News Article | May 10, 2017
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 8, 2017
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
News Article | May 8, 2017
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 10, 2017
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.
News Article | May 26, 2017
— Since 2011, Americans have been planning their estates in accordance with the Economic Growth and Tax Relief Reconciliation Act of 2011, otherwise referred to as, "The Act." The Act succeeded at changing 441 tax laws, making it the first cut in over 25 years since the 1996 Act. The new Act also marks the biggest estate tax reduction in 20 years. Even states without taxes, such as Wyoming, face dramatic changes to their landscape. While the short-term outlook indicates that the estate tax is not slated to go away permanently, there already are, and will be some additional, estate tax relief plans brought forth with this Act over the next decade. There are estate planning techniques which can help you reduce this burden. A more dramatic step is to move your tax domicile to a state with no inheritance taxes. For example, Wyoming does not tax estates. Compare this to New York or California which each take a piece of your hard earned life’s savings. The Act works in favor of individuals planning their estates and their survivors. It lowers the tax rate on a multitude of estate taxes, including: 1) the marginal estate tax 2) the generation-skipping transfer tax (GST) 3) the gift tax. The gift tax alone has seen a major change since 2002. Through 2009, individuals received a steady increase in the amount of gift taxes they were allowed to give from $1 million to $3.5 million. The Act also increases the amount of assets that can be transferred at death and eliminates generation skipping and estate taxes on a portion of assets from years 2012-2019. As of December 31, 2009, the generation skipping tax was repealed permanently so that grandparents can gift portions of their assets directly to their grandchildren and great grandchildren without having to lose a portion of the assets to taxes. Solely for the year 2010, the estate tax will was entirely repealed for the duration of one year. If you had the foresight to die in the year 2010, you were allowed to give your entire estate to your heirs without having to worry about losing a portion of it to taxes. However, if your benefactor passed from 2011 onwards, only $1 million was/is available to be passed on to your heirs without taxes. Even though the estate tax is not slated to be permanently repealed within the foreseeable future, it is important that you plan your estate so that your will can be carried out accordingly once you have passed on or become incapacitated. Understanding the complicated tax system can be a challenge for someone not versed in tax law. For those individuals planning their estates, we recommend enrolling in an estate-planning course or meeting with an attorney who can walk you through the steps needed to ensure your heirs receive as much of your assets that you desire. There are a few options for reducing your overall estate tax bill. The most common is gifting away assets during your life. The second option is to set up a trust. A land trust is a great way to give back to the community, while reaping the benefits of a charitable deduction. Those looking to provide assets for family members in need should explore Special Needs Trusts in WY. These are set up to provide for individuals needing permanent medical assistance, and are designed to avoid disqualifying them from government assistance programs. For more information, please visit https://wyomingestateplanners.com/
News Article | May 25, 2017
VANCOUVER, BC / ACCESSWIRE / May 25, 2017 / GGX Gold Corp. (TSXV: GGX) (the "Company" or "GGX") is pleased to announce GGX Gold Launches AGORACOM Online Marketing and Awareness Program. The Company will receive significant exposure through millions of content brand insertions on the AGORACOM network and extensive search engine marketing over the next 12 months. In addition, exclusive sponsorships of invaluable digital properties such as AGORACOM TV, the AGORACOM home page and the AGORACOM Twitter account will serve to significantly raise the brand awareness of the Company among small cap investors. Barry Brown, Director stated, "GGX Gold has commenced building out its Market awareness strategy which will have several components initiating over the next month. AGORACOM has proven to be a leader in the online marketing space. We are delighted to have retained their services to expand our online presence..." AGORACOM is the pioneer of online investor relations, online conferences and online branding services to North American small and mid-cap public companies, with more than 250 companies served. More than just lip service, AGORACOM achieved two very big milestones on January 22, 2017, surpassing 50 Million Visits from 7 Million investors on AGORACOM. The company also expect to surpass 500 Million page views later this year. AGORACOM traffic ranks within the top 0.5% of all websites around the world. These traffic results are independently tracked and verified by Google analytics. AGORACOM traffic can be attributed to its strategy of maintaining the cleanest, moderated small-cap discussion as a result of implementing the first ever Investor Controlled Stock Discussion Forums. AGORACOM Founder, George Tsiolis, publishes the leading blog on small to mid cap investor relations. His 50 Small-Cap CEO Lessons are a must read for CEO's looking to increase their education and knowledge about online investor relations. GGX GOLD CORP. (TSX-V: GGX) is a gold exploration company primarily focused in southern BC: The Gold Drop mine in Greenwood has permits in place for drilling and trenching. GGX's current strategy is to prove up existing reserves and begin small scale production on the Gold Drop mine. The Company intends to issue shares for services to AGORACOM in exchange for the online advertising, marketing and branding services ("Advertising Services"). Pursuant to the terms of the agreement, the company will be issuing; TERM: June 1, 2017 - June 1, 2018 FEES: $CDN 40,000 + GST to be paid via Shares for Services over the term The number of shares to be issued at the end of each period will be determined by using the closing price of the Shares of the Company on the TSX Venture Exchange on the first trading day following each period for which the Advertising Services were provided by AGORACOM. The agreement/arrangement is subject to TSX-V approval. The term of the Agreement is for 12 months effective June 1, 2017. The Company will issue a press release after the issuance of shares under the terms of the agreement. On Behalf of the Board of Directors This news release includes certain statements that constitute "forward-looking information" within the meaning of applicable securities law, including without limitation, the Company's information and statements regarding or inferring the future business, operations, financial performance, prospects, and other plans, intentions, expectations, estimates, and beliefs of the Company. Such statements include statements regarding the completion of the proposed transactions. Forward-looking statements address future events and conditions and are necessarily based upon a number of estimates and assumptions. These statements relate to analyses and other information that are based on forecasts of future results, estimates of amounts not yet determinable and assumptions of management. Any statements that express or involve discussions with respect to predictions, expectations, beliefs, plans, projections, objectives, assumptions or future events or performance (often, but not always, using words or phrases such as "expects" or "does not expect", "is expected", "anticipates" or "does not anticipate", "plans", "estimates" or "intends", or stating that certain actions, events or results "may", "could", "would", "might" or "will" be taken, occur or be achieved), and variations of such words, and similar expressions are not statements of historical fact and may be forward-looking statements. Forward-looking statement are necessarily based upon several factors that, if untrue, could cause the actual results, performances or achievements of the Company to be materially different from future results, performances or achievements express or implied by such statements. Such statements and information are based on numerous assumptions regarding present and future business strategies and the environment in which the Company will operate in the future, including the price of gold and other metals, anticipated costs and the ability to achieve goals, and the Company will be able to obtain required licenses and permits. While such estimates and assumptions are considered reasonable by the management of the Company, they are inherently subject to significant business, economic, competitive and regulatory uncertainties and risks including that resource exploration and development is a speculative business; that environmental laws and regulations may become more onerous; that the Company may not be able to raise additional funds when necessary; fluctuating prices of metals; the possibility that future exploration, development or mining results will not be consistent with the Company's expectations; operating hazards and risks; and competition. There can be no assurance that economic resources will be discovered or developed at the Gold Drop Property. Accordingly, actual results may differ materially from those currently anticipated in such statements. Factors that could cause actual results to differ materially from those in forward looking statements include continued availability of capital and financing and general economic, market or business conditions, the loss of key directors, employees, advisors or consultants, equipment failures, litigation, competition, fees charged by service providers and failure of counterparties to perform their contractual obligations. Investors are cautioned that forward-looking statements are not guarantees of future performance or events and, accordingly are cautioned not to put undue reliance on forward-looking statements due to the inherent uncertainty of such statements. The forward-looking statements included in this news release are made as of the date hereof and the Company disclaims any intention or obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except as expressly required by applicable securities legislation. Neither TSX Venture Exchange nor its Regulation Services Provider (as that term is defined in the policies of the TSX Venture Exchange) accepts responsibility for the adequacy or accuracy of this release.
News Article | May 23, 2017
Speaking at a conference in Delhi, the food processing minister said said her finance counterpart had agreed to keep foods in the lowest rate band: “I am happy that the GST rate [on food items] has been kept within the 5% range. Lots of things will be cheaper. Food items are definitely not going to be expensive.” Four bands have been set, from 5% to 28%, while some staples have been exempted entirely, including milk, cereals and meat. Processed food items will attract a 5% tax under GST—a drop from the current 15% rate. Also under this band, albeit from a current rate of 3-4%, tea, coffee and masala are expected to become more expensive. Restaurant bills will be cheaper, with their levy dropping from a current 22% to an 18% band. At the other end of the scale, sweetened beverages will attract the 28% GST bracket, along with an additional 12% sin tax. This has prompted manufacturers to plan price increases of 5-10% at a time when some international players are operating at under 40% capacity. Having faced double-digit tax rises over the last three years to 32%, and more recently product boycotts in southern states, the likes of Coca-Cola and PepsiCo are also being troubled by competition from small regional brands that sell their drinks cheaper by 15-30%. According to Euromonitor, their combined share has fallen by three percentage points from 2014-16 to 55.7% cent in a market that has grown from INR446bn (US$6.9bn) crore to INR60.3bn, and continues to rise at a healthy 9.7% per year. A statement issued by Indian Beverage Association on behalf of the two drinks and snacks giants said the companies were “extremely disappointed”, and that the move would have a “negative ripple effect” on bottlers, retailers and distributors. “The effective tax rate of 40% on these products under the GST regime is against the stated policy of maintaining parity with the existing weighted average tax which is significantly below 40%,” the IBA said. The association also expressed its hope for the government to reconsider the levy and set a lower rate for non-sugar sweetened drinks, nutrition beverages and carbonated drinks that contain fruit juice. “[Otherwise] this increase in tax will further limit the growth of the beverage industry,” it added. Chocolate manufacturers will have to fall in line with the constraints of food safety and standards regulations after India’s health ministry placed all types of chocolate under the regulator’s purview. The regulations, which come into effect on January 1, 2018, set the percentage of cocoa and sugar allowed in various kinds of chocolates. They also lay down the minimum percentage of cocoa solids, milk fat, milk solids and acid insoluble ash in different varieties of chocolates. Dark chocolate will be required to contain no less than 35% cocoa solids, including not less than 18% cocoa butter and 14% fat-free cocoa solids. Pralines should be a single mouthful in size and have not less than 25% chocolate component in terms of weight. Edible salts, spices and condiments, and vitamins and minerals will be among permitted ancillary ingredients, as are emulsifying and stabilising agents, and sequestering and buffering agents. Manufacturers will be required to specify on labels the presence of vegetable fat in addition to cocoa butter. All varieties are required to have contain chocolate at at least 25% of total weight.
News Article | May 26, 2017
— At PhoneDoctor+ Mobile Repair in Singapore, the technicians provide customers with as much as three months warranty for parts and six months for labour. If other shops are unable to complete the repairs of a mobile phone, the technicians can get the job done. If there are issues with the work, the firm will either fix the problem quickly or issue a full refund. The company is a team of leading professionals who take pride in the quality of the work. The mobile phone repair Singapore team keeps repairs affordable to the masses as a key to their business model. Motherboard replacement is not required for every repair. A flat rate is standard and there is no GST. The firm has highly professional Apple and Samsung Certified motherboard technicians who can repair chip-level problems. The staff provides customers with reliable advice and accurate estimations of the time and cost taken to get the mobile device fixed. According to a spokesperson for the company, “We provide free in-you-face diagnostics and quote you a fair price for your repairs. We are best at cracked glass replacement for the latest model iPhone 6, iPhone 6S, iPhone 7, S5, S6, S7, Note 4, Note 5 Series. We use Gorilla Glass for our repairs. Come to us for help when the other shops are unable to fix the issues. We can fix what the majority of mobile shops cannot, due to our dedicated motherboard technicians and our ability to get any spare part that the market can offer.” He continues, “Diagnostics is FREE and iPhone 6S logic board issues are diagnosed within 30 minutes. Our turn-around-time is the fastest available. If you need an urgent repair, just tell us. Even a unique part can be ordered to arrive within seven days. We aim to be reliable and to provide cost-effective solutions. iPhone screen cracks can be repaired within thirty minutes. For more information, please visit https://phonedoctor.com.sg/
GST Ltd. | Date: 2014-03-14
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.