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Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes.
The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose–response (0.1–10 µM) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 µM) suppressed the response to isoprenaline, while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 µM) reduced the corrected field potential duration by 45%, tetrodotoxin (10 µM) shortened the minimal field potential by 40%, and E-4031 (50 nM) prolonged field repolarization.
Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine.
Human embryonic stem cells (hESCs),1 with their ability to differentiate into various cell lineages, including cardiomyocytes,2,3 have provided researchers with a unique tool for the study of developmental biology, toxicology, and regenerative medicine.4–6 Cell therapy for cardiovascular repair is being studied in clinical trials.7 However, a major obstacle in the clinical application of hESC-derived cardiomyocytes for myocardial cell transplant is immune rejection of these allogenic cell grafts.
Takahashi and colleagues,8 for the first time, demonstrated that fibroblasts could be reprogrammed by ectopic retroviral expression of transcription factors (Oct-4, Sox2, c-Myc, and Klf4) to generate human induced pluripotent stem cells (hiPSCs) that shared similar pluripotency to hESCs. Likewise, Yu and co-workers9 also demonstrated reprogramming of fibroblasts with Oct-4, Sox2, Nanog, and Lin28 using lentiviral vectors. This virus-based reprogramming technique is able efficiently to generate hiPSCs that could be differentiated into various cell types, including cardiomyocytes.10–12 However, viral vectors are associated with major drawbacks, such as genomic integration,13 insertional mutations, residual transgene expression, and tumourigenesis,14,15 which may limit the clinical application of hiPSC-derived cell types. In the clinical setting, therefore, vector integration-free methods are preferred. Recently, virus-free and vector-free hiPSCs have been derived by employing episomal vectors,13 piggyBac,16 mini-circle,17 and synthetic modified mRNA,18 which may overcome the numerous drawbacks of virus-derived hiPSCs, as well as the ethical and immunological issues surrounding use of hESCs.
There is, however, a paucity of information on the cardiomyogenic potential of virus-free hiPSCs. Our objective in the present study was to generate virus- and vector-free hiPSCs, to characterize their cardiomyogenic differentiation potential, and to define developmental steps involved in the differentiation process. Furthermore, we attempted to study the molecular, structural, and functional properties of hiPSC-derived cardiomyocytes (hiPSC-CMs).
Cell culture and reprogramming of fibroblasts
For detailed methodology of the maintenance of cell lines, please see Supplementary material online. Reprogramming of human foreskin fibroblasts (HFF; Lonza, Basel, Switzerland) were co-transfected with two oriP/EBNA1-based episomal plasmids (pEP4 EO2S CK2M EN2L, pEP4 EO2S ET2K; Addgene Inc., Cambridge, MA, USA) via nucleofection (NHDF – VPD-1001 with U-20 program; Amaxa, Walkersville, MD, USA). Transfected fibroblasts (∼1.0 × 106 cells per nucleofection) were directly plated to 10 cm mitomycin-C inactivated mouse embryonic fibroblast-seeded dishes with foreskin fibroblast culture medium.13 Culture medium was changed every other day. On day 4 post-transfection, the foreskin fibroblast culture medium was replaced with human embryonic stem cell culture medium19,20 containing 100 ng/mL of basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA). Colonies with morphology similar to hiPS colonies were readily visible by day 21–25 post-transfection. Individual colonies were picked manually and plated on 1% Matrigel-coated dishes and maintained in mTeSR1 medium (Stemcell Technologies, Vancouver, BC, Canada). Enzymatic passaging with dispase was employed for expansion, characterization, and differentiation experiments as described in the Supplementary material online.
Embryoid body formation and cardiomyocyte differentiation
Pluripotent stem cell colonies were dispersed into small clumps with dispase (1 mg/mL) and placed in low-adhesion culture dishes in embryoid body (EB) medium,20 with or without 5 µM SB203580 (Calbiochem, La Jolla, CA, USA) for 8 days.21 Subsequently, EBs were plated on 0.1% gelatin-coated dishes in EB medium without SB203580. Beating areas were typically observed around day 11–14 from EB formation. Beating areas were manually cut after day 21 of differentiation and utilized for various experiments.
Colonies of iPSCs and single cells generated from beating clusters were seeded on Matrigel- or gelatin-coated glass slides, respectively. For detailed methodology, please refer to the Supplementary materials online.
Teratoma formation
All animal experiments were conducted following experimental protocols approved by the SingHealth Institutional Animal Care and Use Committee, in full compliance with Singapore laws and regulations, and followed the guidelines of the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. Severe combined immunodeficient (SCID) mice, 6 weeks old, weighing 20–23 g, were obtained from SingHealth Experimental Medicine Centre, and were anaesthetized with 2% isoflurane initially followed by 1% isoflurane with oxygen with a flow rate of 1 ml/min during surgery. Approximately 1 × 106 hiPSCs were injected into the kidney capsule as previously described.22 Mice were killed with carbon dioxide asphyxiation at 8 weeks after cell injections, and tumours were collected, fixed, and processed for haematoxylin and eosin staining following conventional protocols.
Real-time PCR
For quantitative real-time reverse-transcription PCR (qRT-PCR) analysis, adult human heart RNA (Clontech, Mountain View, CA, USA), undifferentiated hiPSCs/hESCs (day 0) and differentiating EBs at different time points were used. The RNA was isolated with the RNeasy kit (Qiagen GmbH, Hilden, Germany). One microgram of total RNA was converted to cDNA by Superscript II first-strand synthesis system (Invitrogen, Carlsbad, CA, USA). The cDNA template (5 ng) was used from each sample, and SYBR green real-time PCR studies were performed using Quantifast kit (Qiagen GmbH) and primer (see Supplementary material online, Table S1) according to the kit instructions. Samples were cycled with Roto Gene Q (Qiagen GmbH) as follows: 5 min at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s extension at 60°C. All experiments were performed in triplicate. Relative quantification was calculated according to the ΔΔCt method for quantitative real-time PCR (using an endogenous control gene, GAPDH). For each gene, the expression on a specific day was then normalized by its baseline values.
For semi-quantitative RT-PCR and genomic PCR please refer to the Supplementary material online.
Microelectrode array recordings
To characterize the electrophysiological properties of the hiPSC-CMs, a microelectrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany) was used. Contracting areas were microdissected and plated on gelatin-coated MEA plates. The clusters were allowed to adjust for 72 h before performing any recording. All clusters were monitored for their beating abilities (beats/min) under the microscope during the 72 h period. Clusters that maintained relatively uniform beating rates were then subjected to drugs. The MEA system allows simultaneous recordings from 60 titanium nitride-coated gold electrodes (30 µm) at high spatial (200 µm) and temporal resolutions (15 kHz). To assess the effects of different drugs on the electrophysiological properties, the stock drugs were diluted in medium (2 mL). The MEA clip along with the beating clusters was maintained at 37°C throughout the duration of the experiments. Care was also taken that all buffers, including the medium used during all experiments, were pre-warmed to 37°C. The tested drugs included isoprenaline hydrochloride (Iso), carbamylcholine (CCh), verapamil (Ver), Bay K 8644 (Bay), tetrodotoxin (TTX), and E-4031 (all from Sigma-Aldrich, St Louis, MO, USA). All extracellular recordings were performed for 180 s at baseline and at 5 min after drug application at 37°C. Data were recorded using MC Rack software (Multichannel Systems) for all drugs; however, for measurement of conduction velocity and generation of local activation maps, Cardio2D software (Multichannel Systems) was used. The recorded electrograms were also used to determine the local field potential (FP) duration (FPD). The FPD measurements were normalized (corrected FPD; cFPD) to the beating rate of the contracting areas with the following Bazzet correction formula: cFPD = FPD/√(RR interval), as described previously.11
Statistical analysis
Comparisons at each time point were conducted using analysis of variance (ANOVA) followed by Tukey's post hoc test, and all data are presented as mean values ± SEM. Differences were considered statistically significant at P ≤ 0.05.
Generation and characterization of human iPSCs
We generated five virus-free hiPSC clones using two episomal vectors (containing Oct-4, Sox2, Klf4, c-myc, Nanog, Lin28, and SV40L) according to the protocol (see Supplementary material online, Figure S1A). The visual morphology of these hiPSCs was similar to hESC colonies (e.g. compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli), on feeders as well as feeder-free culture, with normal karyotype (see Supplementary material online, Figure S1B and C). Strong nuclear staining for Oct-4 and surface antigens, SSEA4, Tra-1-60, and Tra-1-81 (Figure 1A) confirmed the pluripotency of these clones. Furthermore, flow cytometric analysis of these undifferentiated colonies revealed that more than 95% of cells expressed hallmark markers (Oct-4, SSEA4, Tra-1-60, and Tra-1-81) of pluripotent stem cells (see Supplementary material online, Figure S1D).
Real-time gene expression study of pluripotency markers, Oct-4, Sox2, and Nanog, clearly demonstrated that all the five hiPSC clones maintained higher/comparable levels of pluripotency markers in comparison with the standard hESC line, H9 (Figure 1B), while these markers were not detected or very low for foreskin fibroblast cells (Figure 1B). Despite reprogramming of the naive fibroblasts using non-integrating episomal plasmids, it was imperative to rule out genetic incorporation in these hiPSC clones. Genomic DNA PCR indicated no extraneous genetic incorporation of any vector components or exogenous genes in our virus-free iPSC clones (see Supplementary material online, Figure S2A).
One of the hallmarks for pluripotent stem cells is their ability to differentiate into three developmental germ layers in vitro as well as in vivo. In vitro differentiation of hiPSCs was performed by generating EBs (see Supplementary material online, Figure S2B), which demonstrated marked up regulation of genes indicative of ectodermal (Nestin and Pax6), mesodermal (Isl1 and GATA4), and endodermal markers (AFP and HNF4α), confirming ESC-like differentiation capabilities (see Supplementary material online, Figure S2C). Furthermore, transplantation of the undifferentiated hiPSCs into the kidney capsule of SCID mice generated teratomas at 8 weeks, which on haematoxylin and eosin staining demonstrated the presence of neuroepithelial rosettes (ectoderm), cartilage (mesoderm), and secretory tubules (endoderm), representing the three germinal layers (Figure 1C).
In vitro differentiation and characterization of hiPSCs as cardiomyocytes
To induce cardiomyocyte differentiation, the hiPSCs were cultivated in suspension, where they formed three-dimensional differentiating cell aggregates (EBs) in the presence of SB203580 for 8 days. Embryoid bodies were plated, and rhythmically contracting areas appeared as early as 11 days post-differentiation (Figure 2 and Movie 1 on Supplementary file 2). These beating clusters increased with time, and the differentiation efficiency of our clones based on beating frequency ranged from 5 to 25% by day 21 of differentiation. The contracting areas had a diameter ranging from 0.2 to 1.2 mm and continued to beat vigorously for several weeks (45–60 days as observed) in culture. Based on the number of beating clusters, our clone MSnviPSNF3 demonstrated 20–25% contracting efficiency, comparable to the standard hESC line, H9 (20–30%). Interestingly, the appearance of beating clusters was less frequent in other clones (MSnviPSNF2, 10–15%; MSnviPSNF5, 5–10%; and MSnviPSNF1and MSnviPSNF6, 0–5%). Henceforth, all experimental evaluations were performed using the MSnviPSNF3 clone and the hESC line, H9.
Real-time PCR analysis (Figure 2) demonstrated that initiation of differentiation was accompanied by a significant decrease in pluripotency marker (Oct-4) levels by day 7, followed by up-regulation of markers of mesodermal commitment towards cardiogenesis. Cardiac progenitor marker (Isl1, a marker of the secondary heart field) and cardiac-associated transcriptional factors (Nkx.2.5, Hand1, and GATA4) increased significantly by day 7 in comparison to day 0 of differentiation (Figure 2). Concomitant with the increased expression of cardiac transcriptional factors, significant up-regulation was also observed for cardiac-specific structural and sarcomeric proteins (actinin, troponins, MYH7, and MLC2v) by days14 and 21 (Figure 2). Furthermore, ion channel proteins [Cav1.3, encoding the α1D subunit of the L-type calcium channel; KCNH2 (hERG), mediating the rapid delayed rectifier potassium current (IKr); and the hyperpolarization-activated cyclic nucleotide-gated potassium channel (HCN2), responsible for the If pacemaker current]11 were significantly up-regulated by day 21 of differentiation (Figure 2). A similar trend was also observed in the control hESC line, H9.
Molecular and cellular characterization of hiPSC-CMs
Spatial organization for cardiac-specific proteins in hiPSC-CMs was performed by immunostaining. We observed that 61.5 ± 9.9% (range 50–70%, n = 5) of the cells within each beating cluster stained positive with cardiac transcriptional factor, Nkx2.5 (Figure 3A). Furthermore, hiPSC-CMs also stained positive for cardiac α-actinin, myosin light chain 2, β-myosin heavy chain, cardiac troponin C, and titin (Figure 3A). Positively stained cardiomyocytes demonstrated an immature striated pattern, indicative of the early stages of myocyte development. However, Z-bands and A-bands were clearly visible in the cardiomyocytes (Figure 3A; indicated by arrows). Our hiPSC-CMs also stained positively for connexin 43, Na+–Ca2+ exchanger and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2; Figure 3A), suggesting their ability to form cell–cell interactions23 and regulate calcium.24
However, semi-quantitative (Figure 3B) and real-time PCR analysis revealed that sarcomeric structural proteins and ion channels were expressed at lower levels in hESCs and hiPSC-CMs in comparison to the adult heart, while the reverse was true for early developmental markers (Nkx2.5 and GATA4; Figure 3B; Supplementary material online, Table S2).
Functional characterization of cardiomyocytes
Despite the presence of hallmark molecular and cellular characteristics, did these hiPSC-CMs display functional electrophysiological properties and form a functional syncytium? Extracellular ECGs were recorded from electrodes underlying the EBs on a MEA chip (Figure 4A and B). Our hiPSC-CMs demonstrated a conduction velocity ranging from 1.5 to 2.5 cm/s, depending on the EB (n = 9). Electrical activation maps (Figure 4C) demonstrated development of a functional syncytium with stable pacemaker activity and synchronized field potential propagation. The conduction patterns were relatively stable and reproducible within each beating cluster.
Chronotropic responses of hiPSC-CMs to pharmacologically active compounds
One of the most important properties of cardiac tissue physiology is the ability to respond appropriately to pharmacologically active compounds. Human iPSC-CMs demonstrated a dose-dependent increase in spontaneous beating frequency when treated with the L-type calcium activator, Bay K 8644, and the β-adrenergic agonist, isoprenaline (Figure 4D–F). While treatment with 10 µM Bay K 8644 increased the beating rate by 2.5 times [0 µM Bay K 8644 (control) vs. 10 µM Bay K 8644: 0.921 ± 0.002 vs. 2.610 ± 0.040 Hz; P < 0.05; Figure 4E], 1 µM isoprenaline (Iso) doubled the beating rate [0 µM Iso (control) vs. 1 µM Iso: 0.799 ± 0.007 vs. 1.910 ± 0.010 Hz; P < 0.05; Figure 4F]. In contrast, the chronotropic effects of the L-type calcium channel blocker, verapamil (1 µM), reduced the beating frequency by 30–40%, and at 5 µM arrested beating (Figure 4G and H). Similar results were observed with carbamycholine, but at comparatively lower doses (Figure 4I).
Activation–inhibition studies with hiPSC-CMs demonstrated a significant increase in the beating frequency with 10 µM Bay K 8644, which could be significantly reduced by 2.5 µM verapamil (Figure 5A and B). Interestingly, exposure to verapamil also resulted in irregular beating rates, and prolonged exposure arrested spontaneous beating (Figure 5A). Likewise, muscarinic inhibition following adrenergic stimulation was performed. Isoprenaline (1 µM) treatment significantly increased the beating frequency, which could be inhibited with carbamycholine (5 µM; Figure 5C and D).
Furthermore, verapamil demonstrated a dose-dependent effect on the cFPD that at a concentration of 1 µM caused a 45% shortening of cFPD (Figure 6A and B). Likwise, TTX, a reversible, selective blocker of Na+ channels, was observed to shorten the FPmin (40–45%) in a dose-dependent manner and reduce conduction velocity by 33–42% (Figure 6C and D). Finally, E-4031, a methanesulfonanilide class III anti-arrhythmic drug that blocks the hERG-type potassium channel, caused a statistically significant QT prolongation at a concentration of 50 nM, which did not increase significantly at a concentration of 100 nM (Figure 6E and F). These results validated the presence of functional ion channels in our hiPSC-CMs.
The reprogramming of differentiated somatic cells to pluripotency holds great promise for drug discovery and developmental biology. This opens an opportunity to offer hope that patient-specific hiPSCs could generate clinically useful cell types for autologous therapy aimed at repairing defects arising from injury, illness, and ageing. Virus-based methods of generating iPSCs, however, have demonstrated high reprogramming efficiency, viral backbone integration, and continuous expression of reprogramming genes,18 which present formidable obstacles for their eventual therapeutic use.13 Thus, application of reprogramming without incurring genetic changes and subsequent characterization of their derivatives has become a focus of intense research effort. While there are limited studies showing cardiogenic potential for virus-based iPSCs,11,25 no report is currently available for cardiogenic differentiation of virus-free iPSCs. In the present study, we reprogrammed human fibroblast cells using virus-free methodology, and differentiated the hiPSCs to a defined cardiac lineage.
Our initial attempts to use only one episomal plasmid failed to generate any iPSC colonies (data not shown), which probably could be attributed to the lack of sufficient levels of pluripotency genes (Oct-4 and Sox2). Subsequent transfection with two plasmids in combination resulted in generation of iPSCs, suggesting that high levels of these transgenes improve reprogramming. These iPSC colonies exhibited typical hESC morphology (see Supplementary material online, Figure S1B), and exhibited pluripotency markers at gene and protein levels similar to hESC lines (Figure 1). Similar to hESCs, when injected into SCID mice, these iPSCs formed teratomas consisting of differentiated derivatives of all three primary germ layers (Figure 1). Moreover, PCR analysis failed to detect episomal vector integration in the genome (see Supplementary material online, Figure S2A). Exogenous DNA is not integrated into the human iPSC genome with oriP/EBNA1-based episomal vectors, which is gradually lost when cells are cultured in the absence of drug selection pressure.13 However, the reprogramming efficiency with these vectors is reported to be low (∼3–6 colonies per 106 input cells).13 These low frequencies are, however, sufficient to recover iPSCs from a reasonable number of starting cells.
We next investigated the ability of these hiPSCs to differentiate into the cardiomyocyte lineage. Our results demonstrate that reprogrammed somatic cells could differentiate into cardiomyocytes with apt molecular, structural, and functional properties, like hESCs2,26 and viral hiPSC-CMs.11 In order to increase the number of cardiomyocytes in culture, we used a specific p38 MAP kinase inhibitor, SB203580, that could enhance cardiomyocyte generation, probably by favouring early mesoderm formation.21 We observed that the presence of SB203580 enhanced cardiomyocyte differentiation (with SB203580 vs. without SB203580 for MSnviPSNF3: 20–25 vs. 5–7%). Importantly, cardiomyogenesis from hiPSCs mimics the human developmental pathway, and the resulting cardiomyocytes form a functional cardiac syncytium that responds appropriately to pharmacologically active compounds. We also noted that all our five clones demonstrated differential cardiogenic bias, which is in concordance with hESC biology and may be linked to epigenetic and intrinsic properties of each cell line.20,27–30
Temporal gene expression (Figure 2) of our hiPSCs associated with cardiomyogenesis demonstrated a similar trend to that observed in hESC differentiating systems31 and followed the cardiac developmental programme. Down-regulation of Oct-4, the master regulator of pluripotency,20,32 with simultaneous up-regulation of brachyury (marker for primitive streak; data not shown), indicated a trend towards differentiation. Expression of transcriptional factors, GATA4 and Nkx2.5, by day 7–14 suggested commitment towards the cardiac fate. Finally, up-regulated gene expression levels of cardiac-specific structural genes, including sarcomeric-related proteins and ion channels, confirmed terminal differentiation towards cardiomyocytes. These findings may have important implications for future understanding of cardiac developmental pathways and suggest that current differentiation systems and protocols may be subjected to manipulations to trigger known cardio-mesoderm signalling pathways in an attempt to augment cardiomyocyte yield.3,33
Our hiPSC-CMs also demonstrated positive staining for most sarcomeric proteins (Figure 3). However, it was noteworthy that not all cardiomyocytes demonstrated mature cross-striations. While some cardiomyocytes showed Z- and A-bands, suggesting maturity, others did not show clear striations. We believe this may be due to lack of mature myofibril structural organization in de novo cardiomyocytes.34 This may also be deduced from cardiac gene expression levels in hiPSC-CMs and hESC-CMs in comparison with adult human heart (see Supplementary material online, Table S2). Instead, the hiPSC-CMs are phenotypically reminiscent of previously reported 16-week-old fetal hearts.2 Interestingly, despite having irregular sarcomeric structures, these cardiomyocytes expressed functional ion channels coupled with downstream signalling pathways that could be modified by specific cardiac drugs.
Human pluripotent stem-cell-derived cardiomyocytes in various stages of maturity offer the opportunity to create appropriate human test models with high biological relevance in drug safety testing, preventing pharmaceutical attrition.5,35,36 Lack of clinically relevant cell lines and costly animal models further accentuate this problem. In proof-of-concept studies using the MEA mapping technique, we demonstrated that hiPSC-CMs responded appropriately to pharmacological compounds known to affect chronotropy, conduction, repolarization, and QT prolongation (Figures 4–6). Although the conduction velocity of iPSC-CMs was variable (1.0–2.5 cm/s), it was comparable to hESC-CMs.37 We assume that this variability may be due to the unpredictable geometry (size and shape) of the contracting tissue in a beating cluster, resulting in more heterogeneous measurements in different EBs.23 However, within each EB relatively reproducible measurements were observed. This drawback is counterbalanced by the ability to study the same tissue for prolonged periods in different experimental settings, using each EB as its own control.23 We also observed a significant increase in amplitude following Bay K 8644 treatment (Figure 4E). Bay K 8644, a known inotrophic drug and specific L-type Ca2+ channel agonist, increased Ca2+ influx, resulting in an increase in the amplitude of contractions as well as an increase in ICa.38 Furthermore, the response of our iPS-CMs to verapamil (a blocker of ICaL), tetrodotoxin (a blocker of INa), and E-4031 (a blocker of IKr) was consistent with previous findings of virus-mediated iPSC-derived cardiomyocytes,11,39 confirming pharmacological maturity of the derived cardiomyocytes.
Microelectrode arrays permit stable and long-term FP recordings that enable evaluation of relationships between dose dependency and adverse effects in the drug discovery process.40 Although FP does not resolve individual ion currents that contribute to the action potential, we demonstrated that addition of specific channel blockers altered the FP of our iPSC-CMs, indicating expression of functional Na+, Ca2+ and K+ ion channels, which lends credence to its use in drug testing. Indeed, generation of iPSCs from QT patients, resulting in QT-defective CMs,10 could serve as an immensely valuable in vitro model for drug development and related pharmacogenomics.41,42
The most exciting prospects of virus-free iPSC-derived cardiomyocytes are their potential for personalized cell therapy for myocardial repair. Although, in this present study, we have not addressed the regenerative property of these cardiomyocytes, it is conceivable that these cells could circumvent the problem of immune rejections43,44 when derived autologously. Studies from other groups have demonstrated that pluripotent stem-cell-derived cell types do home and engraft in the host tissue,6 without rejections.45 Nevertheless, up-scaling and purification of cardiomyocytes, in vivo maturation, electrical coupling, teratoma formation, and arrhythmogenicity still need to be addressed before this technology becomes a clinical reality.
In conclusion, we generated virus-free hiPSCs that are capable of differentiating into functional cardiomyocytes with similar efficiency to hESCs, and which recapitulate the cardiac developmental process with well-developed electrical properties. This renewable source of cardiomyocytes may serve as an important in vitro tool for drug screening and possibly regenerative medicine.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
This study was supported by National Research Foundation, Singapore grant, NRF2008-CRP001-68.
The authors acknowledge Dr Ralph Bunte, Office of Research, Duke-NUS Graduate Medical School, Singapore for his opinion on tissue pathology in the mouse teratoma assay. G.D. (Principal Research Fellow of NHMRC of Australia) was supported by a grant from the JO and JR Wicking Trust.
Conflict of interest: none declared.


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The aliens return and only the ingenuity of a few brave men and women can bring our world back from the brink of extinction.
It may have been 20 years since the first invasion, but we always knew they were coming back! We were excited to attend a screening of the film in 3D last and we can say that it's cracking !
Director Ronald Emmerich (The Day After Tomorrow, White House Down) reunites with co-writer and producer Dean Devlin for a return to a universe they created more than two decades ago. Independence Day: Resurgence marks Emmerich's first sequel. "This world is very special to me, and I wanted to do right by it and the characters. Enough time has passed that it all felt fresh to me."
We, like millions worldwide, were huge fans of Independence Day, so Roland Emmerich returning to the world of marauding extraterrestrials is welcomed enthusiastically here at Showcase. Also returning to fight the good fight against the alien invaders is Jeff Goldblum. His character David Levinson, scientist and eventual world saviour, was one of the many highlights of the 1996 original. He sounded excited to explore the character again: "David Levinson is complex. He's a wildly romantic person with an abiding love for the planet and for all living things. Most of all, he's humble and curious. So all these things come into play as he receives jaw-dropping, life-changing, species-changing information about where we are and who else is out there and what could happen to us."
Perhaps one of the most memorable speeches in cinematic history was given by U.S. President Thomas J. Whitmore, played by Bill Pullman, in Independence Day. However the intervening twenty years have not been kind to the former President as the aliens have invaded his brain. Pullman explains further, "Whitmore has been exposed, in an unfiltered way, to the entire consciousness of the alien fleet," Pullman explains. "It's like a shockwave to his brain. But he's still a hero to the world, even though he's been sequestered off by his daughter Patricia and a close circle of associates, because Whitmore is convinced, through his visions and intuitions, that the aliens are coming back - and no one believes him."
Journeying with Whitmore into the dark premonitions of the aliens' plans for Earth sounds like another great layer added by Emmerich and Devlin.
Adding to the fun is a new face but one we will recognise! Joining the resistance is Liam Hemsworth as Jake Morrison, a hotshot heroic fighter pilot of alien-human hybrid jets. The original film was a firm favourite of the Hollywood star and he jumped at the chance of joining the cast of Independence Day: Resurgence. "Battling an alien invasion fleet in this film was a dream come true for me," says the actor.
Hemsworth particularly enjoyed exploring the character's motivations to be at the frontline of this new battle against the aliens. "Jake's parents were killed during the war of 1996, so he has a bit of a chip on his shoulder in this fight."
The chip on Jake's shoulder impacts his behaviour with authority. Hemsworth elaborates: "Jake can be a little outspoken, because he's always been the underdog who had to fight for every scrap. As a result of growing up as an orphan, Jake became resentful and jealous of other people's positions."
Also joining the fight against the invasion are William Fichtner, Charlotte Gainsbourg, Judd Hirsch, Maika Monroe and Vivica Fox. An impressive cast for one of the big films of the summer.
Independence Day: Resurgence is released at Showcase Cinemas June 23 in 2D, 3D, IMAX 3D and a special Double Feature.
RealD 3D puts you in the thick of the action, bringing you face to face with your favourite characters. It opens up a world of perspective and detail so rich and lifelike you'll want to reach out and touch it. With RealD Digital 3D, you don't just see what's happening on the screen – you feel it. You need to wear special glasses to enjoy a RealD 3D film, to ensure you have an experience you won't forget.
Featuring up to four times the resolution of traditional Full-HD systems and offering incredible levels of detail and clarity, Sony Digital Cinema 4K projection systems have been installed at all Showcase and Showcase Cinema de Lux locations, offering the ultimate digital cinema experience for both 2D and 3D films, and meaning that all Showcase cinemas are now fully digital.

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Cleaning oven door
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How to Clean the Inside of Your Oven Door - Wipe away years of built on grime with a natural , two-ingredient solution. Check out these great tips!
Cleaning oven glass doesn't have to take all day! This NO SPRAY CHEMICAL tip is so simple, I wish I would have thought of it!
How to Clean Between the Glass on your Oven Doors. Not as tricky as you might think!
bakpoeder en water tot een pasta mengen, oven(deur) insmeren, 20 minuten wachten en eraf spoelen, c'est ca.
clean glass doors screw. There's no big secret to cleaning between the glass panels on your oven door. This tutorial shows you how to do it in less than 30 minutes.
Here's the right way to clean your oven. All it takes are 4 things you already have at home: baking soda, vinegar, water and a sponge. It's so easy.
How to Clean The Oven Glass
This step-by-step tutorial will show you how to clean between oven window glass. All you need is a Windex wipe, rubber band and a wire hanger. You won't believe how easy it is!
The Secret To Making The Glass In Between Your Oven Door Sparklehttp://scavengerchic.com/2015/07/29/cleaning-between-the-glass-on-oven-doors/
Cleaning oven glass doesn't have to take all day! This NO CHEMICAL tip is so simple, I wish I would have thought of it!


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Abhishek Bachchan thinks he wasn't the 'hero' in 'Guru', 'Bluffmaster'on June 11, 2016 at 10:30 am
The actor Abhishek Bachchan says he is not seen in the movies in terms of words like "principal actor" and "one male actor" and is not considered the main advantage in their successful films as 'Guru' and 'Bluffmaster'.
When asked if you have any upcoming projects in which he plays the lead role, he said: "I hope so If you have a good script, I'll be more than happy to read But I do not understand your question…"
He was speaking at a press conference regarding the success of the film 'Housefull 3'.
When referred to as an "actor alone ', she said:" As the only person in the film? It will be a very boring "film.
When the reporter clarified that he meant 'male lead', Abhishek said, "Therefore, act as a female at this time or I'm not understanding your question?".
Asked by films like 'Guru' and 'Bluffmaster' where was the "leader", Abhishek said: "In 'Bluffmaster', which had wonderful fellow actors called Riteish Deshmukh and Boman Irani In 'Guru' I had wonderful fellow actors. as Mithun da (Mithun Chakraborty), Maddy (R. Madhavan), Aishwarya (Rai) and Vidya Balan. What are you trying to say? "
When told about being a "male lead 'in' Guru ', he said:" Mithun da and Madhavan were male actors in that too. "


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Develop innovative and effective ways to improve well being and quality of life. Vice-president Italian Private Practice Psychologist Association (Veneto section).
Academic, Psychology
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O New Providence Carriage House é perfeito para um casal elegante que quer ficar longe de um fim de semana romântico. Mas, apenas no caso de você não pode ficar longe completamente, não há espaço para mais dois no sofá-cama. Faça uma deliciosa refeição na cozinha compacta, mas totalmente funcional com aparelhos de aço inoxidável e grande ilha para o espaço extra contador, ou vala a cozinhar completamente e andar um quarteirão para os melhores restaurantes em Rosemary Beach. O New Providence Carriage House fica a apenas poucas quadras da praia, para que você não vai se sentir muito mal por não ter acesso à piscina privada no pátio.
Melhor. Período de férias. Sempre.
casas de férias de permitir que as famílias para ficar ... juntos. No InvitedHome, pensamos que é muito importante, por isso fazemos tudo ao nosso alcance para tornar as suas férias totalmente épico. Não só escolher as melhores casas em melhores destinos, mas fazemos a experiência sem esforço para que você possa realmente se divertir. Nossa equipe irá abastecer o seu frigorífico, tomar conta das crianças, atender o seu partido, planear a sua viagem de um dia, fazer reservas, e fazer tudo o que pudermos para garantir que você tenha o melhor. Período de férias. Sempre.
We enjoyed an excellent 4 night stay at this property. The location is fantastic: south of 30a and right behind shops and restaurants. Beautiful windows in every spot possible, designer material selections and extra tall ceilings made the space bright and cheery. There were just enough amenities including towels, storage, and kitchen supplies. The kitchen is great for cereal in the mornings and sandwiches at lunch, but maybe not comfortable or stocked enough to cook a gormet meal (which is not necessary since you are a stone's throw away from the best restaurants!). As a designer myself, I really appreciated the efficient uses of space, the upgraded lighting package (with dimmers) and the beautiful color palette! The Invited Home management service was easy to use and provided us with excellent service. We would give it a 5 star rating were it not for these few setbacks: the mattress is NOT comfortable, the lack of window coverings in the bedroom to make the room completely dark at night and an ice maker in the kitchen would have been nice. If the mattress is replaced to something comparable to a Westin Heavenly bed, we would love to return regularly!
***Take Note - Building next door under construction just 100ft away, so mornings can be a bit noisy. They start as early as 6am, but no biggy really***
Great property with a fully appointed kitchen (not that you'll be cooking much, given all the great eating establishments).
Comfortably sleeps 4 adults.
We especially enjoyed the proximity to the beach, beautiful Town Square and community pools. You never have to drive anywhere.
Garage stocked with beach chairs and floating tubes. You can also rent umbrellas and beach chairs from The Pearl for $50/day.
Be sure to check out all the community pools, as they are all unique with their own charm.
Bring lots of sunscreen and enjoy!
Loved this place! It was clean , well appointed and very centrally located. The high ceilings made the space feel much larger than it actually was. The bed was extremely comfortable and the linens were of high quality. Great bathroom w/ plenty of extra towels!The only thing keeping me from giving this a 5 star rating was the loud construction adjacent to this property ( trucks started beeping at 7am daily) and the fact that the throw rugs desperately need to be replaced. We will absolutely book this property again!!
We decided on a last minute weekend out of town and this little space was perfect for the two of us. The architecture in Rosemary Beach is always interesting and this one bedroom carriage house space was no exception. The place felt homey but not cluttered and it was clean and comfortable. Thanks for a perfect weekend away!
Our family of four really enjoyed our stay at this carriage house in Rosemary Beach. Some of our favorite aspects of the house were the proximity to the beach, central location within the Rosemary Beach development and the courtyard pool. The house was well appointed inside with fine furnishings. Being new construction, the house was very new, clean and modern. The bathroom was extremely well done and the shower was fantastic. WIFI service was a nice touch as well.
Only a few negatives to mention...there was construction going on in the neighborhood and the noise of that early in the morning was annoying. Also, the appliances in the kitchen were very nice however there were many basic furnishings that were missing in order to be able to cook properly in the kitchen.
Overall, we had a great stay at your house and would definitely recommend it!
We stayed for three great nights. House and all furnishings are brand new, very nicely done. We didn't have time to use the outdoor fireplace or the pool we were so busy with the beach, music festival and dining. You are on the ocean side of 30A, which is where you want to be. We walked to Edwards for a great dinner, Paradis for drinks, Summer Kitchen for breakfast. And it is short walk to the Donut Truck at the entrance to Caliza, don't miss that Thurs to Sunday mornings. All appliances for those who want to cook in, small balcony to have your coffee in the sun if you don't want to lounge by the pool. We will be back!


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I'll Take You to Mrs Cole!
By (author) Nigel Gray
Illustrated by Michael Foreman
Format: Paperback
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I'll Take You to Mrs Cole! by Nigel Gray
Book DescriptionA touching story about a little boy who lives on his own with his working mother. Whenever he is naughty, his mother threatens him with Mrs Cole, who appears to be a disreputable character living nearby in total squalor. He imagines children being starved, whipped and even stewed. Then one day he runs away from home and despite his fears, finds himself outside Mrs Cole's house. She invites him in and he discovers that Mrs Cole's noisy, chaotic kindly house is welcoming and warm and her house becomes somewhere to go when he is good. ' An intriguing mix of realism and fantasy is the key element in one of the most outstanding young children's picture books for some time...with exceptional illustrations by Michael Foreman' - Financial Times
Buy I'll Take You to Mrs Cole! book by Nigel Gray from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9780862644079
(282mm x 245mm x 3mm)
Pages: 32
Imprint: Andersen Press Ltd
Publisher: Andersen Press Ltd
Publish Date: 6-Jun-1998
Country of Publication: United Kingdom
Books By Author Nigel Gray
Come on Everybody, Time to Play!, Paperback (January 2013)View all books by Nigel Gray
Where is everybody? It's time to play. Lift the flaps to find the family in some very surprising places!
US Kirkus Review » Whenever a young black boy makes a mess, his mother threatens to take him to Mrs. Cole's ramshackle house. One day, disinclined to cio his chores, he runs away and ends up (after some trepidation) at the dreaded neighbor's - where he finds a cheery, red-checked matron in a Mick Jagger apron, a gaggle of friendly children, and a household nearly buried under clutter. He gets such a hearty welcome that thereafter his mother's threat has become a reward - "If you're good, you can go and play at Mrs. Cole's." Foreman darkens his frequently fiery colors to reflect the ominous drift of the boy's imaginings; for more fastidious readers, the reality may seem like a nightmare - and an unsanitary one, at that. This decidedly minor effort was originally published in Great Britain (and briefly available here in 1986); the language has been Americanized for those who can't figure out what a "telly" or a "pram" is. (Kirkus Reviews)
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Author Biography - Nigel Gray
Nigel Gray (Author) Nigel Gray was born in Northern Ireland and brought to England as a child. After leaving school he worked for eleven years in numerous unskilled jobs. He then went to the University of Lancaster as a mature student and was awarded a BA in English in 1974. In 1984 he was awarded an MA in Creative Writing at the University of East Anglia. He has worked as an actor and taught Modern Literature Courses for the University of Liverpool, Lancaster and the WEA. He has taught Creative Writing at several universities and writing workshops in schools, colleges, libraries and prisons.Michael Foreman (Illustrator) Michael Foreman is one of the most talented and popular creators of children's books today. He has won the Kate Greenaway Medal twice and has been the UK nominee for the Hans Christian Andersen Award twice. His highly acclaimed books are published all over the world. He is married and has three sons. He divides his time between St Ives in Cornwall and London.
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CalArts Named One of Top MFA Drama Schools
The Hollywood Reporter recently released a list of "The 25 Best Drama Schools for a Master of Fine Arts," and CalArts has taken a spot on the list. This year,…Continue reading »
The Hollywood Reporter recently released a list of "The 25 Best Drama Schools for a Master of Fine Arts," and CalArts has taken a spot on the list. This year,…Continue reading »
EventAugust 20 - 29, 2015
Above: Trailer for Travis Wilkerson's 'Machine Gun or Typewriter.' Beginning Thursday (Aug. 20), the Brazilian film and art production company Barroca hosts the Fronteira International Documentary and Experimental Film Festival in…Continue reading »
EventAugust 7, 2015
Tomorrow (Aug. 7), the Skirball Cultural Center presents Breakin' Down the Beat: A Night of Spoken Word. The program, produced by Elena Muslar (Theater MFA 14), pays homage to the Beat…Continue reading »
EventAugust 6 - 8, 2015
Above: An excerpt from 'BIT.' The second week of Roy and Edna Disney/CalArts Theater's (REDCAT) New Original Works (NOW) Festival begins on Thursday (Aug. 6) with three premieres of avant-garde…Continue reading »
EventAugust 7 - 31, 2015
Above: Trailer for 'IamI.' For the 12th year, the CalArts Festival Theater returns to the Edinburgh Festival Fringe, the largest arts festival in the world. Three CalArts productions take to the…Continue reading »
Aug. 5 update: We've updated the post to reflect several more CalArtians nominated for Emmys this year. New additions in bold. Good luck to all our alumni! Nominations for the 67th…Continue reading »
On July 10, CalArts president Steven D. Lavine was inducted into the Santa Clarita Shakespeare Festival's Court of the Lord Chamberlain during the opening night event of the LA SummerFest at…Continue reading »
EventJuly 11 - September 6, 2015
Odyssey Theatre Ensemble and Evidence Room present the Los Angeles premiere of French playwright Pierre Carlet de Chamblain de Marivaux's The False Servant on Saturday (July 11) at the Odyssey Theatre…Continue reading »
EventJune 27, 2015
On Saturday (June 27), Automata in Chinatown presents Light in the Dark, a one-night mechanical and metaphorical exploration of the camera. The installation includes several CalArtians displaying visual art and…Continue reading »
Above: Diona Reasonover talks about her 'Boston accent.' Tonight (June 16) is the premiere of TBS' new sitcom Clipped, starring CalArts alumna Diona Reasonover (Theater MFA 09) with High School…Continue reading »


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Latest: ZEISS Presents a Wide Range of Microscopes at PITTCON 2017
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Latest: Pittcon 2017 Attracts More Than 700 Exhibiting Companies
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Your Questions Answered – Clinical Utility of Droplet Digital PCR on Liquid Biopsies from Patients ...
Trends and Perspectives in Gas Chromatography
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Malvern Launches MicroCal PEAQ-DSC for Faster, More Accurate Characterization of Protein and Biomol...
10 Top Tips for Life Science Laboratory Efficiency
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Pittcon Conference and Expo 2...
05 Mar 2017-09 Mar 2017
AACR Annual Meeting 2017
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PerkinElmer's uniquely designed LumaPlates come in 96-well (shallow or deepwell) and 384-deepwell format.
The LumaPlate is a solid scintillator... Read more...
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Hungarians displayed a mixture of disappointment and relief on Thursday after the government ended Budapest's bid to host the 2024 summer Olympic Games in the face of growing popular opposition. The Momentum movement, a new force in Hungarian politics, last week submitted a petition with 266,000
Budapest's withdrawal from bidding for the 2024 summer Olympics, a process already marred by a string of exits, highlights the need for changes to the Games model, insiders say. The Olympic Games were once the most hotly contested sports commodity in the world, but in recent years the IOC have seen
There was a time Lizzy Yarnold looked invincible in the skeleton. During her past two seasons, she won more than half the World Cup races and at the 2014 Winter Olympics she sealed gold by a second in a sport where the title is often decided by hundredths of seconds — tellingly, just 0.1sec separated
London's O2 Arena will be keeping its name for at least another 10 years. The mobile phone operator on Thursday announced that it had struck a deal with site owner AEG, reportedly worth around £125m, to retain the naming rights to the massive venue in East London for another decade. In a press release


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Texas Family Pushes For Cyberbullying Laws After Teen's SuicideFamily members of a Texas high school student who killed herself are rallying for tighter laws against cyberbullying.
Counselors At Plano School After 2 Teens Allegedly Commit SuicideIt will be a somber and tough day for students at Plano East Senior High School. Administrators, staff teachers and students are all mourning the loss of two young women. Police think the girls both committed suicide over the weekend.
Reportedly Suicidal Woman Shot & Killed After Pointing Gun At PolicePolice say a man called 911 from a church to report his wife was suicidal and possibly had a handgun.
Missing Dallas Man Believed To Be SuicidalA missing Dallas man has made suicidal threats and has also threatened to harm anyone that attempts to stop him from taking his own life.


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Don't Miss Washington's San Juan Islands
The San Juan Islands are a very unique spot in the beautiful Pacific Northwest. They are a must-see for any Washington itinerary.
Nomadic Life Made Me Appreciate Home
While preparing to switch from nomadic life to a more traditional one, I discovered a new-found appreciation for my home country and having a "normal" job.
Beer, Bats, and Salmon in Olympia
Olympia is one of Washington's underrated places to visit. It really deserves some attention.
One month since reentry
We've been back in the US for a month now. How are things going?
Banos de Agua Santa
Baños de Agua Santa is known as the adrenaline capital of Ecuador and for good reason. Here are some fun activities to do in this wonderful city.


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Create innovative and addictive games with C++
Learn C++ and start building your very own games
Learn C++ from scratch and get started building your very own games.
Learn C++ game development is a course I made for everyone who knows how to program, but doesn't know where to start with game development. The course teaches you how to use the SFML library for C++, to start working with graphics, events and sound to create a 2D game. Everything is done step by step with the help of videos, so it's easy to follow along and learn. At the end of the course you will know what you need for game programming and will be able to start making your own games.


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Blogging Hours: How Much Should You Work on Your Blog?
The obvious answer is: "As many as it takes!" This is your business you're working on, your entire future rides on this. Anything worth having is worth fighting for. However, if working on your blog is the only thing you're doing, ignoring family, friends and your health, and you're still not getting any closer to your goals, then you're probably doing something wrong. How many hours per week should you work on your blog? If you're doing it right you won't even need to ask.
Subscribe to my exclusive newsletter, and learn how to grow your blog like the pros»
If It Was Easy, Everyone Would Be Blogging
Sure you hear other bloggers talking about lounging on the beach or working in their jammies with the music pumping in the background. They sound like they're having fun and like blogging is the easiest thing in the world. Talk to those bloggers though and you'll find out they're lying so you'll think they're an authority figure and if they really are hanging out on the beach every day they're not really successful at all.
Blogging is not the lazy man's answer to his dreams. It takes a lot of hard work and long hours of concentrated effort. If you're only phoning it in then of course you're going to spend a lot of non-productive hours sitting at your desk.
You Do Sometimes Have To Conform
Successful blogger all have a little bit of a rebel inside and that's good. You need to be able to think outside the box and take risks. But there are times when, whether you like it or not, you need to suck it up and follow the rules.
I've seen so many bloggers who think they can get by without doing keyword research, who think they can game Google instead of doing the necessary legwork. I've also seen bloggers who refuse to join social networks or network with other bloggers. They refuse to work on linkbuilding or building a list. They think that they're different, they're better and they'll just automatically rise above all those other blogs because they're brave enough to make their own way in the blogosphere. Everyone else is just a simpering fool.
Eventually, they either fail miserably or the realize they need to conform. Some rules are made to be broken but most are made for a reason. You can spend hours a day, day after day, trying to forge your own path, or you can follow the path that others have already paved and reach your goal faster.
You Have To Admit Your Weaknesses
Are you the stubborn type who refuses to admit defeat? If you're doing the same thing over and over again and the results never change, then yes, you are, and you need to get over it. Every successful blogger will tell you that there's always something new you need to learn and sometimes it takes more than one attempt before you're successful.
Admitting defeat doesn't mean you can't eventually be successful and it doesn't mean you failed. It simply means you need to change your approach and try again. Learn to recognize when something isn't working and learn to admit defeat and you won't have to spend so many hours working on your blog. You can join those other bloggers on the beach, but you'll actually be successful.
When you're learning how to make money off a blog, you need to understand that the very first step is to create a website. If you're interested in starting your own blog, I have written a step-by-step guide that will show you how to start blogging for money for as little as $3.49 per month (this low price is guaranteed only through my link). You will also receive your own domain name for free ($15 value) by clicking on this link and purchasing at least 12 months of hosting with BlueHost. Keep in mind that if you're learning how to make money blogging, the first thing you need is your own self-hosted website. It will help you look more professional in front of your visitors, clients, companies, and everyone else.
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2 Comments
I normally "blog" an article or picture, once every two hours in a time lap of ten hours, and I do agree, with what you've written, it's really hard at times to find the right balance between blogging and dealing with daily basis stuff, because the tend to to overcome one another.


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Clean Sound – Logo Reveal 11842362 Videohive – Free Download After Effects Template
After Effects Version CC 2015, CC 2014, CC, CS6 | 1920×1080 | No Plugin | 589 mb
Here is a stylish, modern, fresh and very useful sound design of circle logo. This After Effects Templates is suitable for any business, company and corporation or for any kind of your brand or product promotion and advertising. It's perfectly fits for your intro, opener, logo reveal or any idea you have. Just add your logo, tagline, change colors , it's easy to change it for classical white and black of colorful and render it, minimal work for you to do!
There are flexible modifications for your sound, you can create any variation you want using minimum time to design it on your own needs. Just move markers, and all animations will look different. With the customization panel it's all possible and ease. Enjoy and don't forget to rate.
In Project was used "Ray-traced 3d", so depends for your configurations, render time can be around 1 hour. And you also need check your "Cuda Drivers". How install it you can watch here.
- Full HD 1920×1080p;
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- Drag and Drop Logo Customization;
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- Editable with After Effects CS6, and above;
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Sound for this project was exclusively written by Brait.
You can buy it here!
Video tutorial will safe your time and give you a result only with few simple steps! You can create any variation you want using minimum time to design it on your own needs. Just move markers, and all animations will look different.
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British energy company Centrica plc announced that it has signed a contract with Cheniere Energy Partners, L.P., to purchase 89 Bcf (approximately 1.75 million metric tons) per year of LNG from the Sabine Pass liquefaction plant in Cameron Parish, La. The contract is for an initial 20 year period with the purchase price indexed to the Henry Hub natural gas price plus a fixed component. Centrica will purchase the LNG on a "Free on Board" (FOB) basis, giving it destination rights for the cargoes. As it has done in the past, Cheniere filed a complete copy of the contract with the Securities and Exchange Commission.
Eversheds Sutherland is an international law firm helping the Fortune 100, industry leaders, sector innovators and business entrepreneurs solve their biggest challenges and reach their business goals. Dedicated to unfaltering excellence in client service, we are known for our business savvy and industry intelligence, providing creative and custom solutions for each of our clients. Industry and business experience makes the difference for our clients.
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Kiper-McShay mock: Browns take CooperIf the Browns are looking to upgrade at guard, it should be Warmack and not Cooper. Kiper has Cleveland taking North Carolina's Jonathan Cooper with the No. 6 overall pick. During Cooper's absence, bullpen coach Bobby Thigpen filled in as the team's pitching coach, while Curt Hasler, the organization's pitching coordinator, has served as the bullpen coach. The team is eager for his return to the dugout, where he spent 11 of his 26 seasons with the organization as pitching coach.Source: ESPN.com
Pitching coach Cooper set to rejoin White SoxPitching coach Don Cooper will rejoin the White Sox in Chicago on Friday for the start of a 10-game homestand that begins with a three-game set against the Twins.Source: MLB.com


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White plastic mounting bracket used for 4108 or 3290 curtain track. Can be used for wall or ceiling mount. Top of track slides into slot. Mounting screws not included.
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This is your resource to discuss support topics with your peers, and learn from each other.
I have a simple app that does nothing more than display an icon under up to two touchpoints on the screen. In my screen's touchevent I have:
protected boolean touchEvent(TouchEvent touchEvent) { synchronized (this) { if (touchEvent.getEvent() == TouchEvent.MOVE || touchEvent.getEvent() == TouchEvent.DOWN) { GameModel.touchX = touchEvent.getX(1); GameModel.touchY = touchEvent.getY(1); GameModel.touch2X = touchEvent.getX(2); GameModel.touch2Y = touchEvent.getY(2); } } return true; }
Then I just draw the icon at touchx,touchy and touch2x,touch2y.
When I run the app on my Storm I test by making circular motions with two fingers on different sides of the screen. It works for a few seconds, but then it seems to get mixed up. Each icon has it's X and Y values controlled by separate fingers! I.E. icon 1's X value correctly matches finger 1's touch point, but it's Y value is driven by finger 2's Y point, and vice versa.
This confusion will switch back and forth seemingly at random.
Any ideas would be appreciated!
Kallin Nagelberg
Random Axis
What BlackBerry device software version are you testing on? You can find this under Options, About on the BlackBerry Smartphone. This was an issue with early releases of BlackBerry device software version 4.7.0. An upgrade may resolve this issue with your BlackBerry Storm.
I reverted my phone to 188.8.131.52 because the firmware that came installed did not work well with my carrier. It was a Verizon Phone and I am using it unlocked on the rogers network. The problems I had were outlined here: http://supportforums.blackberry.com/t5/Java-Develo
Do you know what versions were affected? If I have to upgrade, could you tell me what to do about my networking troubles? I'm hoping 5.0 resolves that. Could I install it?
10-27-2009 11:14 AM - edited 10-27-2009 11:14 AM
I have upgraded to the latest Verizon OS, 220.127.116.118 . Unfortunately multi-touch still doesn't work on this version. Here is what happens:
When one finger's X or Y coordinate crosses with another they may become confused. IE., in the picture above when the Y values of the touch points converge touch Point 1 is assigned touchpoint 2's Y value and vice versa. The X values do not switch and remain bound to the correct touch points. The same problem is observed on the X axis if touch points cross in that direction.
I tried using getX,getY, getGlobalX, getGlobalY, and touchpoint.getMovePoints functions and the problem was the same with all. In order to verify what I was seeing on the screen I logged the values in the arrays from touchPoint everytime a new touchEvent.move or touchevent.down event was triggered using the following code:
for (int i = 0; i < oldPointsSize; i++) { int x1 = xArray1[i]; int y1 = yArray1[i]; int x2 = xArray2[i]; int y2 = yArray2[i]; System.out.println(x1 + "," + y1 + " " + x2 + "," + y2);
The results show that the data in the arrays is bad:
New Touch Event New Touch Event New Touch Event 291,176 74,254 New Touch Event 291,176 74,254 290,199 73,199 New Touch Event 291,176 74,254 290,199 73,199 289,210 71,210 New Touch Event 291,176 74,254 290,199 73,199 289,210 71,210 290,190 60,249 New Touch Event 291,176 74,254 290,199 73,199 289,210 71,210 290,190 60,249 291,173 57,275 New Touch Event 291,176 74,254 290,199 73,199 289,210 71,210 290,190 60,249 291,173 57,275 291,162 56,288 New Touch Event 291,176 74,254 290,199 73,199 289,210 71,210 290,190 60,249 291,173 57,275 291,162 56,288 292,151 52,301 New Touch Event 291,176 74,254 290,199 73,199 289,210 71,210 290,190 60,249 291,173 57,275 291,162 56,288 292,151 52,301 292,151 50,313
In the final event log you can see the behavior described in the image:
The Y value of the first touch point increases from 176 to 210 as the first touchpoint moves down the screen.The Y value of the second point decreases from 254 to 210 as the second touchpoint moves up the screen. At this point the Y values of both are the same. After here they are incorrectly reported. touchpoint 1 receives touchpoint 2's Y values and vice versa. It may be that this only happens when an axis value is the same for both points.
If there is any suggestion on how to address this it would be appreciated. Otherwise it appears that multi-touch cannot be reliably implemented even on the latest OS versions.
Kallin Nagelberg.
I want to make sure this issue isn't ignored. Multitouch support is critical to an app we have in development and we need to know whether or not the Storm actually supports it.
Kallin Nagelberg.
I am also seeing this for os188.8.131.520. What did you end up doing? I am pretty shocked to see this as I would have thought that having little icons follow your fingers would have been one of the very first test for multitouch.
I know this is an old topic - but is there any news on this? Or has anyone found a workaround?
I filed a bug report.
I'm developing for the Storm 1 and 2. I'm using the JRE 5.0.0.
This issue is not solved. On the simulator it seems to work, but on the device it exchanges 'y' values when the points meet at the horizontal axis.
import net.rim.device.api.ui.Field;
import net.rim.device.api.ui.Graphics;
import net.rim.device.api.ui.TouchEvent;
import net.rim.device.api.ui.UiApplication;
import net.rim.device.api.ui.container.MainScreen;
import net.rim.device.api.util.MathUtilities;
public class MultiTouchTest extends UiApplication {
public static void main(String[] args) {
MultiTouchTest instance = new MultiTouchTest();
public MultiTouchTest(){
pushScreen(new MyScreen());
class MyScreen extends MainScreen{
int x1=-100,y1=-100,x2=-100,y2=-100;
int ang0=-1, ang1, angF;
int d0=0, d1=0;
double dR=0;
public MyScreen(){
Field f = new Field(){
protected boolean touchEvent(TouchEvent message) {
switch(message.getEvent()) {
case TouchEvent.MOVE:
if(message.getX(1) > 0 && message.getY(1) > 0) {
x1 = message.getX(1);
y1 = message.getY(1);
if(message.getX(2) > 0 && message.getY(2) > 0) {
x2 = message.getX(2);
y2 = message.getY(2);
if (ang0 == -1){
ang0 = (int)angle(x1, y1, x2, y2);
d0 = (int)distance(x1, y1, x2, y2);
ang1 = (int)angle(x1, y1, x2, y2);
angF = ang1-ang0;
if (angF < 0)
d1 = (int)distance(x1, y1, x2, y2);
dR = (double)d1/(double)d0;
return true;
return false;
protected void layout(int width, int height) {
setExtent(360, 480);
protected void paint(Graphics graphics) {
graphics.drawLine(x1+10, y1+10, x2+10, y2+10);
graphics.drawText("1 ", x1, y1);
graphics.drawText("2 ", x2, y2);
graphics.drawText(""+ang0, 10, 5);
graphics.drawText(""+ang1, 10, 20);
graphics.drawText(""+ angF, 10, 35);
graphics.drawText(""+d0, 100, 5);
graphics.drawText(""+d1, 100, 20);
graphics.drawText(""+ dR, 100, 35);
public double angle(int x1, int y1, int x2, int y2) {
double dx = x2 - x1;
double dy = y2 - y1;
double angle = 0.0d;
if (dx == 0.0) {
if(dy == 0.0) angle = 0.0;
else if(dy > 0.0) angle = Math.PI / 2.0;
else angle = (Math.PI * 3.0) / 2.0;
else if(dy == 0.0) {
if(dx > 0.0) angle = 0.0;
else angle = Math.PI;
else {
if(dx < 0.0) angle = MathUtilities.atan(dy/dx) + Math.PI;
else if(dy < 0.0) angle = MathUtilities.atan(dy/dx) + (2*Math.PI);
else angle = MathUtilities.atan(dy/dx);
return (angle * 180) / Math.PI;
public double distance(int x1, int y1, int x2, int y2){
double dx = x2 - x1;
double dy = y2 - y1;
return Math.sqrt( dx*dx + dy*dy);
Does anyone have a solution?
Vitor Jesus
ps - copy the code to notepad to read it better.
I just read the issue on the bug tracker. It seems it won't be fixed as it is a hardware limitation. It seems that the touch electronics report the points' coordinates, but not necessarely in the right order... this sure explains why I can't replicate the problem in the simulator.
I just want to throw my project to the bin....
How the hell is this possible? Somehow I find this so hard to believe.... such a major part of supporting multi-touch relies on correctly identifying where each finger is.... on the other hand it sure explains why the only multitouch feature that comes with the storm is the text selection.
I'll have to correct this in software. I'll post the solution here if and when I can fix this.


text:
Randle Golf
Golf, schools, Teacher
John Randle is the Academy Director at the Bear Mountain Golf Academy. He provides award-winning golf instruction in Victoria, BC. John is a two-time PGA of BC teacher of the Year. He uses cutting edge learning technology to provide the very best lesson experience. Trackman, K-Vest 3D and SAM Puttlab are three of the tools John uses to help his students.
If you are looking for more consistency, to fix a specific problem or you have big goals for your game, give John a call and get started now!
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The Marines need to set up a secure zone for civilian workers. First they must defeat an elusive enemy that wages a hit-and-run campaign from its labyrinthine mud compounds
Serenaded only by the howl of jackals in the dark of predawn, nearly 200 Royal Marines waited in uncertain silence for their attack on the Taleban to begin.
They clustered in cover beneath the rock-strewn slopes of a hill northeast of Kajaki where, only 24 hours earlier, a similar attack mission had been called off at the last moment because bad weather interfered with their air cover.
But this time, as the final minutes closed to their 0615 H-hour and the waning moon broke through the clouds, the voice of a Marine troop commander crackled softly into their headsets.


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Sherwin-Williams Paint Store, Paint - Retail, listed under "Paint - Retail" category, is located at 2032 Tiffin Ave Findlay OH, 45840 and can be reached by 4194232712 phone number. Sherwin-Williams Paint Store has currently 0 reviews.This business profile is not yet claimed, and if you are the owner, claim your business profile for free. If you are not the owner you can invite this business to join by email or by phone.Browse all Paint - Retail in Findlay OH. Discover census data for Findlay, OH.
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Gateway launched the search for their 2017 ambassadors at a prestigious media launch on Thursday, 19 January – where they..
Easily accessible off the M4 and one of the first places you'll see as you enter Ballito, is the continental..
The High Tea at the Oyster Box is literally heaven. From freshly baked scones, pastries, quiches and cakes, to finger..
Ladies and gentlemen! The world-famous Great Moscow Circus in association with M-Net and East Coast Radio is coming to SUNCOAST,..
The much-anticipated 11th Annual Sunflower Fund Ladies Breakfast in association with The Smile Emporium will take place on Saturday, 11..
Perfectly balanced wines with elegance and texture – that is the philosophy of guest speaker Gerhard Smith, winemaker extraordinaire at..
The Gooderson Kloppenheim Country Estate Hotel is a luxurious hotel for all families who are in need of a fun filled..
Our family of four was recently invited to spend 2 nights, dinner, bed and breakfast included at the Gooderson Natal..
Gateway launched the search for their 2017 ambassadors at a prestigious media launch on Thursday, 19 January – where they..
Overview It's difficult to imagine a better spot for a Festival than the Amphitheatre Backpackers, with its amazing backdrop of..
With panoramic views of the Drakensberg mountains as our backdrop, tents of all colour, shapes, and sizes started to spring..


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20 Ways to Fix Your Trip
Our "Travel Intelligence" provides road-tested secrets for planning and fixing your trips at home and abroad. From packing essentials to tips for surviving travel snafus, our consumer advocate Christopher Elliott and fellow National Geographic travel experts share their insider secrets for the smart traveler.
Packing Tips From Our Experts
9 Tips to Remember When Traveling to U.S. National Parks
To celebrate the centennial of the National Park Service, our experts share their tips for planning the perfect national park trip.
The 10 Rules of Packing
Intelligent Travel's Good Traveler, Aric S. Queen, explains what to take, what to leave, and where to put it.
Travel Essentials for a Digital Nomad
Find out what Andrew Evans packs for his journeys around the world.
How to Pack for Weekend Trips
Our Urban Insider, Annie Fitzsimmons, shares seven things to consider while you're packing for your next weekend trip.
Travel Gear Guide
Gear Tips for Tech-Savvy Travelers
Our six picks of the latest tech travel gear that will make your next trip virtually stress-free.
Tips to Travel Bliss
How to Be a Smart Traveler
Travel Photo Advice
Stay Healthy
Traveling With Kids
Columns by Travel Consumer Advocate Chris Elliott
- Mission Creep at the TSA?
- A Tale of Two Airlines
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- Got a Beef? Don't Count on a Tweet
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- Power to the Real People
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- Scammed Again
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text: The Art of Monitoring New York City's Telephone ConversationsLISA KATAYAMA2/21/08 11:20amFiled to: SurveillanceArtMomaNYTEMitsenseable city labsocial control2EditPromoteShare to KinjaToggle Conversation toolsGo to permalink You can gage how busy New York City is by looking at all the people swarming in the streets, or by smelling the giant piles of trash they've left at the curbs. But there are ways to take stock of the city's populace that are far more revealing. For a new MoMa exhibit this month, MIT's Senseable City Lab chose to expose how talkative New York is by tracking lines of electronic communication into and out of the city. Their project is aptly named the New York Talk Exchange (NYTE). It's also inadvertently a portrait of digital surveillance, showing exactly how easy it is for people to use phone records to monitor which countries New Yorkers are ringing up. AT&T is a sponsor of the project, and handed over reams of phone records to the group so they could link NYC with cities around the world based on phone calls and IP traffic. No, it's not a surveillance spree, though it's hard not to wonder about that given AT&T's recent eagerness to hand over its phone data to the government without warrants. But this project merely aims to show how busy NYC can be. And just how pretty busy can be. This will be part of the MoMa's "Design and the Elastic Mind" exhibition starting February 24th.Images by senseable city lab Advertisement Advertisement New York Talk Exchange main pageReply2 repliesLeave a reply


text:
When did the story of the soul of the great bell action take place
In the city of Peking......
This Is a Certified Answer
Certified answers contain reliable, trustworthy information vouched for by a hand-picked team of experts. Brainly has millions of high quality answers, all of them carefully moderated by our most trusted community members, but certified answers are the finest of the finest.
In the country of China,at the capital(Peking or Beijing City)


text:
*Please contact dealer to verify price, options, and other vehicle details.
* While every reasonable effort is made to ensure the accuracy of this information, we are not responsible for any errors or omissions contained on these pages. On occasion some vehicles in inventory may have dealer installed options and accessories that may or may not be reflected in the online pricing. Please verify pricing and configuration with with Pat Lobb's Toyota Of McKinney. All vehicles subject to prior sale. Dealer discounts reflected on our website are only applicable to in stock units. Please see dealer for details.
MPG Disclaimer: *Based on 2014 EPA mileage and driving range ratings. Use for comparison purposes only. Your MPGe/MPG and driving range will vary depending on driving conditions, how you drive and maintain your vehicle, battery age/condition, and other factors.
Pat Lobb Peace of Mind Purchase Plan
Used Toyotas
This outstanding example of a CERTIFIED 2014 Toyota Venza XLE w/ NAV is offered by Pat Lobb Toyota.
Drive home in your new pre-owned vehicle with the knowledge you're fully backed by the CARFAX Buyback Guarantee. The CARFAX report for this 2014 Toyota Venza XLE w/ NAV highlights that it's a One-Owner vehicle, and for good reason. A One-Owner vehicle gives you the feel of owning a new vehicle without paying the new car price.
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You demand the best in everything, so why would your automobile be any different? Enjoy the best features available in this stunning Toyota Venza. You'll quickly realize how much you will need a navigation system once you are stopping for directions or looking up addresses on your phone instead of looking at the road. This Toyota Venza XLE w/ NAV features a navigation system that will free you of these unnecessary headaches.
A unique vintage vehicle that exudes taste and sophistication. This Toyota is equipped with some of the most amazing wheels the market has to offer! Once you take this Toyota Venza XLE w/ NAV for a test drive, you'll immediately realize how much of an added bonus this vehicle's upgraded wheels will provide.
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The Venza starts at under $28,000, offering a lot of space and utility for the money. However, the vehicle can be optioned up fairly substantially for those wanting a bigger dose of luxury and increased utility. Prices eventually top out at just under $40,000 for a fully loaded V6 version with all-wheel drive. The Venza's main trick, is offering up the space and utility of an SUV without the compromised handling and fuel economy that would accompany such a vehicle.
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All prices plus Tax, Title, License and $150 doc fee.
Images of vehicles are for illustration purposes only.


text:
Bangladesh Police job Circular
Bangladesh Police job Circular.
Organization Name: Bangladesh Police
Number of job vacancies: N/A
Type of Jobs: Full Time
Job category: government Jobs
salary: see circular , Jobs Deadline: 24 aug, 2016
Recently Bangladesh Police has published an appointment circular for following positions. The circular has published 3-8 , 2016 and application last date is 24-8, 2016. Job circular details are given below :


text:
Disc 1
- Carolina In My Mind
- 111
- Nine Pound Hammer
- The Kind of Place
- Climb To Safety
- Looking Out My Window
- Shake Your Hips
- Dark Star Jam/Mexico
- Opium
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How many musicians would kill to get a peek into Warren Haynes' address book? He has been holding his annual benefit Christmas jam concerts in Asheville, North Carolina since 1988, and each time, the lineup is more impressive than the year before. This one, recorded December 21, 2002 but not released until nearly nine years later, documents his 14th such gig, featuring Robert Randolph, moe., John Hiatt, Bob Weir & Friends, and a closing free-for-all with Haynes' Gov't Mule and guests such as DJ Logic and Widespread Panic's Dave Schools. Haynes lends his talents to about half the tracks on this nearly two-and-a-half-hour, double-disc set as he shares the spotlight with Hiatt, moe., Jerry Joseph, Weir, and others. With Weir around, there's a heavy Dead vibe to much of the music, as moe. even covers Dark Star (sounding arguably better and tighter than vintage Dead) and Weir rolling out the Dead's "Truckin'," "Shakedown Street," and "The Other One" with somewhat surprisingly sprightly results. Since Haynes was touring as a temporary member of that band for a while, the connection is logical. Randolph lights a fire under all that noodling with a propulsive nine-minute take on Slim Harpo's "Shake Your Hips" that just about burns the stage down with its intensity. Bluegrass is usually present at these Haynes gatherings, and for this one, he invites Ashville's Sons of Ralph. The band features some lightning-fast mandolin playing from Don Lewis that will have most listeners Googling the group to learn more about them. John Hiatt delivers inspired versions of some of his better-known songs including a ten-minute romp through "Memphis in the Meantime" (with Haynes sitting in). Having the always stellar Sonny Landreth on slide helps raise the temperature substantially. The final 30 minutes are spent on three Gov't Mule tunes, including a raw cover of Lynyrd Skynyrd's "Simple Man," with that group's drummer Artimus Pyle, that closes the concert by paying tribute to Haynes' 70s and Southern rock roots. These shows usually run over eight hours, so even at a hefty 150 minutes, this is just a sampler of the music soiree that night. As with the three previous double-disc packages, the recording is pristine and the live setting adds a sharp edge to every performance. The CD's profits, as well as those from the shows, goes to Habitat for Humanity. That gives the proceedings an even more positive vibe, making this a logical purchase for any American roots rock or Dead fan.
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Disney punk edits
Explore related topics
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disney punk edit || Punk Merida awright (she's kind of a punk anyway, though) || sparklepunk
"Disney Punk Princesses" by fashiongirlz143 ❤ liked on Polyvore
This is ash shes 17 and has the power fire shes up for adoption just comment down below if you want her
Punk disney ariel. Day of the dead
this is jessie she is 15.and this is jase he is 16 .both have no powers .also their a couple and r always kissing
Disney punks!!!! I don't know how I feel about Elsa and Snow White, though.
I don't know who's idea it was to turn Disney characters "punk" but I think it's kind of super cute. :S


text:
Ever since Google Earth was first released, users have been spotting aircraft in flight in the imagery.When an aircraft is captured in flight in Google Earth it is not uncommon for it to have a … [Read more...]
Planes in flight and the rainbow effect
Google Maps 'Earth' view FOV
We recently noticed that if you look at a given place in both Google Earth and Google Maps' 'Earth' view, they look a bit different, with distant objects looking closer in Google Maps than in Google … [Read more...]
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We have recently been looking at some of the features exclusive to the now free Google Earth Pro. Today we are looking at the 'regionate' feature.The regionate tool is a way to make large datasets … [Read more...]
Measuring in Google Earth Pro
Last month Google removed the US$399 a year price tag from Google Earth Pro and released it for free. There is no longer any need to sign up for a licence key, simply login with your email address and … [Read more...]
Google Earth Pro importing addresses
Google recently made Google Earth Pro available for free. As a result, we have been having a look at some of the premium features of Google Earth Pro. So far we have looked at the movie maker, … [Read more...]
Google Earth Pro map making tool
Now that Google Earth Pro is fee we are having a look at some of the features it has that are not found in the standard version of Google Earth. We have already looked at the movie maker and Viewshed … [Read more...]
Google Earth Pro: Viewshed tool
On Tuesday we had a look at Google Earth Pro's Movie Maker. Today, we are looking at another Google Earth Pro exclusive feature, the Viewshed tool.The Viewshed tool shows the line of sight from any … [Read more...]
Google Earth Pro Movie Maker
Now that Google Earth Pro is free it is worth exploring some of the features it has that are not available in the standard version of Google Earth.Google Earth Pro comes with a built in tool for … [Read more...]
Syncing Google Earth's 'My Places'
We have in the past shown you how to move Google Earth's 'My Places' from one computer to another. However, if you regularly use Google Earth on more than one computer, it can be useful to sync your … [Read more...]
Problems with the new search in Google Earth
Last week we noted that Google had changed the way search results are displayed in Google Earth (desktop version). Several GEB readers have noted, however, that there are some problems with the … [Read more...]


text:
Lacrosse In Belgium
Lacrosse was originated by the native Americans, the game was originated in the st.Lawrence valley area in north America.
How the game lacrosse is played
The team with the most goals at the end of the game wins. The scoring of the game is manly how they tell who wins or losses.
In a game 10 players can be involved at once. one goalkeeper three defensemen, three middle fielders and three attack men. The length of a game is 60 mins.
Scoring occurs during the game when a person throws the ball from there goal to a teammates.
A lacrosse field is 110 yards long, and can be from 53 1/3 to 60 yards wide.
Offense strategies are to never be standing still or your doing something wrong, all passes should be made over hand.
Defense strategies try to keep the player's moves
to be lateral and away from the net. and Control what the offensive can do with the ball.
In lacrosse they use
Please use Google Chrome to obtain the best export results.
by 22mmallen
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Public - 4/12/16, 6:15 PM


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Need a Titer Test in Dallas, TX? Simply order online 24/7 and get a fast, affordable Titer Test at a lab near you (Find More Locations):
What is a Titer Test?
A Titer Test is a blood test that checks your titer level for one or more diseases. People in Dallas, TX order a Titer Test for compliance with hospital, vendor credentialing, schools, and other organizations.
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text:
Melbourne's Hidden Boutiques & BarsPosted on: 25 June 2008 by Gareth Hargreaves
There are plenty of hidden gems in Australia's cultural capital.
One of the best things to do in Melbourne is take a tour of the laneways and arcades.
You can happily get lost for hours in the maze of bars, cafes, street art and boutiques.
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English Meaning
That which is educed, as by analysis.
- That which is educed
- A reactant; see also product
Malayalam MeaningTransliteration ON/OFF | Not Correct/Proper?
വിദ്യാഭ്യാസം - Vidhyaabhyaasam | Vidhyabhyasam ; ;വിദ്യ - Vidhya ;
This service is a free English - Malayalam Dictionary with English & Malayalam meaning of more than 125000 words. You can find out equivalent Malayalam meaning, definitions, Synonyms & more of any English word by using this service. The malayalam meaning is displayed with transliterated output (Manglish) as well & that will help people who doesn't know to read Malayalam language. Please support this free service by just sharing with your friends. If you find any bugs in this program please report me at firstname.lastname@example.com
That which is educed, as by analysis.
വിദ്യാഭ്യാസം - Vidhyaabhyaasam | Vidhyabhyasam ; ;വിദ്യ - Vidhya ;
The Usage is actually taken from the Verse(s) of English+Malayalam Holy Bible.×


text:
Ted & Company
In this scene, David has returned from his meeting with Nathan. He tells Bathsheba the story, revealing the truth about how her husband Uriah died, and the consequences that their son will now suffer as a result of David's actions. Based on 2 Samuel 12. Featuring Ted Swartz and Ingrid DeSanctis.
The video clip above is a 2 minute preview.
This clip appears on the Deliverance, Dysfunction & Destiny DVD. This DVD as well as downloads of individual scenes and scripts are available for purchase on the Ted & Company website tedandcompany.com


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Note: Product may take up to 12 weeks to ship.
Once purchased you cannot return or cancel your order.
See Our Policies
PROJECT PRICING available for Trade Professionals
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Vanity: W 35.38" x D 18.75" x H 21.75"Medicine Cabinet: W 29.5" x H 26" x D 5"
Yes it does come with a sink. It comes with everything you see in the photo.


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Added: Monday, September 21, 2009
Daring Scorching Sex Scenes Of Bitch Teener
This one exceptional teenage whore has her sex move in her own way. She's daring and she's always up into wild, scorching hot sex session. See her humps over this enormous, thick-headed cock until it squirts a thick. gelatinous-like semen in her pleasured mouth.
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text:
*Transform these sentences into the past.
1.Lee uses chopsticks when eating rice and sushi.
2.James often stays up late at night.
3.Reginald goes camping every fortnight in summer.
*Insert "may","can"or"must".Use the negative form where necessary.
1.I................eat anything . I've got a stomachache
2.You ......................go to the doctor's office.You have a bad cough.
3.............................. you tell me where the fishmonger's is?
*Use the Future Simple instead of the going to-Future.
1.I'm going to build a snowman.
2.The twins are going to sled near the chalet tomorrow.
3.Marge is going to have a snowball fight soon.
de Kamy


text:
Women's Professional Soccer announced the starting 22 players for its 2010 WPS All-Star Game
presented by U.S. Coast Guard Reserve which kicks-off at 8pm ETÂ on June 30 at KSU Soccer Stadium
in Kennesaw, Ga. The starting 22 players were selected on ballots from players (25%), coaches
(25%), media (25%) and online fan voting (25%).
Pitch Invasion
Like Jennifer Doyle, I have only questions about the FIFA U-20 Women's World Cup from watching
it so far.
And my main question so...
Pretty Tough - Soccer Week 14 was a pretty predictable week. FC Gold Pride won (I think it's getting old now). The
Breakers, who have recently come...
Pretty Tough - Soccer The starting lineups for the 2010 WPS All-Star Game presented by U.S. Coast Guard Reserve were
selected Monday evening in front of a...
Jared Montz's Weblog Nice put back by Marta from the shot off the crossbar!
Jared Montz's Weblog Chicken dance goal celebration... love it!
The skill by Marta at the 3:15 mark is awesome!


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Floor Plans & Pricing
Prices and availability subject to change without notice.
Property Details
14808 - 14932 E Burnside
2 Bedroom with Washer/Dryer hookups and fenced backyard! Pets ok! - Lovely brick garden walkup in SE portland! Located directly on the max line this home is ideal for those that don't want to drive a car everywhere. Unit features New stainless appliances and hardwood floors! 15 min to downtown PDX and close to tons of shopping, parks and great golf courses like Glendoveer! Homes have nice back patio area with room for flowers and small garden. Pet friendly with a $500.00 deposit per animal. (RLNE2032799)
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text:
Orangeburg is located in Orangeburg county and has a population of 12,765.
Business Writing schools in Orangeburg charged students an average tuition of $4,806.00 if they were South Carolina residents, and $5,949.00 if they were non-residents, during the 2009 - 2010 school year.
The cost, on average, for books and supplies needed for business writing schools in Orangeburg is $1,166.00. Students living on campus at business writing schools in Orangeburg paid an average of $7,834.00 for room and board during the 2009 - 2010 school year. Students living at home incurred costs of $4,240.00. There is one business writing college in Orangeburg. In the 2008 - 2009 school year, reportedly 0 students completed business writing programs in Orangeburg.
|Average Salary
|Number Employed in City
|Business Operations Specialists, All Other
|Public Relations Specialists