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News Article | May 9, 2017
Site: www.prnewswire.com

Oracle ERP Cloud is a complete, modern, and proven financial platform delivered seamlessly through the Oracle Cloud. A modern user interface driven by the latest design innovations delivers embedded analytics, contextual social collaboration and a device-independent mobile experience that makes Oracle ERP Cloud familiar and easy to use. Importantly, Oracle ERP Cloud will now enable Indian firms to administer and leverage the significant GST process changes, including tax calculations, liability accounting, recovery, settlement and reporting. The availability of Oracle ERP Cloud in India reflects Oracle's support of the government's Make in India initiative. "Global adoption of our leading Oracle ERP Cloud continues to accelerate," said Safra Catz, CEO, Oracle. "We expect this trend to grow in India as customers can now take advantage of Oracle ERP Cloud's comprehensive tax management capabilities, fully integrated with core ERP functions, to help make better business decisions and address regulatory changes driven by the new GST." To learn more about Oracle ERP Cloud, follow @oracleerpcloud on Twitter, and join the Facebook conversation at Oracle ERP Cloud. The Oracle Cloud delivers hundreds of SaaS applications and enterprise-class PaaS and IaaS services to customers in more than 195 countries while processing 55 billion transactions a day. For more information about Oracle (NYSE: ORCL), please visit us at http://cloud.oracle.com. Oracle and Java are registered trademarks of Oracle and/or its affiliates. Other names may be trademarks of their respective owners. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/oracle-announces-availability-of-oracle-erp-cloud-in-india-300453593.html


News Article | May 10, 2017
Site: www.nature.com

HEK293 cells stably transfected with the STF plasmid encoding the firefly luciferase reporter under the control of a minimal promoter, and a concatemer of 7 LEF/TCF binding sites32, were obtained from J. Nathans. Mouse L cells stably transfected with the STF plasmid and a constitutively expressed Renilla luciferase (control reporter) were obtained from C. Kuo. L cells transfected with a mouse WNT3A expression vector to produce conditioned media were obtained from the ATCC. A375, SH-SY5Y and A549 cells were stably transfected with the BAR plasmid encoding the firefly luciferase reporter under the control of a minimal prompter and a concatemer of 12 TCF/LEF binding sites and a constitutively expressed Renilla luciferase (control reporter) using a lentiviral-based approach33. All reporter cell lines were cultured in complete DMEM medium (Gibco) supplemented with 10% FBS, 1% penicillin, streptomycin, and l-glutamine (Gibco), at 37 °C and 5% CO and cultured in the presence of antibiotics for selection of the transfected reporter plasmid. C3H10T1/2 cells were obtained from the ATCC. Human primary MSCs were obtained from Cell Applications, Inc. Mouse primary MSCs were obtained from Invitrogen. Cell lines have not been tested for mycoplasma contamination. The coding sequence of B12 containing a C-terminal 6×His-tag was cloned into the pET28 vector (Novagen) for bacterial cytoplasmic protein expression. Protein expression was performed in transformed BL21 cells, expression was induced with 0.7 mM IPTG at an OD   of 0.8 for 3–4 h. Cells were pelleted, lysed by sonication in lysis buffer (20 mM HEPES, pH 7.2, 300 mM NaCl, 20 mM imidazole), and soluble fraction was applied to Ni-NTA agarose (QIAGEN). After washing the resin with lysis buffer containing 500 mM NaCl, B12 was eluted with 300 mM imidazole, and subsequently purified on a Superdex 75 size-exclusion column (GE Healthcare) equilibrated in HBS (10 mM HEPES, pH 7.2, 150 nM NaCl). XWnt8 was purified from a stably transfected Drosophila S2 cell line co-expressing XWnt8 and mouse FZD8 CRD–Fc described previously4. Cells were cultured in complete Schneider’s medium (Thermo Fisher Scientific), containing 10% FBS and supplemented with 1% l-glutamine, penicillin and streptomycin (Gibco), and expanded in Insect-Xpress medium (Lonza). A complex of XWnt8 and FZD8 CRD–Fc was captured from the conditioned media on Protein A agarose beads (Sigma). After washing with 10 column volumes of HBS, XWnt8 was eluted with HBS containing 0.1% n-dodecyl-β-d-maltoside (DDM) and 500 mM NaCl, while the FZD8 CRD–Fc remained bound to the beads. All other proteins were expressed in High Five (Trichoplusia ni) cells (Invitrogen) using the baculovirus expression system. To produce the B12-based surrogate, the coding sequences of B12, a flexible linker peptide comprising of 0, 1, 2 or 3 GSGSG-linker repeats, followed by the C-terminal domain of human DKK1 (residues 177–266), and a C-terminal 6×His-tag, were cloned into the pAcGP67A vector (BD Biosciences). To clone the scFv-based surrogate ligand, the sequence of the Vantictumab was retrieved from the published patent, reformatted into a scFv, and cloned at the N terminus of the surrogate variant containing the GSGSG linker peptide. To produce recombinant FZD CRD for crystallization, surface plasmon resonance measurements, SEC-MALS experiments and functional assays, the CRDs of human FZD1 (residues 113–182), human FZD4 (residues 42–161), human FZD5 (residues 30–150), human FZD7 (residues 36–163), human FZD8 (residues 32–151) and human FZD10 (residues 30–150), containing a C-terminal 3C protease cleavage site (LEVLFQ/GP), a biotin acceptor peptide (BAP)-tag (GLNDIFEAQKIEWHE) and a 6×His-tag were cloned into the same vector. The human FZD8 CRD used for crystallization contained only a C-terminal 6×His-tag, in addition to a Asn49Gln mutation to mutate the N-linked glycosylation site. FZD1/FZD8 CRD for inhibition assay contained a C-terminal 3C protease cleavage site, Fc-tag (constant region of human IgG), and a 6×His-tag. Human DKK1 (residues 32–266) with a C-terminal BAP-tag and 6×His-tag, and the two furin-like repeats of human RSPO2 (residues 36–143) with a N-terminal Fc-tag and a C-terminal 6×His-tag, were cloned also into the pAcGP67A vector. All proteins were secreted from High Five insect cells grown in Insect-Xpress medium, and purified using Ni-NTA affinity purification, and size-exclusion chromatography equilibrated in HBS (10 mM HEPES, pH 7.3, 150 nM NaCl). Enzymatic biotinylation was performed in 50 mM bicine, pH 8.3, 10 mM ATP, 10 mM magnesium acetate, 0.5 mM d-biotin with recombinant glutathione S-transferase (GST)-tagged BirA ligase overnight at 4 °C, and proteins were subsequently re-purified on a Superdex 75 size-exclusion column to remove excess biotin. We attempted to mimic the native Wnt–FZD lipid–protein interaction with a de novo designed protein–protein binding interface. A 13-residue alanine helix was docked against the lipid-binding cleft using Foldit34. This structural element was grafted onto a diverse set of native helical proteins using the Rosetta Epigraft35 application to discover scaffolds with compatible, shape-complementary backbones. Prototype designs were selected by interface size and optimized using RosettaScripts36 to perform side-chain redesign. 50 selected designs were further manually designed to ensure charge complementarity and non-essential mutations were reverted to the wild-type amino acid identity to maximize stability. DNA was obtained from Gen9 and screened for binding via yeast surface display as previously described with 1 μM biotinylated FZD8 CRD pre-incubated with 025 µM SAPE (Life Technologies)37. A design based on the scaffold with PDB code 2QUP, a uncharacterized four-helix bundle protein from Bacillus halodurans, demonstrated binding activity under these conditions, whereas knockout mutants Ala52Arg and Ala53Asd made using the Kunkel method38 abrogated binding, verifying that the functional interface used the predicted residues. Wild-type scaffold 2QUP did not bind, confirming that activity was specifically due to design. To improve the affinity of the original design, a full-coverage site-saturation mutagenesis library was constructed for design based on the 2QUP scaffold via the Kunkel mutagenesis method38 using forward and reverse primers containing a ‘NNK’ degenerate codon and 21-bp flanking regions (IDT). A yeast library was transformed as previously described39 and sorted for three rounds, collecting the top 1% of binders using the BD Influx cell sorter. Naive and selected libraries were prepared and sequenced, and the data was processed as previously described37 using a Miseq (Illumina) according to manufacturer protocols. The most enriched 11 mutations were identified by comparison of the selected and unselected pools of binders and were combined in a degenerate library containing all enriched and wild-type amino acid identities at each of these positions. This combination library was assembled from the oligonucleotides (IDT) listed below for a final theoretical diversity of around 800 k distinct variants. This library was amplified, transformed, and selected to convergence over five rounds, yielding the optimized variant B12. The B12–FZD8 CRD(N49Q) complex was formed by mixing purified B12 and FZD8 CRD(N49Q) in stoichiometric quantities. The complex was then treated with 1:100 (w/w) carboxypeptidase A (Sigma) overnight at 4 °C, and purified on a Superdex 75 (GE Healthcare Life Sciences) size-exclusion column equilibrated in HBS. Purified complex was concentrated to around 15 mg ml−1 for crystallization trials. Crystals were grown by hanging-drop vapour diffusion at 295 K, by mixing equal volumes of the complex and reservoir solution containing 42–49% PEG 400, 0.1 M Tris, pH 7.8–8.2, 0.2 M NaCl, or 20% PEG 3000, 0.1 M sodium citrate, pH 5.5. While the PEG 400 condition is already a cryo-protectant, the crystals grown in the PEG 3000 condition were cryoprotected in reservoir solution supplemented with 20% glycerol before flash freezing in liquid nitrogen. Crystals grew in space groups P2 (PEG 400 condition) and P2 (PEG 3000 condition), respectively, with 2 and 4 complexes in the asymmetric units. Cell dimensions are listed in Supplementary Table 1. Data were collected at beamline 8.2.2 at the Advanced Light Source (ALS), Lawrence Berkeley National Laboratory. All data were indexed, integrated, and scaled with the XDS package40. The crystal structures in both space groups were solved by molecular replacement with the program PHASER41 using the structure of the FZD8 CRD (PDB code 1IJY) and the designed model of a minimal core of B12 as search models. Missing residues were manually build in COOT42 after initial rounds of refinement. Several residues at the N terminus (residues 1 to 16/17/20/21), at the C terminus (residues after 117) and several residues within loop regions were unstructured and could not been modelled. Furthermore, we observed that in both crystal forms, B12 underwent domain swapping, and one B12 molecule lent helix 3 and 2 to another B12, resulting in a closely packed B12 homodimer. The density of the loops connecting helixes 1 and 2, and 3 and 4 were clearly visible, and folded into helical turns. Yet, SEC-MALS experiments confirmed that B12 existed as a monomer in solution, and complexed FZD8 CRD with a 1:1 stoichiometry. PHENIX Refine43 was used to perform group coordinate refinement (rigid body refinement), followed by individual coordinate refinement using gradient-driven minimization applying stereo-chemical restraints, NCS restraints, and optimization of X-ray/stereochemistry weight, and individual B-factor refinement. Initial rounds of refinement were aided by restraints from the high-resolution mouse FZD8 CRD structure as a reference model. Real space refinement was performed in COOT into a likelihood-weighted SigmaA-weighted 2mF  − DF map calculated in PHENIX. The final model in the P2 space group was refined to 3.20 Å with R and R values of 0.2002 and 0.2476, respectively (Supplementary Table 1). The quality of the structure was validated with MolProbity44. 99.5% of residues are in the favoured region of the Ramachandran plot, and no residue in the disallowed region. The structure within the P2 space group was refined to 2.99 Å with R and R values of 0.2253 and 0.2499, respectively, with 99.2% of residues in the favoured region of the Ramachandran plot, and no residues in the disallowed region. See Supplementary Table 1 for data and refinement statistics. Structure figures were prepared with the program PYMOL. Binding measurements were performed by surface plasmon resonance on a BIAcore T100 (GE Healthcare) and all proteins were purified on SEC before experiments. Biotinylated FZD1 CRD, FZD5 CRD, FZD7 CRD and FZD8 CRD were coupled at a low density to streptavidin on a SA sensor chip (GE Healthcare). An unrelated biotinylated protein was captured at equivalent coupling density to the control flow cells. Increasing concentrations of B12 and scFv–DKK1c were flown over the chip in HBS-P (GE Healthcare) containing 10% glycerol and 0.05% BSA at 40 μl ml−1. The chip surface was regenerated after each injection with 2 M MgCl in HBS-P or 50% ethylene glycol in HBS-P (scFv–DKK1c measurements), or 4 M MgCl in HBS-P (B12 measurements) for 60 s. Curves were reference-subtracted and all data were analysed using the Biacore T100 evaluation software version 2.0 with a 1:1 Langmuir binding model to determine the K values. To characterize the FZD-specificity of B12, the yeast display vector encoding B12 was transformed into EBY100 yeast. To induce the display of B12 on the yeast surface, cells were growing in SGCAA medium45, 46 for 2 days at 20 °C. 1 × 106 yeast cells per condition were washed with PBE (PBS, 0.5% BSA, 2 mM EDTA), and stained separately with 0.06–1,000 nM biotinylated FZD1/4/5/7/8/10 CRDs for 2 h at 4 °C. After washing twice with ice-cold PBE, bound FZD CRDs were labelled with 10 nM strepdavidin-Alexa647 for 20 min. Cells were fixed with 4% paraformaldehyde, and bound FZD CRD was analysing on an Accuri C6 flow cytometer. FZD8 fused to an N-terminal HaloTag47 and LRP6 fused to an N-terminal SNAP-tag48 were cloned into the pSEMS-26m vector (Covalys Biosciences) by cassette cloning49, 50. The template pSEMS-26m vectors had been coded with DNA sequences of the SNAP-tag or the HaloTag, respectively, together with an Igκ leader sequence (from the pDisplay vector, Invitrogen) as described previously50. The genes of full-length mouse Fzd8 or human LRP6 without the N-terminal signal sequences were inserted into pSEMS-26m via the XhoI and AscI or AscI and NotI, restriction sites, respectively. A plasmid encoding a model transmembrane protein, maltose-binding protein fused to a transmembrane domain, fused to an N-terminal HaloTag was prepared as described recently13. HeLa cells were cultivated at 37 °C, 5% CO in MEM Earle’s (Biochrom AG, FG0325) supplemented with 10% fetal calf serum and 1% nonessential amino acids. Cells were plated in 60-mm cell culture dishes to a density of 50% confluence and transfected via calcium phosphate precipitation49. 8–10 h after transfection, cells were washed twice with PBS and the medium was exchanged, supplied with 2 μM porcupine inhibitor IWP-2 for inhibiting maturation of endogenous Wnt in HeLa cells51. 24 h after transfection, cells were plated on glass coverslips pre-coated with PLL-PEG-RGD52 for reducing nonspecific binding of dyes during fluorescence labelling. After culturing for 12 h, coverslips were mounted into microscopy chambers for live-cell imaging. SNAP-tag and HaloTag were labelled by incubating cells with 50 nM benzylguanine-DY649 (SNAP-Surface 649, New England Biolabs) and 80 nM of HaloTag tetramethylrhodamine ligand (HTL-TMR, Promega) for 20 min at 37 °C. Under these conditions, effective degrees of labelling estimated from single molecule assays with a HaloTag–SNAP-tag fusion protein were ~40% for the SNAP-tag and ~25% for the HaloTag13. After washing three times with PBS, the chamber was refilled with MEM containing 2 μM IWP-2 for single-molecule fluorescence imaging. Single-molecule fluorescence imaging was carried out by using an inverted microscope (Olympus IX71) equipped with a triple-line total internal reflection (TIR) illumination condenser (Olympus) and a back-illuminated EMCCD camera (iXon DU897D, 512 × 512 pixel from Andor Technology). A 561-nm diode solid state laser (CL-561-200, CrystaLaser) and a 642-nm laser diode (Luxx 642-140, Omicron) were coupled into the microscope for excitation. Laser lights were reflected by a quad-line dichroic beam splitter (Di R405/488/561/647, Semrock) and passed through a TIRF objective (UAPO 150×/1.45, Olympus). For simultaneous dual-colour detection, a DualView microimager (Optical Insight) equipped with a 640 DCXR dichroic beamsplitter (Chroma) in combination with bandpass filters FF01-585/40 and FF01 670/30 (Semrock), respectively, was mounted in front of the camera. The overlay of the two channels was calibrated by imaging fluorescent microbeads (TetraSpeck microspheres 0.1 μm, T7279, Invitrogen), which were used for calculating a transformation matrix. After channel alignment, the deviation between the channels was below 10 nm. For single-molecule imaging, typical excitation powers of 1 mW at 561 nm and 0.7 mW at 642 nm measured at the objective were used. Time series of 150–300 frames were recorded at 30 Hz (4.8–9.6 s). An oxygen scavenging system containing 0.5 mg ml−1 glucose oxidase, 40 mg ml−1 catalase, and 5% (w/v) glucose, together with 1 μM ascorbic acid and 1 μM methyl viologene, was added to minimize photobleaching53. Receptor dimerization was initiated by incubating with 100 nM Wnt proteins or surrogates. Images were acquired after 5 min incubation in the presence of the ligands. All live-cell imaging experiments were carried out at room temperature. A 2D Gaussian mask was used for localizing single emitters54, 55. For colocalization analysis to determine the heterodimerization fraction, particle coordinates from two channels were aligned by a projective transformation (cp2tform of type ‘projective’, MATLAB 2012a) according to the transformation matrix obtained from microbead calibration measurement. Particles colocalized within a distance of 150 nm were selected. Only co-localized particles, which could be tracked for at least 10 consecutive frames (that is, molecules co-locomoting for at least 0.32 s) were accepted as receptor heterodimers or hetero-oligomers, which has been previously found to be a robust criterion for protein dimerization13. The fraction of heterodimerization or hetero-oligomerization was determined as the number of co-locomotion trajectories with respect to the number of the receptor trajectories. Since the receptor expression level of FZD8 or LRP6 was variable in the transiently transfected cells, only cells with similar receptor expression levels were considered (less than three times the excess of one subunit over the other). The smaller number of trajectories of either FZD8 or LRP6 was regarded as the limiting factor and therefore taken as a reference for calculating the heterodimerized/hetero-oligomerized fraction. Oligomerization values were not corrected for the degree of labelling. Single-molecule trajectories were reconstructed using the multi-target tracing (MTT) algorithm56. The detected trajectories were evaluated with respect to their step length distribution to determine the diffusion coefficients. For a reliable quantification of local mobilities, we estimated diffusion constants from the displacements with three frames (96 ms). Step-length histograms were obtained from all single molecule trajectories and fitted by a two-component model of Brownian diffusion, thus taking into account the intrinsic heterogeneity of protein diffusion in the plasma membrane57, 58. A bimodal probability density function p(r) was used for a nonlinear least square fit of the step-length histogram: where is the percentage of the fraction, contains the diffusion coefficient of each fraction (nδt = 96 ms). Average diffusion coefficients were determined by weighting the diffusion coefficients with the corresponding fractions. Single-molecule intensity distribution of individual diffraction-limited spots was extracted from the first 50 images of the recorded time lapse image sequence, in which photobleaching of dyes was kept below 10%13. Oligomerization of receptors was evaluated by fitting the obtained single molecule intensity with a multi-component Gaussian distribution function59. To ensure a reliable analysis, monomeric receptors were first distinguished based on the observation that monomers diffused much faster than oligomers. Therefore, the characteristic intensity distribution of monomeric receptor subunits was obtained by tracking of the fast mobile fraction. Fractions of the monomer, dimer, trimer and higher oligomers were then de-convoluted from the single molecule intensity distribution, presuming that intensities of clusters were multiples of the monomer intensity distribution. Immortal cells were seeded in triplicate for each condition in 96-well plates, and stimulated with surrogates, XWnt8, WNT3A conditioned media, control proteins, or other treatments for 20–24 h. After washing cells with PBS, cells in each well were lysed in 30 μl passive lysis buffer (Promega). 10 μl per well of lysate was assayed using the Dual Luciferase Assay kit (Promega) and normalized to the Renilla luciferase signal driven constitutively by the human elongation factor-1 alpha promoter to account for cell variability. A375 BAR, SH-SY5Y BAR, L STF and HEK293 STF cells were plated at a density of 10,000–20,000 cells per well, and treatment was started after 24 h in fresh medium. A549 BAR cells were plated at a density of 5,000 cells per well in the presence of 2 μM IWP-2 (Calbiochem) to suppress endogenous Wnt secretion, and treatment was started after 48 h in fresh medium containing fresh IWP-2. To induce β-catenin accumulation, SH-SY5Y BAR cells were treated for 2 h with scFv–DKK1c, WNT3A conditioned media (positive control), B12 (negative control protein) and mock conditioned media (from untransfected L cells, negative control) at 37 °C, 5% CO . After, cells were washed twice with PBS. For β-catenin stabilization assay, cells were scraped into hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 0.2 mM MgCl , supplemented with protease inhibitors), incubated on ice for 10 min, and homogenized using a hypodermic needle. Sucrose and EDTA were added to final concentration of 0.25 M and 1 mM, respectively. For LRP6 phosphorylation assay, cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.5% sodium deoxylate, 1% Triton X-100), supplemented with protease inhibitor and phosphatase inhibitor for 1 h at 4 °C. Lysates were centrifuged at 12,000g for 1 h at 4 °C. Supernatants were then diluted into SDS sample buffer. For immunoblotting, samples were resolved on a 12% Mini-PROTEAN(R)TGX precast protein gel (Bio-Rad) and transferred to a PVDF membrane. The membranes were cut horizontally approx. at the 64 kDa mark of the SeeBlue plus 2 molecular mass marker (Invitrogen). Top half of the blot was incubated with anti-β-catenin primary antibody ((D10A8)XP, rabbit, Cell Signaling 8480), LRP6 antibody ((C47E12), rabbit, Cell Signaling 3395), and P-LRP6 (S1490) antibody (rabbit, Cell Signaling 2568), and the bottom part with the anti-α-tubulin primary antibody (mouse, DM1A, Sigma) in PBS containing 0.1% Tween-20 and 5% BSA overnight at 4 °C. Blots were then washed, incubated with the corresponding secondary antibodies in the same buffer, before washing and developing using the ECL prime western blotting detection reagent (GE Healthcare). To induce β-catenin accumulation, K562 and cells were stimulated for 0, 15, 30, 45, 60, 90 and 120 min with 10 nM scFv–DKK1c, recombinant Wnt3a (R&D Systems), B12 (negative control protein) or plain complete growth medium at 37 °C, 5% CO . After, cells were washed twice with PBS, fixed with 4% PFA for 10 min at room temperature, and permeabilized in 100% methanol for at least 30 min at −80 °C. The cells were than stained with Alexa-647 conjugated anti-β-catenin antibody (L54E2) (Cell Signaling Technology, 1:100–1: 50 dilution). Fluorescence was analysed on an Accuri C6 flow cytometer. Total RNA was isolated using either TRIZOL (Invitrogen) or RNeasy plus micro kit (QIAGEN) according to manufacturer’s protocols. A total of 2 μg RNA were used to generate cDNA using the RevertAid RT kit (Life Technologies) using oligo(dT)18 mRNA primers (Life Technologies) according to manufacturer’s protocol. 12 ng of cDNA per reaction were used. qPCR was performed using SYBR Green-based detection (Applied Biosystems) according to the manufacturer’s protocol on a StepOnePlus real-time PCR system (ThermoFisher Scientific). All primers were published, or validated by us. Transcript copy numbers were normalized to GAPDH for each sample, and fold induction compared to control was calculated. The following gene-specific validated primers were used: human FZD1: F: 5′-ATCCTGTGTGCTCCTCTTTTGG-3′, R: 5′-GATTGCTTTTCTCCTCTTCTTCAC-3′; human FZD2: F: 5′-CTGGGCGAGCGTGATTGT-3′, R: 5′-GTGGTGACAGTGAAGAAGGTGGAAG-3′; human FZD3: F: 5′-TCTGTATTTTGGGTTGGAAGCA-3′, R: 5′-CGGCTCTCATTCACTATCTCTTT-3′; human FZD4: F: 5′-TGGGCACTTTTTCGGTATTC-3′, R: 5′-TGCCCACCAACAAAGACATA-3′; human FZD5: F: 5′-CCATGATTCTTTAAGGTGAGCTG-3′, R: 5′-ACTTATTCAAGACACAACGATGG-3′; human FZD6: F: 5′-CGATAGCACAGCCTGCAATA-3′, R: 5′-ACGGTGCAAGCCTTATTTTG-3′; human FZD7: F: 5-TACCATAGTGAACGAAGAGGA-3′, R: 5′-TGTCAAAGGTGGGATAAAGG-3′; human FZD8: F: 5′-ACCCAGCCCCTTTTCCTCCATT-3′, R: 5′-GTCCACCCTCCTCAGCCAAC-3′; human FZD9: F: 5′-GCTGTGACTGGAATAAACCCC, R: 5′-GCTCTGCTTACAAGAAAGACTCC-3′; human FZD10: F: 5′-CTCTTCTCTGTGCTGTACACC, R: 5′-GTCTTGGAGGTCCAAATCCA-3′; mouse Fzd1: F: 5′-GCGACGTACTGAGCGGAGTG, R: 5′-TGATGGTGCGGATGCGGAAG-3′60; mouse Fzd2: F: 5′-CTCAAGGTGCCGTCCTATCTCAG, R: GCAGCACAACACCGACCATG-3′60; mouse Fzd3: F: 5′-GGTGTCCCGTGGCCTGAAG-3′, R: 5′-ACGTGCAGAAAGGAATAGCCAAG-3′60; mouse Fzd4: F: 5′-GACAACTTTCACGCCGCTCATC-3′, R: 5′-CAGGCAAACCCAAATTCTCTCAG-3′60; mouse Fzd5: F: 5′-AAGCTGCCTTCGGATGACTA-3′, R: 5′-TGCACAAGTTGCTGAACTCC-3′60; mouse Fzd6: F: 5′-TGTTGGTATCTCTGCGGTCTTCTG-3′, R: 5′-CTCGGCGGCTCTCACTGATG-3′60; mouse Fzd7: F: 5′-ATATCGCCTACAACCAGACCATCC-3′, R: 5′-AAGGAACGGCACGGAGGAATG-3′60; mouse Fzd8: F: 5′-GTTCAGTCATCAAGCAGCAAGGAG-3′, R: 5′-AAGGCAGGCGACAACGACG-3′60; mouse Fzd9: F: 5′-ATGAAGACGGGAGGCACCAATAC-3′, R: 5′-TAGCAGACAATGACGCAGGTGG-3′60; mouse Fzd10: F: 5′-ATCGGCACTTCCTTCATCCTGTC-3′, R: 5′-TCTTCCAGTAGTCCATGTTGAG-3′60; human AXIN2: F: 5′-CTCCCCACCTTGAATGAAGA-3′, R: 5′-TGGCTGGTGCAAAGACATAG-3′; human GAPDH: F: 5′-TGAAGGTCGGAGTCAACGGA-3′, R: 5′-CCATTGATGACAAGCTTCCCG-3′; mouse Gapdh: F: 5′-CCCCAATGTGTCCGTCGTG-3′, R: 5′-GCCTGCTTCACCACCTTCT-3′. Differentiation of C3H10T1/2, and human and mouse primary MSCs were performed essentially as described previously61. In brief, approximately 10,000 cells cm−2 were plated in normal culture medium (αMEM + FBS + penicillin/streptomycin), and allowed to adhere overnight. The following day, the medium was replaced with osteogenic medium (αMEM, 10% FBS, 1% penicillin/streptomycin, 50 μg ml−1 ascorbic acid, 10 mM β-glycerol phosphate (βGP), and replaced every other day. To determine alkaline phosphatase enzymatic activity, cells were fixed for 10 min with 10% formalin in PH7 PBS, before incubation in NBT-BCIP solution (1-Step(tm) NBT/BCIP Substrate Solution (Thermo Fisher Scientific, 34042) for 30 min. qPCR reactions were done with the SYBR method using the following primers: human ACTB F: 5′-GTTGTCGACGACGAGCG-3′, R: 5′-GCACAGAGCCTCGCCTT-3′; human ALPL: F: 5′-GATGTGGAGTATGAGAGTGACG-3′, R: 5′-GGTCAAGGGTCAGGAGTTC-3′; mouse Alpl: F: 5′-AAGGCTTCTTCTTGCTGGTG-3′, R: 5′-GCCTTACCCTCATGATGTCC-3′; mouse Actb: F: 5′-GGAATGGGTCAGAAGGACTC-3′, R: 5′-CATGTCGTCCCAGTTGGTAA-3′; mouse Col2a1 F: 5′-GTGGACGCTCAGGAGAAACA-3′, R: 5′-TGACATGTCGATGCCAGGAC-3′. P26N, normal adult human colon organoids, were established from a tumour-free colon segment of a patient diagnosed with CRC as described18, 62, 63. CFTR-derived colorectal organoids were obtained from a patient at Wilhelmina Children’s Hospital WKZ-UMCU. Informed consent for the generation and use of these organoids for experimentation was approved by the ethical committee at University Medical Center Utrecht (UMCU) (TcBio 14-008). Human stomach organoids, derived from normal corpus and pylorus, were from patients that underwent partial or total gastrectomy at the University Medical Centre Utrecht (UMCU) and were established as described19, 64, 65. Pancreas organoids were obtained from the healthy part of the pancreas of patients undergoing surgical resection of a tumour at the University Medical Centre Utrecht Hospital (UMC) and were established as described66, 67. The liver organoids were derived from freshly isolated normal liver tissue from a patient with metastatic CRC who presented at the UMC hospital (ethical approval code TCBio 14-007) and were established as described20, 68. For the performance of 3D cultures, Matrigel (BD Biosciences) was used and overlaid with a liquid medium consisting of DMEM/F12 advanced medium (Invitrogen), supplemented with additional factors as outlined below. 2% RSPO3-CM (produced via the r-PEX protein expression platform at U-Protein Express BV), WNT3A conditioned medium (50%, produced using stably transfected L cells in the presence of DMEM/F12 advanced medium supplemented with 10% FBS), and Wnt and Wnt/RSPO2 surrogates at different concentrations were added as indicated. Single-cell suspensions of normal human organoids were cultured in duplicate or triplicate in round-bottom 96-well plates to perform a cell viability test using Cell Titer-Glo 3D (Promega). In brief, organoids were trypsinized to single-cell suspension and plated in 100 μl medium in the presence of the different reagents. 3 μM IWP-2 was added to inhibit endogenous Wnt lipidation and secretion. After 12 days, 100 μl of Cell Titer-Glo 3D was added, plates were shaken for 5 min, incubated for an additional 25 min and centrifuged before luminescence measurement. All animal experiments were conducted in accordance with procedures approved by the IACUC at Stanford University. Experiments were not randomized, the investigators were not blinded, and all samples/data were included in the analysis. Group sample sizes were chosen based on (1) previous experiments, (2) performance of statistics analysis, and (3) logistical reasons with respect to full study size, to accommodate all groups. Adenoviruses (E1 and E3 deleted, replication deficient) were constructed to express scFv–DKK1c or scFv–DKK1c–RSPO2 with an N-terminal signal peptide and C-terminal 6×His-tag (Ad-scFv–DKK1c or Ad-scFv–DKK1c–RSPO2), respectively. Adenoviruses expressing mouse IgG2α Fc (Ad-Fc), human RSPO2–Fc fusion protein (Ad-RSPO2–Fc) and mouse WNT3A (Ad-Wnt3a) were constructed and described in the companion paper by Yan et al.26 The adenoviruses were cloned, purified by CsCl gradient, and titred as previously described69. Adult C57Bl/6J mice were purchased from Taconic Biosciences. Adult C57Bl/6J mice between 8–10 weeks old were injected intravenously with a single dose of adenovirus at between 1.2 × 107 p.f.u. to 6 × 108 p.f.u. per mouse in 0.1 ml PBS. Serum expression of Ad-scFv–DKK1c or Ad-scFv–DKK1c–RSPO2 were confirmed by immunoblotting using mouse anti-6×His (Abcam ab18184, 1:2,000) or rabbit anti-6×His (Abcam ab9108, 1:1,000), respectively. All experiments used n = 4 mice per group and repeated at least twice. qRT–PCR on liver samples were performed as following. Total cDNA was prepared from each liver sample using Direct-Zol RNA miniprep kit (Zymo Research) and iScript Reverse Transcription Supermix for RT-qPCR (BIO-RAD). Gene expression was analysed by -ΔΔC or fold change (2−ΔΔCt). Unpaired Student’s t-test (two tailed) was used to analyse statistical significance. Primers for mouse Axin2 and Cyp2f2 were previously published70. Additional primers used were listed as below: For the parabiosis experiment, age- and gender-matched C57Bl/6J mice were housed together for at least 2 weeks before surgery. At 2 days before surgery, the ‘donor’ mice were injected intravenously with a single dose of adenovirus at between 1.2 × 107 pfu to 6 × 108 pfu per mouse in 0.1 ml PBS and were separated from the ‘recipient’ mice until surgery. The parabiosis surgery was performed as described previously71. The establishment of shared circulation was confirmed at day 5 after surgery by presence of adenovirus-expressed proteins in the serum of both donors and recipients. Mouse livers were collected and fixed in 4% paraformaldehyde. 5 μm paraffin-embedded sections were stained with the following antibodies after citrate antigen retrieval and blocking with 10% normal goat serum: mouse anti-glutamine synthetase antibody (Millipore MAB302, 1:200), mouse anti-PCNA (BioLegend 307902, 1:200), and rabbit anti-HNF4α (Cell Signaling 3113S, 1:500). The immunostained tissue sections were analysed and images were captured on a Zeiss Axio-Imager Z1 with ApoTome attachment. Atomic structure factors and coordinates have been deposited to the Protein Data Bank (PDB) under accession numbers 5UN5 and 5UN6. All other data are available from the corresponding author upon reasonable request.


HOUSTON, May 10, 2017 /PRNewswire/ -- Gastar Exploration Inc. (NYSE MKT: GST) ("Gastar") announced today that it has declared monthly cash dividends on its 8.625% Series A Preferred Stock ("Series A Preferred Stock") and its 10.75% Series B Preferred Stock ("Series B Preferred Stock") for...


News Article | May 10, 2017
Site: www.prnewswire.com

HOUSTON, May 10, 2017 /PRNewswire/ -- Gastar Exploration Inc. (NYSE MKT: GST) ("Gastar" or the "Company") today reported financial and operating results for the three months ended March 31, 2017.  First quarter 2017 highlights include: Average daily production 5,700 Boe per...


News Article | May 9, 2017
Site: www.prnewswire.com

"We're pleased to serve the residents of Sydney, Melbourne, their surrounding areas, plus their visitors," says Soothe Founder and CEO Merlin Kauffman. "Everyone deserves to have a massage after a long day at the office or while on vacation, either as a gift to oneself or to another." Soothe offers deep tissue, Swedish, sports, and prenatal massage for singles and couples, and operates daily between 8 a.m. and midnight. Massages are fixed-price per person. The prices in Australian dollars, which exclude GST, are: $119 (60 minutes), $169 (90 minutes), and $229 (120 minutes). "Our rapid growth is really due to the high-quality treatments our massage therapists deliver, combined with flexible work opportunities and higher wages that Soothe provides for massage therapists," adds Kauffman. "Our therapists earn two to three times more than the industry standard." Kauffman created the company in 2013 while at Harvard Business School, a period when he was frequently travelling, and found it difficult to schedule a massage appointment outside standard business hours. What started as a solution to his own problem, however, has grown into a flourishing business. Clients can book a Soothe massage by downloading the app (soothe.com/app), choosing the type of massage, treatment length, and therapist's gender. They can also book via soothe.com, or call 1800.838.276 from within Australia. All booking options are available 24/7. Soothe clients can even book the same therapist on future occasions. Soothe's global network includes over 10,000 certified and registered massage therapists, each interviewed in-person. Only therapists who meet the requisite level of certification and have a history of customer excellence are accepted into the Soothe network. Soothe, the on-demand massage service and app, lets users schedule a same-day massage in the privacy of their home or hotel. For more information, visit soothe.com, call 1800.838.276 from within Australia, 800.960.7668 from within the US and Canada, or 808 178 0086 from within the UK.  For a full list of locations, visit http://soothe.com/locations/. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/leading-on-demand-massage-provider-soothe-arrives-in-australia-300452093.html


T-TEN was launched in 1986 to provide a strong pipeline of talented, career-minded, highly trained service technicians for Toyota and Lexus dealerships. The program typically offers a class size of 20 to 25 students per year and leverages Toyota's industry-leading training with both a classroom and hands-on laboratory setting over a period of 24 months. In order to enter the program, students must demonstrate a passion for the career and an aptitude to learn the skills necessary to perform as a service bay technician.  The training also includes a paid internship experience for students at a local dealership as they pursue certifications for Toyota and Lexus vehicles. T-TEN has placed thousands of certified technicians in the service departments of nearly 1,500 dealerships around the country. "T-TEN was conceived more than 30 years ago to ensure our dealers have a source of highly-trained, entry-level technicians and to support America's Career and Technical education system," said Chris Nielsen, executive vice president of product support, and chief quality officer of Toyota Motor North America. "Over 11,000 graduates have started their career through T-TEN, and, today, TCC joins a community of 37 schools providing industry-leading and occupationally-aligned automotive training." The event held on Monday to announce the new program was attended by executives from TMNA, GST, TCC, and other state and local dignitaries, including U.S. Representative Marc Veasey. At the event, it was announced that Toyota has already provided 12 training vehicles and plans to provide an additional 12 vehicles over the next 18 months for the program, allowing students access to modern vehicle technology. The TCC program is the fifth school in the region and its graduates are destined to be placed where GST and Lexus Southern dealers are located in a five state region that includes Texas, Arkansas, Louisiana, Mississippi, and Oklahoma. "Our mission, in partnership with Toyota dealers, is to support programs that create tomorrow's Toyota technician, acknowledging that a skills gap truly exists and it's a challenge we have to overcome," said Jeff Parent, the president of Gulf States Toyota. "Partnering with TCC to bring this curriculum to Fort Worth is one way to overcome that challenge, and we want to help interested, qualified students pursue a career in the technical field by providing them with the resources and skillset that Toyota believes are essential for success." Toyota (NYSE: TM) has been a part of the cultural fabric in the U.S. and North America for 60 years, and is committed to advancing sustainable, next-generation mobility through our Toyota and Lexus brands. During that time, Toyota has created a tremendous value chain as our teams have contributed to world-class design, engineering, and assembly of more than 33 million cars and trucks in North America, where we operate 14 manufacturing plants (10 in the U.S.) and directly employ more than 46,000 people (more than 36,000 in the U.S.).  Our 1,800 North American dealerships (nearly 1,500 in the U.S.) sold almost 2.7 million cars and trucks (2.45 million in the U.S.) in 2016 – and about 85 percent of all Toyota vehicles sold over the past 15 years are still on the road today. Toyota partners with community, civic, academic, and governmental organizations to address our society's most pressing mobility challenges. We share company resources and extensive know-how to support non-profits to help expand their ability to assist more people move more places. For more information about Toyota, visit www.toyotanewsroom.com. Tarrant County College is a public two-year college with campuses in Fort Worth and surrounding communities. TCC is one of the 20 largest higher education institutions in the United States based on annual enrollment, with more than 100,000 students in academic, career training and noncredit Community & lndustry Education programs. The College offers both on-campus and online learning, through six campuses throughout Tarrant County. Visit tccd.edu for more information. To view the original version on PR Newswire, visit:http://www.prnewswire.com/news-releases/toyota-partners-with-tarrant-county-college-to-launch-industry-leading-t-ten-training-program-300453512.html


News Article | May 8, 2017
Site: www.businesswire.com

DUBLIN--(BUSINESS WIRE)--Research and Markets has announced the addition of the "Overview of the Goods and Services Tax (GST) in India" report to their offering. The Government of India is all set to introduce the Goods and Services Tax (GST) in the country to create a uniform indirect tax levy. This deliverable provides an overview of GST, it's benefits, the timeline for its implementation and how it will affect some of the key sectors in the economy. For more information about this report visit http://www.researchandmarkets.com/research/q8k7hk/overview_of_the


Patent
Gst Co. and Hwang | Date: 2017-09-27

The present invention is directed to a hydrolysis apparatus for organic waste using a hydraulic crushing effect, and more particularly to a hydrolysis apparatus for organic waste using a hydraulic crushing effect that includes: a housing having an open top surface and an inner lodging space; a reaction tank being lodged in the inner lodging space of the housing and having an open top surface and an inner space for accommodating a defined liquid chemical; a heating section connected to a bottom surface of the reaction tank and integrated into the reaction tank; and a driving means for moving the housing to put the housing in a linear reciprocating motion. The present invention has an effect to greatly reduce the physical crushing time and the dissolution time using a liquid chemical in the hydrolysis of animal carcass and other organic wastes, such as food waste, sewage/waste water sludge, animal/plant residue, and so forth, thereby increasing the work efficiency and reducing the cost.


A system, apparatus, device, tools, kit and method is provided for the preparation of the jawbone and insertion of dental implants. The apparatus includes a universal and reusable clamping device and removable components for the clamp which allow for precision surgical preparation and implantation of dental implants into the jawbone. According to some embodiments, an apparatus is provided that includes a platform; one or more frames connected to the platform, wherein the frames include a clamp arm that extend to opposing sides of the bone; and one or more fixation cleats on each arm; wherein the apparatus includes one or more features for positioning the platform, for changing a position of the platform, for changing an angle of a component of the platform or any combination thereof, following the securing of the clamp arms to the bone and prior to the procedure.


A system, apparatus, device, tools, kit and method is provided for the preparation of the jawbone and insertion of dental implants. The apparatus (1) includes a universal and reusable compact clamping device and removable components for the damp which allow for precision surgical preparation and implantation of dental implants into the jawbone. According to some embodiments, an apparatus is provided that includes a swivel platform (3) having a guide member for receiving a tool for preparing an osteotomy and/or for implanting a dental implant into a jaw bone; an orientation member for positioning of the platform and one or more frames (2,12) connectable to the platform.

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