the Pathology Laboratory at the University of Colorado Hospital.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
We created four adenoviruses expressing Oct4, Sox2, c-Myc, and Klf4 under control of the CMV promoter. In an initial experiment, human fetal fibroblasts, IMR90, were plated at a density of 50,000, 500,000, or 5,000,000 cells per 100-mm dish. On day 2, the four adenoviruses plus Ad-GFP were added at a concentration of 250 pfu/cell. On day 7, about 45%–55% of the fibroblasts were GFP+ (Table 1). The fibroblasts were dissociated and replated on mouse embryonic fibroblasts and cultured with human ES cell medium. No ES cell-like colonies were formed under these conditions. Because adenoviruses lose their expression after cell division, we decided to repeat adenovirus treatment on day 4.
In a new set of experiments, the IMR90 cells were plated at a density of 500,000 cells per 100-mm dish. On day 2, the four adenoviruses plus Ad-GFP were added at a concentration of 50-500 pfu/cell. On day 4, adenovirus exposure was repeated at the same concentrations. On day 7, IMR90 cells were replated on mouse embryonic fibroblasts in human ES cell medium (Fig. 1A-1C). On days 25-30, ES cell-like colonies emerged (Fig. 1D). All colonies had lost intrinsic Ad-GFP expression, as expected for transient expression of the adenoviral vectors (Fig. 1E). We picked up a subset of iPS colonies showing good ES cell-like morphology, and excluding those colonies with apparent differentiated cells, dark centers, or colonies that appeared too large or too small in size. The ES cell-like colonies were expanded, and stable iPS cell lines were established (Table 1). A third series of iPS cell induction experiments was performed using 5 × 104 to 5 × 106 cells per 100-mm dish with adenovirus infections on day 2 and day 4. The results confirmed that human iPS cells can be generated by repeated adenovirus infections at a concentration of 250 pfu/cell with 500,000 cells per 100-mm dish, as shown in Table 1. Of 12 ES cell-like colonies, three independent human iPS cell lines were established. The iPS cell induction efficiency is low, about 0.0002% (one iPS cell clone per 500,000 starting fibroblasts).
As shown in Figure 1F, human iPS cells demonstrated morphology typical of human ES cells and were positive for ALP staining (Fig. 1G). Human iPS cells expressed ES cell markers such as Oct4, Nanog, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 1H–1M). Results of RT-PCR analysis showed that all three iPS clones expressed mRNA for endogenous Oct4, Sox2, Klf4, c-Myc, and Nanog (Fig. 1O). By contrast, we could not detect any adenoviral vector-derived mRNA for Oct4, Sox2, Klf4, or c-Myc (Fig. 1O). We analyzed the methylation status of cytosine guanine dinucleotides (CpGs) in the Oct4 promoter in the three iPS clones. Results showed that CpGs were highly unmethylated in all three clones, compared with the highly methylated CpGs in parent IMR90 cells (Fig. 1P). This finding indicates that the Oct4 promoter was activated in the iPS cells.
STR analysis of three iPS clones revealed identical results as with IMR90 cells, indicating that all three iPS cells were derived from parent IMR90 cells (Table 2). Cytogenetic analysis demonstrated that each human iPS cell line had the same karyotype (46, XX) as the parent IMR90 cells (an example is shown in Fig. 2). All three iPS cell lines were diploid, in contrast to previous findings that adenovirus-generated mouse iPS cells were tetraploid in three of 13 iPS cell lines [19].
To check for viral integration into genomic DNA, we performed Southern blotting and PCR analysis. Genomic DNA from iPS cells, IMR90 cells, and HEK293 cells was digested with BamHI. To detect viral DNA integration, adenoviral Klf4 plasmid DNA (pAd-Klf4) was digested with BamHI-HindIII, and all fragments were purified for probe labeling. To detect any transgene integration, the whole cDNA fragments of Oct4, Klf4, c-Myc, and Sox2 (1.5-2.0 kb) were used for probe labeling. In all five Southern blots, there were no viral DNA or transgene-specific bands in any of the three iPS cell lines (Fig. 3A–3E). In genomic DNA PCR analysis, all three iPS cell lines amplified a 650-bp human engrailed one (En1) promoter fragment, the same size as from positive controls using the human En1 promoter plasmid (Fig. 4B). The amplification of a randomly chosen DNA fragment helps demonstrate that genomic DNA from the three iPS cell lines is intact. By contrast, all seven PCR reactions were negative for adenoviral gene integration in all three human iPS cell lines (Fig. 4B). These results demonstrate that there was no adenoviral integration in iPS cells generated by adenoviral vectors.
We differentiated human iPS cells to test their pluripotency. The iPS cells formed embryoid bodies after 7 days in suspension cultures (Fig. 5A). Embryoid bodies were allowed to differentiate further in adherent cultures. After 8 days in adherent culture, neural cells appeared (Fig. 5B). Immunofluorescent staining showed that human iPS cells can differentiate into all three germ layers—mesoderm (SMA, Fig. 5C), endoderm (AFP, Fig. 5D), and ectoderm (Nestin, Fig. 5E).
To produce dopamine neurons, iPS cells were plated on PA6 cells for 2 weeks [24–26]. Immunostaining revealed a large number of neurons positive for MAP2 and βIII-tubulin/TuJ1 (Fig. 5F, 5G). Subsequent immunostaining for TH, the rate-limiting enzyme in dopamine synthesis, showed clusters of cells with morphology compatible with dopamine neurons (Fig. 5H). Optimization of midbrain dopamine neuron production from iPS cells is now being pursued in our laboratories.
To examine the differentiation potential of human iPS cells in vivo, we injected 2 × 106 iPS cells into NOD/SCID mice s.c.. After 5-6 weeks, large teratomas formed from all three iPS clones, weighing about 1 g by 6 weeks. Histology revealed a wide range of tissue types including neural tissue (Fig. 5I), gut-like epithelium (Fig. 5J), cartilage (Fig. 5K), and muscle (Fig. 5L).
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
In this paper, we believe that we are the first to report that an adenoviral vector can reprogram human embryonic fibroblasts to iPS cells. These iPS cells show features typical of human ES cells, including surface markers and highly unmethylated Oct4 promoter. We have shown that adenovirus-generated human iPS cells are pluripotent and can be differentiated into all three germ layers in vitro and in vivo. Most important, we have demonstrated that human iPS cells generated by adenovirus are free from viral DNA integration into host chromosomes. Therefore, iPS cells created by the adenovirus method eliminate the risk for malignant transformation associated with retrovirus or plasmid transfection methods. Because we would like to use iPS cells to develop cells for transplant into patients with Parkinson's disease, we have shown that iPS cells can be differentiated into cells with markers compatible with dopamine neurons, as we have described with human ES cells [23, 26]. Refinement of the differentiation and purification protocols could yield cells appropriate for human therapeutic trials.
We have found that the optimal adenoviral concentration for making human iPS cells is 200-250 pfu/cell in a fibroblast density of 5 × 105 cells/65 cm2 with infections applied at day 2 and day 4. The overall efficiency for generating human iPS cells is quite low, about 0.0002%, similar to that reported for adenoviral induction of mouse iPS cells [19]. The low efficiency of human iPS cell formation may be a result of a low percentage of cells coexpressing all four genes and dilution of the viral titer with progressive cell replication. Because IMR90 cells were continuously dividing, adenoviral vectors were lost after each cell division. To improve induction efficiency, we are developing an adenoviral vector expressing all four genes in a single vector, as demonstrated by retroviral and plasmid transfection methods [10-12, 27, 28].
We have not found any tetraploid human iPS cells in our adenovirus experiments. In mice, using an adenovirus-based iPS cell induction method, Stadtfeld et al. [19] have found tetraploid iPS cell clones that were derived from mouse fetal liver cells (one of nine clones) and mouse adult hepatocytes (two of three clones), but not from mouse fibroblasts. Others have shown that 53% of adult liver cells are tetraploid, whereas nearly all fetal hepatocytes are diploid [29, 30]. Because the frequency of tetraploidy in mouse iPS cells matched the ratio of tetraploidy in fetal and adult liver cells, it is most likely that tetraploid mouse iPS cells were derived from starting tetraploid liver cells and not from cell fusion induced by adenovirus.
With improved induction efficiency, our adenoviral method should be useful for reprogramming fibroblasts derived from adults and even elderly people. We plan to use these vectors to generate iPS cells from patients with genetic forms of Parkinson's disease and other neurodegenerative disorders. These integration-free iPS cells will be valuable for studying disease mechanisms, drug screening, and toxicity testing.
In summary, we successfully generated human iPS cells using adenoviral vectors without viral gene integration into the genome. The adenoviral method can create patient-specific iPS cells that can be converted into specific cell types for modeling disease and for treating individual patients without the risk for chromosomal disruption.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
The study was supported by the Leopold Korn and Michael Korn Professorship in Parkinson's Disease at the University of Colorado School of Medicine and the Edward D. and Anna Mitchell Family Foundation. Wenbo Zhou, Ph.D., is a Coleman Faculty Fellow at the University of Colorado. We thank Bette K. DeMasters, M.D., for assistance with histology of the iPS cell teratomas.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
The authors indicate no potential conflicts of interest.
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
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- 29Evidence for a terminal differentiation process in the rat liver. Differentiation 1995; 59: 35–42., , et al.
- 30Hepatic polyploidy and liver growth control. Semin Cancer Biol 2000; 10: 161–171..
Supporting Information
- Top of page
- Abstract
- Acknowledgements
- Supporting Information
Additional supporting information available online.
|Supplemental Table 1 Primers used for RT-PCR Analysis.
|Supplemental Table 2 Primers used for genomic PCR Analysis.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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Kelly Osbourne in Armani Exchange
Kelly Osbourne stepped out for an event for Armani Exchange, wearing a denim motorcycle jacket by Armani Exchange. I love Kelly's purple hair, I think it really suits her. You can buy Kelly's jacket here.
Mark Sanchez Shopping at Armani Exchange
New York Jets quarterback, Mark Sanchez was caught after doing a little shopping at Armani Exchange in New York. Looks like Armani Exchange has gotten quite popular with the male celebrities. Gavin Rossdale, Derek Hough and Twilight's Alex Meraz have all been spotted shopping at the store. Are any of you male readers also a fan of the store?
Images Courtesy of StarTraksPhotos
Rachel Bilson in Citizens Of Humanity
Rachel Bilson was spotted on her way into an awaiting car. She wore her Citizens of Humanity Slick Jeggings in Sedona again. I really love Rachel's style. She really has an eye for putting things together perfectly. Hmmm… I wonder if Rachel is part of the "Secret Chanel Club." I have never seen any other celebrity carry Chanel more than her. Click here to purchase these jeans at RonHerman for $216.
Jessica Alba in MiH
Jessica Alba carries coffee to her car after having breakfast with her daughter, Honor. Jessica is wearing MiH jean leggings, the Vienna jean in Basin wash. I love that Jessica is wearing combat boots! I've seen a lot of celebs wearing them but I'm not sure if I like this trend for summer!

subset: commoncrawl


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The Sixth Annual West Coast Joint Graduate Liberal Studies Symposium calls for papers for presentation at the symposium to be held June 22-24, 2012 at the University of Southern California.
This interdisciplinary symposium provides an opportunity for students and alumni from West coast graduate liberal studies programs to come together to share their work and experiences, and to foster growth in the intellectual community at the core of GLS programs. Approximately 45 students are selected to present papers prepared in a GLS course to an audience of their peers, alumni, faculty, and friends. We also encourage non-presenting students and alumni to register and attend the symposium.
Students submit 250-word proposals to their program directors using the proposal form by March 1, 2012 (unless specified otherwise by your program director).
Selected students will give a 20-minute presentation at the symposium, followed by brief Q&A. Sessions run concerrently throughout the day on Saturday, June 23, and Sunday morning, June 24.
Click here for a Symposium and Call for Papers flyer.
E-mail Kim Allen or call the USC Master of Liberal Studies Program at (213) 740-1349.


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The lights are on
Electronic Arts and developer DICE provided a tantalizing glimpse of Faith and a next-gen version of Mirror's Edge, courtesy of these new screens and a trailer.
DICE isn't saying a lot about the project other than that it's on the next-gen systems and PC, and that it's still in the early stages of development.
Visit our E3 Headquarters for all the news from the show.
Email the author Matthew Kato, or follow on Twitter, and Game Informer.
Dude this looks amazing. Hopefully it won't be 6-7 hours. So much eye candy =D
Cannot wait for this! I'm guessing Spring 2014 or Fall.
Hell yes. Top 3 anticipated games for me.
I love the release date 'when it's ready'.
This makes me happy :)
Future architecture is so damn pristine :D
YES! Although Mirror's Edge had some frustrating gameplay and a mediocre story, I've always seen the massive potential for a sequel. Glad to see that this is actually happening!
Cool beans.
Doing parkour myself I can't wait to see how this turns out. I hope they put in more vaults like kong, double kong, dash, speed, reverse, and maybe even turn, plus lazy and thief. Perfect ga,e for rainy days when I can't train. Can't wait
I like that they kept the bright color palate of the first. Can't wait to see what improvements they have in store!
I hope it's longer. I hope it goes into more details about the world and Faith. I hope it's ready soon. I've never really played anything that gave the feeling of the first one. Like one epic chase scene in a movie. Beautiful and symbolist if colors. Very striking. I look forward to this a lot.


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Poly 5-gal. Fire Pump
This Indian® poly fire water pump allows you to enjoy all the benefits of steel fire water pumps with less weight and no rusting. Features a 52"L spray hose, adjustable shoulder straps and a brass pump that includes both a stream and fan nozzle. Also features padded shoulder straps, a waist strap and a lumbar support for extra support and comfort. 5-gal. capacity. Weighs 9 lbs. empty. USA made.
2016 Master Catalog: Page 345


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Maine Attorney General Under Fire Over Elvers
Maine and Burundi communities begin to heal through storytelling
Join in spiritual solidarity today
Preparing the way for truth and reconciliation in Maine
Conversation with Denise Altvater on Truth and Reconciliation in Maine
The following are excerpts from a recent conversation with Denise Altvater, AFSC's Wabanaki Program Coordinator in Maine. Keith Harvey, AFSC's regional director in New England, hosted the telephone conversation, and several friends and supporters joined the call.
Keith: Denise, would you introduce yourself and your work?
Community Phone Call: Truth and Reconciliation
TimesRecord (ME).com Opinion: Wiscasset students step out to build the beloved community
KEITH 'BEAR' HARVEY was a scholarship football player on the Miami University of Ohio football team when its team name was the "Redskins." His acceptance of that nickname changed following a conversation with a Native American classmate. Harvey's commentary below explains why he joined the effort to change the team's nickname.


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Veal watches from sideline: Bulldog Notebook
J.T. Wall holds Terrence Edwards as they celebrate Edwards' second-quarter touchdown.
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A "star-spangled" Fourth of July kicks summer into high gear in New Braunfels. With attractions like cold rivers and lakes, cool caverns, and Schlitterbahn – 35 years of "the hottest, coolest time in Texas," it's the best place to "summer" in Texas!
Come enjoy the spectacular Fourth of July fireworks and choose New Braunfels for your summer vacation. July is the perfect time – school starts again mid-August and you'll be busy getting ready in about 30 days!
Here's what's happening over the Fourth of July weekend and you can still find places to stay. Keep up with New Braunfels all year long, like us on Facebook.
2015 Old Time 4th of July Patriotic Parade and Program
Saturday, July 4, 2015: 9:15 a.m., Main Plaza, Downtown, Fun for the entire family! Everyone is welcome to walk or ride! More Info…
Join us for the largest Independence Day celebration in New Braunfels featuring music by Adam Johnson Band and fireworks over Landa Lake. Come early, bring a picnic and enjoy the show! NEW: This year we will also have a band at Fischer Park, UpriZing will play at the Fischer Park Amphitheater starting at 6:30 p.m.
Fireworks will be shot from Landa Lake in Landa Park. The Fireworks display can be viewed from Fischer Park. July 4th Spectacular sponsored by H-E-B.
Free Concerts at 6:30PM
Fireworks Start at 9:15PM
Tune in to KGNB 1420 AM to listen to the live broadcast of patriotic music timed to accompany the fireworks display.
Viewing Information: Seating will be available on the Landa Park Golf Course for fireworks viewing this year. Seating will only be available in designated areas only. Firework viewers will NOT be allowed to sit on greens or putting areas (these areas will be roped off). We are encouraging people to view the fireworks from Fischer Park this year. The fireworks will be shot from Landa Lake but can be viewed at Fischer Park. We will have a band at Fischer Park (UpriZing) and will broadcast KGNB 1420AM with the fireworks show.
Gruene Hall: July 3rd – Gary P. Nunn, July 4th – Jack Ingram
Whitewater Music Amphitheater: July 4th Amos Lee & David Gray
Lazy L&L Hall: July 3rd – Keen Country Band, July 4th – County Line Band
Phoenix Saloon: July 3rd – Junior Nash, July 4th – The Georges
Conways Dancehall: July 3rd – Chris Salinas, July 4th – DJ & Open Dance Floor
Natural Bridge Caverns – Bracken Bat Flight
Just a few miles from Natural Bridge Caverns is another amazing natural wonder: The Bracken Bat Flight, and for a limited time we are offering the opportunity to witness the evening emergence of the world's largest bat colony. Be there as millions of free-tailed bats spiral out of Bracken Cave and take to the skies on their nightly hunt for insects, in a truly awe-inspiring reminder of how amazing nature can be.
A private tasting and 4 course meal of our award winning new wines. This fabulous event has been expanded this year with a children's ticket including their own meal and children's area including water slide bounce house and other fun activities. More Info...


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Ty Wentz, 8, hammers a nail into a small birdhouse as his father, Brent, steadies the frame. Ty and his brother, Levi, 12, along with their dad, took part in Saturday's annual father/son breakfast at the Kinston-Lenoir County Public Library. Attendees enjoyed a breakfast provided by Chick-fil-A followed by a small woodworking project, courtesy of Lowe's Home Improvement.
Location, ST | website.com


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Calculating your target heart-rate zone is done by subtracting your age from 220. This will be your maximum heart rate. Then multiplying that number by 0.7. If you would like a little higher end, then use 0.85. Here is an example. For a 55-year-old the maximum heart rate would be 165 BPM, and the range during exercise is 116 and 140 beats per minute. Of course these numbers are approximate and it is important to note that individual targets might vary.
Aggressive exercise might yield greater health benefits than moderate workouts. But working out too vigorously isn't a great idea. A heart rate monitor can help you stay in your safety zone while helping you reach your target heart rate.
There are many different types of heart monitors including wrist and chest strap models. The Rhythm+ is accurate like the chest strap monitors, but it is much more comfortable like the wrist versions. The key to a quality heart rate monitor is the consistent reading of the user's heart-rate and the Rhythm+ delivers.


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On Blogger since June 2009
Profile views - 232
My blogs
Blogs I follow
- ADDICTED to VEGGIES
- Aida's 42-days Juice Fasting Diary
- barefoot and frolicking
- Breuss & Juice Fasting Essentials
- Bunnies By The Bay
- Chronicles of Heather
- cook.eat.think.
- Farmgirl Gourmet: Delicious Recipes for the Home Cook.
- Foxglove and Folk Songs
- Ginger is The New Pink
- In Mama Joe's Shadow~'Cooking With Adele's' Best Recipes
- In the Fields
- Inside Storey
- letters from the end consumer
- Little Bites Of Joy
- Mod Vintage Life
- my freedom is now
- Nourishing Cuisine
- Parisienne Farmgirl
- Return to Sunday Supper
- Simple Pleasures
- Simply life
- Simply So Good
- Still Living Well
- The Country Farm Home
- the happy raw kitchen
- The Vintage Bag Lady
- Wild Carrot Farm Blog
|United States
|I'm also known as Ali. My favorite things include cooking and reading... and eating! Most people like to shop for clothes... I love to shop in natural health food stores! I am interested in Macrobiotics and the Raw Food Diet. I've received training from the Kushi Institute and the Institute for Integrative Nutrition. I'm going through menopause. Despite that, I live a quiet, but quite wonderful, life with my boyfriend and my cat!


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This is my very favorite place to stay, and one of my very first TripAdvisor Reviews. We arrived and were surprised to be upgrade to a suite without extra charge. We were surprised ! a well appointed room, with a spacious bathroom with a large jet tub, separate bedroom, patio and exit from our room directly to it. Thank heavens... More
What is Certificate of Excellence?
TripAdvisor gives a Certificate of Excellence to accommodations, attractions and restaurants that consistently earn great reviews from travellers.


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Key things you need to know aboutDr. Bourey:
Dr. Bourey's Experience
Research training, expertise and qualifications
Dr. Bourey's Care Philosophy
Dr. Bourey's Specialties
- Endocrinology, Diabetes & Metabolism
- View all 2 Specialties and 4 board certifications
Procedures Dr. Bourey Performs
- Continuous Glucose Monitoring
- Exercise Counseling
- See all 4 procedures
Conditions Dr. Bourey Treats
- Abnormal Thyroid
- Adrenal Insufficiency
- See all 43 conditions
More About Dr. Bourey's Background
Hospital Quality
The right hospital is as important as the right doctor
- SSM Health Saint Louis University Hospital
- 3635 Vista Ave
- Saint Louis, MO 63110
9 Facts About Hospital Quality
Patients' risk of dying during a hospital stay for heart bypass surgery could be 87.4% lower, on average, at a hospital rated 5 stars compared to a hospital rated 1 star for that procedure. Choosing the right hospital is critical to your health. View Slideshow
Patient Satisfaction
Patients' feedback on their experience with Dr. Bourey
Likelihood of recommending Dr. Bourey to family and friends is 3 out of 5 5 1 4
See More Survey Results
8 Facts About Patient Satisfaction Surveys
Patient satisfaction surveys provide a standardized way of reporting about healthcare experiences. But there can be more than meets the eye when reviewing scores. Here's what you should know
Appointments & Offices
Check insurance plans, locations and make an appointment
Accepting new patients?
- Yes
- Saint Louis University School of Medicine
- Doctors Office Building
- 3660 Vista Ave # 204
- Saint Louis, MO 63110
- (314) 977-6157
- Directions
- Prepare for Your Appointment
- Saint Louis University Sleep Disorders Center
- Saint Louis University Sleep Disorders Center
- 3545 Lafayette Ave # 1100
- Saint Louis, MO 63104
- (314) 977-5337
- Directions
- Prepare for Your Appointment
7 Tips to Prepare for Your Appointment
The key to a successful appointment is communication. To get the most out of your visit, prepare ahead of time so you can lead the conversation with your doctor. These 7 tips will get you ready
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Greenville's Convention Center will be going through an expansion.
The Greenville City Council voted unanimously to expand the Greenville Convention Center. The plan would increase the space from about 11-thousand square feet, to 52-thousand square feet.
The total cost for the project is 4.1 million dollars, with a groundbreaking expected in May. The projected economic impact to Pitt county of the Convention Center expansion is estimated to increase from $24 million this year, to approximately $34 million by 2020, a net gain of $10 million.


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Spongebabe-The guys.. by NeoSlashottWatch
©2006-2016 NeoSlashott
I didn't know Timmy Turner was in SpongeBob
whos the paul guy?
lol mr.puff what happen to mrs.puff?
haha Dusty cheeks looks cute XD
wow nice work
I think Randy would have made more sense
But its still great
But its still great
Cool pic. I've been looking at your art ever since I discovered DA (which was a year or sumfin ago), and it gets better every time.
who is paul based off of and WHOO SPONGEBABE!!
cool XDD paul's wearing pink XDD but i think mr. puff looks too young... sorry...
It makes me wonder about Plankton, Larry (the lobster-guy), and [oh my god] Mermaid Man and Barnacle Boy. XDDD
I like SpongeBob a lot. So can you do Plankton in SpongeBabe as well as Gary?
Arenita es la unica que salva
es decir que es la mas bonita ^^
es decir que es la mas bonita ^^
This must be Mr. Puff BEFORE he got turned into the pull chain lamp. XD
*giggle* I love it! What would Plantion look like I wonder? *giggle*
dusty rox! he looks quite a bit like timmy turner though
he's sooo cute!!!!
he looks ten!!!!
he's sooo cute!!!!
he looks ten!!!!
Dusty has a striking resemblence with Timmy Turner of the Fairly Oddparents
Dusty has a striking resemblence with Timmy Turner of the Fairly Oddparents
pual looks gay... not that theres anthing wrong with that! ^__^
oops i meant pearl, that annoying whale that reminds me of someone i know eeeeekkk
wow u made 'paul' look cute (personally i hate pear but paul? you wouldnt think he was a whale )
I must say Dusty is very cute (Why he uses the suit by the way?)
Too great. Just too great XD *glomps Dusty* I said it once and I'll say it again, your a genius, man.
Pretty cool, But I like Dusty the best.
you should have drawn paul having a tantrum or something (thats what pearl is famous for)
otherwise, great job!
otherwise, great job!
well since the creator has done pearl male maybe it means heill have an opposite personality
i mean mr puff looks a lot less anxious and may be a lot more tolerant and admirable of spongebob err i mean spongebetty (lol)
i mean mr puff looks a lot less anxious and may be a lot more tolerant and admirable of spongebob err i mean spongebetty (lol)
Dusty is the cutest thing on two legs!
Who is Paul sopposed to be...Its so cool Its confusing
its pearl outta spongebob, i was confused at first especially the way hes hot and got a nice bod unlike pear whos just an annoying bag of jelly
Shouldn't Pearl-Paul be Earl?!
I wonder if you are going to do Sheila (Plankton)? And Lisa (Larry)? And.... *thinks* Crabby Patty! (the opposite of the Krabby Patty!) Okay, maybe I am getting a bit obsessive o.o
Paul... Is that Pearl? I'm racking my brain, haha. Dusty's cute ^^
lol, i'm just thinking about the relationship between mr puff and sponge. I think he gets frustrated at her for some different reasons, XD *bad yazzy, bad*
Paul looks a little bit... um... Gay, but that could be because he's wearing pink... other than that, they look great! Your art is always so nice, I love it! Would it be too much to ask for good ol' masculine Ardee back though? I like him, you are cool just the way you are!
Dusty looks awesome!
How come dusty looks like Timmy
coz timmy looks like a squirrel!!!
Good Job, very original idea, but and Plankton.
Dusty rules!!!


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Jul 01st 2015
Taqueria 27: A Taco's Natural Habitat
Mary's review of Taqueria 27's new digs Read More
Mary's review of Taqueria 27's new digs Read More
We are on the hunt for Utah's best tattoo artist and want your pics. Read More
The best of Utah's outdoors, fashion, dining, arts and family fun Read More
Of course, it's not all butterflies and red balloons in the Beehive State. Along with our picks for the best of everything in Utah, we would be remiss if we didn't include a list of losers as well. Read More
Utah's beauty is found in its outdoors, and here are our favorite ways to experience that beauty in 2015. Read More
Here are our picks for the very best in Utah's arts and entertainment for 2015. Read More
Our breakdown of the best shopping and services in Utah for 2015. Read More


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I'm going through the initial configuration of Citrix XenApp 6.5. I have two servers, a DC and the XenApp server.
I created an OU XenApp, and a user "ctxadmin" on the DC. When configuring the XenApp farm I specific the admin user as DOMAIN\ctxadmin. Now when I'm logged in as Administrator and launch AppCenter, it says:
This user account is not an administrator of this farm, or there was a problem contacting the data store. Check that the data store server for the Citrix XenApp farm is online, and verify that your account is configured and enabled as an administrator on the farm.
If I log into the server as ctxadmin I cannot launch AppCenter without putting in the Administrator credentials, and then discovery doesn't work.
I seem to be in an endless loop. How can I fix this?


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I so miss them!
really cool
I Like the perceived brutality and it's , i think one could call it, grittyness?
Adorei, muito bom.
Zica !
LOVING the character here man, so cool.
I love the mouth! I make a lot of drawings with mouths as well! Very cool! neat!
So awesome! I love the mouth!
very nice


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Do You Know Your Bev-O-Metrics?
Sodergren, Rebecca, Pittsburgh Post-Gazette (Pittsburgh, PA)
Do you have a preference for particular types of coffee? Or wine? Subha R. Das, assistant professor of chemistry at Carnegie Mellon University, will explain what's behind those flavors in a Slow Food Pittsburgh presentation at 6:30 p.m. Tuesday, Feb. 4, at Harvard & Highland above Union Pig & Chicken in East Liberty.
The energetic Mr. Das will lead a discussion about the basics of taste and taste perceptions, focusing on coffee, soda and wine.
In fact, it was over an afternoon cup of coffee that Mr. Das and Roberto Gil, director of Carnegie Mellon's Nuclear Magnetic Resonance (NMR) facility, began to mull the idea of analyzing beverages to create flavor profiles using NMR, a technique that allows scientists to determine a substance's molecular chemistry, structure and dynamics.
Other scientists have already developed techniques for pinpointing various additives or substances in beverages, so that's not what these two are trying to do. Instead, they're trying to create "fingerprints" for different beverages, so that each type of beverage has its own unique NMR fingerprint that distinguishes it from every other type of beverage. Thus, you could identify a beverage based solely on its NMR line graph, which (for the non- scientists among us) looks a bit like an electrocardiogram printout. Peaks on the graph represent sugars, fats and other compounds in the beverage.
They're calling it Bev-O-Metrics.
So far, the professors have found that they seem to be able to detect one type of coffee over another. They also have students working on wines, and they've developed a database of flavor profiles for a number of different wines.
From a consumer's point of view, here are some of the questions the professors expect to be able to answer:
* If you like a certain type of wine, which other types might you also like, based on similarities in their flavor profiles?
* How does a particular coffee's flavor profile change if you change the method of roasting the beans? How does it change when you make the coffee a couple different ways in your home kitchen?
* Why does decaf coffee taste weak?
* How much caffeine is in different types of beverages?
* What's the best time to harvest grapes used in winemaking?
Mr. Das believes there's a certain appeal to creating an NMR profile of beverages because while the scientific profile never changes, people's tastes actually can change -- a beverage might not taste the same to a person from one day or year to the next.
He moved from Chicago to Pittsburgh seven years ago and gradually is making inroads into the food scene here. He combines chemistry and food in more than one way; he also teaches the popular "Kitchen Chemistry Sessions," a five-week CMU mini-course that was previously featured in Food & Flavor. …


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Almost 69 years ago today, Norman Turgel, a soldier in the British Army, met the woman whom he immediately knew he would marry. Just days later, they were engaged.
When a four-year-old evacuee returned to his Hackney home after fleeing the Blitz, he received a letter from the well-to-do prep school boy who had become his wartime best-friend.
But despite his mother's insistence, Barry Spencer never replied. It was a decision he came to regret for more than 70 years.
Two years before the Second World War broke out, Otto Meyer prepared five potassium cyanide capsules, one for himself, one for his wife, and one for each of his three children.
But luck was on his side. By the end of 1937, he managed to wind up his affairs, sell his machine factory in Rheda and take his family to Palestine before the onset of the Holocaust. The capsules remained unused.
Year Zero: A History of 1945
By Ian Buruma
Atlantic Books, £25
Several books on the end of the Second World War have been published recently, but Ian Buruma's is distinctive by virtue of its scope and personal tone. Buruma adopts a global perspective, framed by the story of his own family.
There can rarely have been more of an innocent abroad than Lance-Corporal Ron Jones at the start of the Second World War. Born near Newport, he was working in a Cardiff steel forgings factory when he was called up by the South Wales Borderers as a result, he claims, of a clerical error, a fact that still nettles him at the age of 96.
Most people know of Ajex —the Association of Jewish Ex-Servicemen and Women — as the people who parade down Whitehall every November to commemorate fallen comrades.
Yad Vashem honoured the relatives of five British PoWs this week by naming them Righteous Amongst the Nations in a ceremony at the House of Lords.
Also receiving the awards, presented by Israeli envoy Daniel Taub, were the relatives of a Lithuanian and a Polish citizen.
The funeral of a Nazi war criminal was cancelled at the last minute in Italy yesterday, after protesters broke into the compound where the ceremony was taking place.
Former Leeds United chairman Leslie Silver has been belatedly honoured for wartime services.
Mr Silver has received the new Bomber Command Clasp in recognition of his contribution to the RAF during the Second World War. He took part in some 44 operations in Europe and 20 in the Far East.
A new group is launching a UK-wide campaign to collect stories and memorabilia from Jewish men and women who served in the British army during the Second World War.
The Association of Jewish War Heroes has been set up to amass material to fill a new section of the Latrun military museum in Israel that will be dedicated to war heroes from Britain.


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U.S. Navy veteran Anthony Davis escaped injury during 18 months on duty in the Middle East only to become paralyzed after he returned to the United States.
Three weeks before his discharge, Davis broke his back in a traffic accident on Interstate 5 in California on his way home to Vancouver. Doctors told him he would never walk again.
That didn't stop him. Six years later, the 29-year-old is a contender for the Paralympic Games to be held at the 2012 Olympics in London. Davis, who never rowed before 2009, qualified for the Paralympic/Adaptive National rowing team at trials June 29 at Mercer Lake in West Windsor, N.J.
He will represent the United States at the August world championship in Slovenia. That could lead up to the Paralympic Games at the 2012 Olympics in London.
"It was awesome," he said.
Davis joined the Navy after graduation in 2000 from La Center High School, where he played football and baseball. War didn't seem imminent at the time, and the Navy offered travel abroad and help paying for college. He was trained as a rescue swimmer and submarine hunter. With 9/11, things changed. Davis was deployed to Afghanistan in 2002 and then, to Iraq. He spent 18 months altogether in both countries.
Three weeks short of his discharge in August 2005, he and his friends were driving a Jeep up Interstate 5 through California to go home to Vancouver. Davis was asleep in the backseat when the vehicle rolled multiple times on the freeway. He was ejected from the window in a fetal position and landed on his spine on the ground. The impact broke his back, ribs and collar bone and paralyzed him from the waist down.
He spent four months at a veteran's hospital in Seattle, where doctors informed him he would never walk again. Then, his wife, who was pregnant with their son, filed for legal separation while he was still in the hospital — and later, divorce.
For more than a year, Davis was immobile and struggled with depression. He lived with his grandmother in La Center because her house was wheelchair accessible.
"I didn't do anything except hang out with friends," he said. "Before, I was athletic. Now, I couldn't move and had to take my wheelchair wherever I went. I was not happy. I decided I didn't want to be like that."
He began to go to the gym daily to build strength in his back. His son Ethan served as an inspiration and a lesson on how to take baby steps toward his goal of walking again. Davis would closely observe Ethan as the tyke was learning to walk and would imitate his movements.
"My son taught me how to walk," Davis said. "I couldn't control my ankles, so I would just throw my feet forward." Slowly, Davis worked up to a walker and, later, a cane.
"The nerves started growing back, and I started experiencing pain I could never imagine," he said. "But I was getting stronger every day."
Soon, he was walking unassisted. He got back into sports and coached football at La Center High School.
His first experience rowing was at a winter sports camp in 2009 for disabled veterans in Aspen, Colo. The grand prize for the camp's rowing competition was a shirt.
"It was a nice shirt," Davis said. "I thought, 'I'll try it.'"
Davis sat down at the rowing machine and sailed past his competitors, covering 500 meters in two minutes.
"I guess that was really fast, but I just wanted the shirt," he said.
An hour later, he received an invitation to a military adaptive sports clinic in Fort Lewis, where he rowed for the first time on water.
"I had no idea what to do," he recounted. "I was flying through the water. I felt like I could run again."
He found a coach in Vancouver to help him hone his skills. He rowed three times a week on Vancouver Lake, battling severe pain in his body but building strength as well. He went to competitions around the country.
In April, USRowing invited him to train in Oklahoma City, the official Paralympic training site for rowing. He has spent the past three months in Oklahoma City preparing for the national trials and practicing morning and night. He returned to Vancouver June 30 to spend the July Fourth weekend his son, now 5. His practice regimen for the world championship resumed this week in Oklahoma City.
Davis said after he wins all the rowing competitions he can, he would like to go on to be a public speaker.
"I hope people who hear my story realize they can do whatever they want with hard work," he said. "God gave me a second chance, but I had to work hard."
Paris Achen: 360-735-4551, firstname.lastname@example.com, Twitter@Col_Trends, www.Facebook.com/#!/pages/Paris-Achen


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[Interview] Micky Yoochun "Sea Fog", like a sponge
While two-character (in Korean) movies worth 10 billion won (~US$10 million) have been flooding the theaters these days, the audiences are highly excited. "KUNDO : Age of the Rampant" and "Pirates" has been out for a while and currently "The Admiral: Roaring Currents" is causing a syndrome. It"s not that much of a great news for "Sea Fog" but Micky Yoochun says, "I am happy to have just been in that movie because the film itself is amazing".


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The purpose of this research study is to find better ways to measure and monitor kidney (renal) disease activity and damage in people with Systemic Lupus Erythematosus (SLE). The study will look for indicators of kidney disease as well as determine the relationship between the levels of biomarkers in urine and blood and the presence, severity and future development of SLE renal disease.
You or your child may be eligible to participate if you have been diagnosed with SLE and have one of the following:
and your child will be in the research study for 6 months. Participation in
this research study will involve a study visit about every 3 months and when/if
you or your child has worsening of kidney involvement (a renal flare). Study
visits will be combined with regularly scheduled rheumatology appointments at
Cincinnati Children's.
detailed list of procedures will be provided to anyone interested in knowing
more about this study. Here are some of
the procedures:
Your child will be asked to:
or your child may not receive a direct medical benefit. However, the information gained from this
study may help other patients with SLE in the future.
as a participant or parent interested in having their child participate, will
be given a consent form that thoroughly explains all of the details of the
study. The form covers all of the
procedures, the risks, the benefits, the pay, who to contact with questions or
concerns and more. A member of the study
staff will review the consent form with you and will be sure that all of your
questions are answered.
There are no physical risks associated with
this study. Other possible discomforts or risks will be discussed with you or
your child if interested in learning more about the study.
or your child will receive $15 for each completed study visit.
Kasha Wiley, M.Ed., CCRP
Children's Hospital Medical Center
of Rheumatology
3333 Burnet Ave.
Cincinnati, OH 45229-3039
Brunner, MD
Division of Rheumatology
Children's Hospital
Medical Center
3333 Burnet Avenue, Cincinnati, Ohio 45229-3026 | 1-513-636-4200 | 1-800-344-2462 | TTY:1-513-636-4900
New to Cincinnati Children's or live outside of the Tristate area? 1-877-881-8479
© 1999-2016 Cincinnati Children's Hospital Medical Center. All rights reserved.


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Added space gives research operations more room to grow
[caption id="attachment_23875" align="alignleft" width="216" caption=" BioMarin is expanding its administration staff into 20,000 square feet in the Wood Hollow Building in Novato."][/caption]
NOVATO – BioMarin Pharmaceutical Inc. is expanding across Hwy. 101 into 20,000 square feet on Wood Hollow Drive.
The move will free up space in the pharmaceutical developer's eight-building campus on Bel Marin Keys, said Mark Wood, director of human resources.
Initially, about 30 employees in administrative positions will make the move, less than 10 minutes away from the main buildings, no later than November, he said.
The former GreenPoint Mortgage building at 100 Wood Hollow Lane has been vacant since the mortgage company went out of business three years ago.


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"City in the Sand Dunes" Top 5 Page for this destination Florence by Camping_Girl
Florence Travel Guide: 109 reviews and 518 photos
Florence is a really lovely little city, about midway down the Oregon coast. It is surrounded by sand dunes, which stretch from here all the way to Coos Bay. I really liked the scenery in this area!
Florence is also home to another Mo's Restaurant, we were actually able to find this one and had dinner there. It was very good, especially the bucket of clams - yum!
Dinner at Mo's was great - clam chowder, a bucket of clams to shuck and eat with butter, the biggest crab salad I have ever seen in my life and, of course, fish and chips. The kids rounded out their meal with the biggest pieces of cheesecake I have ever seen! Even with 4 beer for me and hubby the bill was under $50 USD for the five of us.
This photo is of the coastline just north of Florence.
Reviews (4)
We stayed at the Horsfalls Campground, just across the bay from Coos Bay. It was a lovely, peaceful campground with... more travel advice
Another of the old historic lighthouses along highway 101. This one is a little smaller than the first one we looked... more travel advice
There are many camping spots along the dunes, but be forewarned, most of them are party central! For some reason... more travel advice
There are many old lighthouses along the Oregon coast and Heceta Head is the most widely photographed of them all. There... more travel advice
Comments (3)
i really liked florence when i was there. and i have an olmost identical photo myself of the heceta head lighthouse.
It's been almost 20 years since I was last down that way. Thanks for the more recent photos.
What a fantastic coastal location to slow down and enjoy! I can see why the Heceta Head Lighthouse area is so popular and those dunes along the coast are also amazing! Great stuff.
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Anyone who thinks that fairies aren't real hasn't seen your little girl dressed up like one. She believes, and this Plum Pixie child costume is the only thing she needs to live out her dreams. It's perfect for trick or treating on Halloween or for everyday playtime on her own or with her fairy friends. This magical costume comes with a beautiful purple dress with an ultra soft bodice, sheer puff sleeves, and layered tulle skirt. The neckline and the skirt are trimmed with pink, white, and yellow flowers, as is the included head piece. There's also a pair of lightweight gossamer wings decorated with glittery swirls and a long plastic wand decorated with colorful ribbons and a golden butterfly. Add a pair of matching slippers for a fun experience that will be hard to top.
Not yet rated. Be the first to Write a Review


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Edit Article
How to Have Insects As Pets
Mom said no pets, but how could you count insects? Keep these little critters for pets by following the steps below.
This article has been tagged as a stub.
That means it's off to a good start, but still has room to grow into a more helpful resource. Until the article reaches its full potential, it will be hidden from search results. Can you help it flourish? If you think the article offers complete and accurate instructions, feel free to remove this tag.
1Get a small terrarium. You need a place to keep your insects. You can also punch holes in a bottle top or plastic bin for a handmade terrarium.
2Fill your terrarium with leaves, sticks, plants, and dirt. This will create a lifelike habitat for your insect.
3Go outside and find your insect. It should be small, and if your terrarium is big enough, capture two insects.
4Once you've put your insects inside their terrarium, find them food. Other smaller insects work, as well as leaves and plants.
5Place the terrarium in the sunniest place you can find. This is good for them, as they need natural sunlight.
Community Q&A
Ask a Question
- Do not keep your insects for too long. Capture them, study them, and release them as soon as possible. The longest period of time you should keep an insect as a pet is two days, after which you must set them free and capture new insects.
- If one insect dies but another is still alive, remove the dead one instantly or the other insect will start to eat it.


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Sunday, July 4, 2010
For July 4th
"On this Independence Day, I will remember the fire of liberty that burned in the Virginia House of Burgesses, when Patrick Henry, a Southern man, rose to declaim, "Give me liberty or give me death." Virginia Burgess Peter Poythress was there to pledge his fortune and faith to his countrymen." Read the rest HERE.
Posted by Brock Townsend at Sunday, July 04, 2010
Labels: Diversity, God, Liberty, Multi-Culturalism, Southern Men
Subscribe to: Post Comments (Atom)


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Burlap Lined Cabinet With Drapery Panel Doors
Materials and Tools:
pry bar
table saw
staple gun and staples
hot glue gun and glue sticks
spray adhesive
sewing machine, needle and thread
1/4" thick medium-density fiberboard (MDF)
two different fabrics (one to cover the interior back of bookcase and one for sewing drapery panels for outer front coverage)
wooden buttons
industrial-strength hook-and-loop tape
cup hooks
1. With a pry bar peel the thin, cardboard backer off of the bookcase and disassemble the sides, shelves and bottom of the unit with a screwdriver.
2. Use a table saw to cut down a sheet of MDF to create a new back panel for the bookcase (replacing the flimsy cardboard backer).
3. Spray the interior side of the new back panel with adhesive spray and wrap with batting. Secure with a staple gun.
4. Wrap the same panel in fabric and staple into place.
5. Wrap the interior and exterior of the side panels with burlap, use spray adhesive to secure. Fold over hem and secure with a hot glue gun rather than staples so that the edges lay flat and do not fray. (No batting was used under the burlap; it is under the back of the upholstered panel only.)
6. Reassemble the bookcase with a screwdriver and finish by attaching the upholstered back panel.
7. Cut and sew drapery panels to cover the front of the bookcase. Hand-sew buttons onto the left panel. Then, on the right panel, machine-stitch small loops to create tabs that will hook around the buttons. Also, add a loop twice as long as the button loops to the mid-section of each panel to use as tiebacks when the drapery panels are open.
8. Attach the two panels with industrial strength hook-and-loop tape along the top and sides of the front of the bookcase.
9. Screw cup hooks onto the side exterior at a point where they will line up with the tieback loops on the drapery panel so the panels can be held open.


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I had read a couple of bad reviews recently and so was a bit wary about my appointment. I'd heard that they missed the conditioning treatment for a few people so I specifically mentioned that I'd booked this when checking in for my appointment and being handed over to my hairdresser and it did not seem to be a problem.
All staff I met were incredibly friendly, particularly the receptionist. I waited around 5 minutes to be taken to have my hair washed, which they were very apologetic about. The treatment was from the Nioxin range which I was pleased about as I've been keen to try the brand. They told me they select the type of treatment specific to your hair type, which is nice.
My stylist was Paula, who was lovely and very informative. She went through what I wanted very thoroughly and told me that I don't need appointments every 6-8 weeks if I wanted to grow my hair (as I've always expected). She told me to come back every 6 months. She actually cut very little off my hair saying that there weren't really many split ends to get rid of and to cut any more would simply just be shortening my hair unnecessarily (as I'm keen to grow it).
Unlike many stylists, I was pleased that Paula didn't give me the usual spiel about not bleaching and straightening my hair at home and instead recommended methods and products of keeping my hair in good condition despite my styling. Initially I thought this may have been to entice me to purchase additional products from them, but she actually told me I could by them in Boots anyway and that she genuinely just wanted to advise me as these are the products she's tried and tested on herself (Wella).
Overall the appointment was a very good experience and I will likely return for a follow up appointment.
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Tracey became our Acting Chief Executive in September 2015. She has previously been Director of Supervision and Authorisations and prior to that Director of Enforcement and Financial Crime. Tracey joined our predecessor the Financial Services Authority (FSA) in 2001.
Tracey graduated in Law from Queen Mary and Westfield College, University of London and qualified as a solicitor in 1995. She specialised in commercial litigation, working for law firms in the UK, US and Brussels.
Copyright © 2016 FCA. All Rights Reserved.


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Check Out Ronnie Eaton Performing in the Townsquare Media Studios in Midland / Odessa (VIDEO)
Recently, Rooster of KBAT came by the Townsquare Media studios with a good friend of his who is on the verge of making it big in the rock scene.
Recently, Rooster of KBAT came by the Townsquare Media studios with a good friend of his who is on the verge of making it big in the rock scene.
The 2nd Annual Harvest Festival at Grapevine Farms in Abilene is scheduled for Nov. 1st. This will be an afternoon and evening of wine tasting, cooking lessons, socializing, organic food and live music. Grapevine Farms is a non-profit organization that focuses on organic chemical free foods. It's Abilene's only 'all natural' farm. Here are the details.
Cassadee Pope just released a video that features footage from her sold out headline show in Columbus Ohio. Her fans made an impressive showing too, lining up early and in a line that wrapped around the venue. The 2012 "Voice" winner jammed out to her latest single "I Wish I Could Break Your Heart" and so did her fans, take a look.
The Wylie Christian Church is a church that I've often referred to, as the little church on the hill. So when I was asked by Jon and Don a couple of church elders, to help get the word out, I jumped at the chance. Wylie Christian Church is in dire need of a "real playground" for the children, so the church is hosting a party of with lots of live bands this Saturday, September 29th from 2 - 6 p.m. on the churches parking lot at 6250 Buffalo Gap road.
Photo by: Rudy F.
Business Mercado was this past Saturday at the Abilene civic center. What I expected was a very subdued and quite affair. Was I surprised when I walked into the civic center and was greeted with the wonderful sounds of a Mariachi band. Mariachi Alma Mexicana set the mood and made me feel like I was in a real Mercado (Market) in Mexico. The air was filled with sound and the smell of fresh tamales, compliments of our local United Super Markets. This Free to the public event was attended by people of all walks of life and all ages. One thing we all had in common, was that, when the Mariachi Alma Mexicana played we all stopped to listen, sing along, danced or swayed to the beat of the music. Oh and yes there were over one hundred businesses represented and y es they were talking business and how they could better serve us.
This will be the 29th Annual KEAN 105 Cruise Night and the route we will be taking a more nostalgic night. Taking it old school the cruise for 2011 will be from the old K-Mart on North 1st to the old Mac Eplen's at 1st and Shelton, around Mac's back on to North 1st heading west to the old K-Mart and back around again. WOW! That just brought back old memories of when I was in high school and we'd get some Eats and cold soft drinks at Mac's or Milano's on North 1st street. I wished I could still fit into my old Abilene High School letter jacket. I would be wearing it this Saturday during the cruise from 5 till 7 PM. After the cruise is over we will all go to the new K-Mart parking lot on South 1st street for the 29th Annual Sock Hop Dance and Car Show.


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Maybe you're giving because of a professor who enhanced your life, or a service trip that gave you a new outlook on the world, or perhaps because of the role St. Ambrose plays in our community.
Our donors give for many reasons, and all are proud supporters of a place where students are enriched by a Catholic education committed to academic excellence, the liberal arts, social justice, and service.
St. Ambrose University has announced the $18.5 million campaign for the Wellness and Recreation Center - the priority project of the Building our Future campaign.
You did it! On April 26, with suppport from alumni and friends, we surpassed our goal andraised over $70,000 through the 2016 SAU Day of Giving #50K2day! What a great day it was for St. Ambrose University.
The St. Ambrose experience lasts a lifetime. It prepares students to take their places as responsible members of their business communities, their towns, their parishes and their families. It prepares them to be leaders. You can help prepare them.
Support from Alumni and friends means the difference between a university that simply meets its students' needs and one that exceeds the expectations of its students, its faculty and its community.


text: 19 February 2012 archive See all show photos BODYSUITS, dresses and then more bodysuits – Pam Hogg knows what she does best and if she had it her way we'll all be in head-to-toe skintight looks by Christmas. A front row including Jo Wood, Janice Dickinson, Duran Duran's Nick Rhodes and Alexandra Burke watched as models strutted down the catwalk in panelled red, white and black; or gold, silver and copper versions – with the number of sheer panels increasing as the show went on, until one model appeared with only the tiniest triangle of opaque PVC to conceal her modesty. There were dresses and skirts too – dramatically flared out with layers of netting beneath – as modelled by Jaime Winstone, whose geometric red and black version was paired with a matching knit. Mary Charteris also made an appearance on the catwalk to whoops and cheers from the audience – first in thigh-high PVC boots and a skimpy playsuit and finally in a cutaway sequinned dress. Models wore drawn-on monobrows – a tribute to Frida Kahlo? – and false lashes at the centre of their upper lids, whilst hair was styled into lacquered rolls on the top of their heads and emphasised further by the dramatic mesh and sequin bonnets attached to each model's neck. It was classic Pam Hogg – tight, bright, daring and extremely revealing – and is bound to delight her loyal fans. SEE THE PAM HOGG SHOW ARCHIVE Lisa Niven Continue reading Back to top


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Our professors are distinguished scholars, active researchers, published writers, and faithful Christians who demonstrate what it means to be both Christ-followers and leaders in their academic fields. As mentors and role models, they play a huge role in the integration of faith and learning at Bethel.
David Nah
Associate Professor | firstname.lastname@example.com | Started at Bethel: 2006
Teaching Specialty
Systematic Theology, Christian Social Ethics, Theology in Global Context, Theology of Religions, World Religions


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You limited your search to:
Partner: UNT Libraries
Country: Australia
Creating Impact - Feed-In Tariff Laws: Making clean energy a reality for all
Date: March 2009
Creator: World Future Council
Description: The brochure describes evidence from fifty governments that Feed-In Tariff (FIT) policy speeds the transition to clean energy production and use.
Contributing Partner: UNT Libraries
Heading for 100 % renewable urban transport: Policy recommendations
Date: unknown
Creator: Gust, Iris & Meusel, Titus
Description: The brochure promotes urban transportation policy to increase the use of renewable energy to 100%. Seen globally, transport is one of the main sources of greenhouse gas emissions. Yet fossil fuels are becoming scarce, will become increasingly expensive and will eventually stop being viable as transport fuels. Before this happens, climate change will have begun to have a serious impact on human lives. The authors believe that it is crucial to replace fossil fuels with renewable energy as soon as possible, especially in the transport sector. Making urban transport independent of fossil fuel is a great challenge, but the authors cite growing evidence that it can be achieved.
Contributing Partner: UNT Libraries
Policies to Change the World: Energy Sufficiency - Eight Policies towards the Sustainable Use of Energy
Date: 2009
Creator: Rohde, Anja & Bee, Hilmar
Description: This booklet discusses how energy sufficiency is the best solution for reducing energy consumption and waste. It presents policies for reducing global energy consumption such as energy auditing, phasing out incandescent light bulbs, combined heat/cooling energy and power, carbon-negative cooking, smart metering, area road pricing, and other measures.
Contributing Partner: UNT Libraries
Winds of Change: East Asia's Sustainable Energy Future
Date: May 2010
Creator: World Bank
Description: This report outlines the strategic direction of the energy sector to meet its growing energy demand in an environmentally-sustainable manner over the next two decades, and presents a pathway of policy frameworks and financing mechanisms to get there. This study found that large-scale deployment of energy efficiency and low-carbon technologies can simultaneously stabilize East Asia's CO2 emissions by 2025 and significantly improve the local environment and enhance energy security, without compromising economic growth.
Contributing Partner: UNT Libraries
Global Trends in Sustainable Energy Investment 2010: Analysis of Trends and Issues in the Finacning
Date: 2010
Creator: United Nations Environment Programme
Description: This report shows that in spite of the global economic downturn, investment in sustainable energy is still strong.
Contributing Partner: UNT Libraries
Tunza: The UNEP Magazine for Youth, Volume 4, Number 3, 2007
Date: 2007
Creator: Lean, Geoffrey
Description: Tunza is a UNEP magazine for and by young people. This issue is devoted to the value of forest ecosystems.
Contributing Partner: UNT Libraries
Dead Planet, Living Planet: Biodiversity and Ecosystem Restoration for Sustainable Development
Date: 2010
Creator: Nelleman, Christian & Corcoran, Emily
Description: This report discusses some vital services that natural ecosystems contribute to human health and development.
Contributing Partner: UNT Libraries
Clearing the Waters: A focus on water quality solutions
Date: March 2010
Creator: Palanaippan, Meena; Gleick, Peter H.; Allen, Lucy; Cohen, Michael J.; Christian-Smith, Juliet; Smith, Courtney et al.
Description: This report discusses global water issues and offers a variety of proposals for countering the degradation of freshwater ecosystems for the benefit of public health and the environment.
Contributing Partner: UNT Libraries
Purchasing Power Parity and the Efficient Markets: the Recent Empirical Evidence
Date: December 1988
Creator: Yuyuenyongwatana, Robert P. (Robert Privat)
Description: The purpose of the study is to empirically determine the relevance of PPP theory under the traditional arbitrage and the efficient markets (EPPP) frameworks during the recent floating period of the 1980s. Monthly data was collected for fifteen industrial nations from January 1980 to December 1986. The models tested included the short-run PPP, the long-run PPP, the EPPP, the EPPP with deviations from expectations, the forward rates as unbiased estimators of future spot rates, the EPPP and the forward rates, and the EPPP with forward rates and lagged values. A generalized regression method called Seemingly Unrelated Regression (SUR) was employed to test the models. The results support the efficient markets approach to PPP but fail to support the traditional PPP in both the short term and the long term. Moreover, the forward rates are poor and biased predictors of the future spot rates. The random walk hypothesis is generally supported.
Contributing Partner: UNT Libraries
South Pacific Regional Environment Programme Agreement : message from the President of the United States transmitting agreement establishing the South Pacific Regional Environment Programme, done at Apia on June 16, 1993
Date: 1997
Creator: United States. President (1993-2001 : Clinton) & Albright, Madeleine Korbel
Description: The South Pacific Regional Environment Programme is to promote cooperation in the South Pacific islands region and to provide assistance in order to protect and improve the environment and to ensure sustainable development.
Contributing Partner: UNT Libraries


text:
Top Definition
To throw something entirely unreasonable, such as a mattress, microwave, or live animal, out of the window for no good reason.
I didn't feel like calling up the geek squad and getting my computer fixed, so I just jagoed that shit.
από ppppppppppppppppp 6 Μάιος 2007
1) An acronym for the independent film production company, "JAGO Productions" LLC.. Which stands for Just Authentic Genuine and Original Productions.
2) A popular character in the video game, Killer Instinct.
2) A popular character in the video game, Killer Instinct.
JAGO Productions, LLC are the creators of great independent films.
από Solomon Tubbs-Foster 12 Νοέμβριος 2006
To jerk off in a sock while driving on long road trips of atleast 100 miles. After cumming, throw it out the window onto the road.
On the way home I pulled a JAGO thinking about your mom.
από Elliot206 16 Οκτώβριος 2007
Italian slang word for a sweaty horse pussy.
You'll never be anything more than a filthy Jago!
από brave juice box 29 Μάιος 2011
A legend in their own right. However does tend to knob children.
That Jago just grabbed my willy.
από Jamesie Dick 22 Οκτώβριος 2004
Fittest newsreader ever. It's now become an adjective.
You're a bit Jago!
από Anonymous 18 Οκτώβριος 2003
a spoilt, middle class, white boy who thinks he was born and raised in Compton, wears his clothes in a backward ass manner, bombards anyone in range with his loveless Spotify playlists and whines about anything and everything, including spliff politics.
Stop being such a Jago man!
από BadMan678 5 Νοέμβριος 2010


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Color Flash Cards
Learn about colors and their names with these simple free printable color flash cards. The first set includes 12 flash cards: red, yellow, blue, green, pink, orange, violet, purple, grey, black, brown and white. The classics!
We also use them for find n' seek games around the house, matching the color cards to objects in the house and garden or a fun challenge : everyone picks just one random color card and then writes down as many objects that they can find in 5 minutes or so. Fun at parties – You decide the prizes! (perhaps something yummy the same color…). For more activities with colors try our Ice Cream color matching file folder game or our coloring pages.
We'll add more colors to these flash cards later… peacock green, peach or salmon pink, lemon yellow, olive, charcoal grey, scarlet red… What's your favorite color?
These color printable flash cards are best printed on card stock paper as they feel nicer to handle and survive longer after repeated use.
Check the printer and run a nozzle check if necessary before printing.
Colors are sometimes tricky to print correctly. If the colors seem to be wrong, try color correction with your screen and printer. As every printer has different settings.
Strictly for personal use only.
Color Flash Cards (A4)
Color Flash Cards (Letter)
SPANISH Color Flash Cards
DUTCH Kleuren Flash Cards
RUSSIAN Color Flash Cards
ARABIC Color Flash Cards
All design © Mr Printables
By downloading these files,
you agree to our terms of use.
You will need
Papers (preferably card stock for durability)
Scissors or craft knife & ruler
We have a growing selection of children's resources for preschool, homeschool & classroom learning activities. Please check back soon to see what's new!


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This is the part of our Learn WordPress series and in this post you will learn how to set up WordPress after the fresh installation. So far you have learned:
- Difference between WordPress.com and Self-hosted WordPress
- How to buy Webhosting and Domain for WordPress Blog
- How to install WordPress blog on Bluehost hosting.
Now the next in the series is to setup your WordPress blog in the right manner. You can download WordPress Guide eBook for A-Z of setting up WordPress.
This is the list of essential WordPress settings you need to follow after installing WordPress.
Essential WordPress settings for beginner:
When you install WordPress, it's just a stock installation with few stuff like a dummy post, dummy page, dummy comment. Rest there are many things that you need to do to make it ready for yourself and public. So, let's get started with the essential settings that you need to make:
Delete default Posts and page:
Login to your WordPress dashboard and go to Posts> All posts and delete the default "Hello world" post. Similarly go to Pages > All pages and delete the default "Sample page".
Set up WordPress Permalink:
The default WordPress permalink is like http://yourdomain.com/p=123 This permalink is short but not friendly for search engines.
You need to change it to make it search engine friendly.
Go to settings > Permalink and select the post name and click on save. This ensures that your permalink when appearing in the search engine, it will contain some keywords, and your blog posts will rank better in the search engine.
Here is an example from my live blog:
Set your time Zone:
Remember at the time of installing WordPress on Bluehost, we added site-name & tagline. You can change it anytime from this setting page.
This is where you will configure your homepage name and tag line. This is very important, as by default this is what shows up in Google search. Email address will be your site admin Email address, and this is where you will get all the Email notifications related to your site. Make sure you set up the time zone to your local time, this will make sure your scheduled post will go according to your time zone.
Disable users registration
It's always a good idea to keep your registration open if you are creating a WordPress site like ShoutMeloud or any other Websites which allow guest posting, but spam registration will be a pain. But, if you are sole writer and don't need people to register, I recommend you to disable the WordPress registration Disable it under Settings > General.
WordPress discussion/comment settings:
This is another section that you should setup just after installing WordPress. Just follow the screenshot and put the checkmark in front of the field as shown in the screenshot.
WordPress Ping list:
By default WordPress ping only one ping service, you can notify many more services by extending the ping service. Add more ping list here at Settings> Writing. You can get a big list of Ping list on this page: WordPress ping list.
Quick Recap
- WordPress Permalink Structure.
- WordPress time zone.
- Threaded comment.
- WordPress ping list
For now if you are installing WordPress for the first time you can refer to this post, for quickly check if you have done the basic settings for WordPress settings. You can also bookmark this post on delicious or stumble upon for future reference. Make sure you subscribe to Shoutmeloud feeds to keep receiving more wordpress updates.
What next?
Once you are done setting up WordPress, it's time to install plugins. I have created two lists of plugins, and you can read the detail of each plugin and install the one that make more sense to you.
- Install Yoast SEO plugin and here is a guide to help you get started.
- Install W3 Total Cache plugin for making your blog load faster. Here is a guide to get started.
- Check out this guide to learn how to setup Google Analytics for your WordPress blog
You can subscribe to my Email newsletter, to stay tuned with latest update in WordPress.