News Article | May 8, 2017
News Article | January 27, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Human HOIP and HOIL-1L cDNA were purchased from Open Biosystems (cloneIDs 4653017 and 3877587, respectively). HOIP RBR (residues 696–1,072), HOIP RING2L (residues 853–1,072) and full-length HOIL-1L were cloned into the pET-NKI-6xHis-3C-LIC vector30 coding for an N-terminal 6×His tag with a 3C protease cleavage site. HOIP UBA–RBR (residues 475–1,072) was cloned into a pET-NKI-6×His-eGFP-3C-LIC vector that codes for a 3C-cleavable His-tagged enhanced green fluorescent protein (eGFP) followed by the HOIP sequence. Human UbcH5B and Cdc34 DNA were a gift from M. Petroski. Coding sequences for UbcH13 and Uev1a were extracted out of a human cDNA library (Agilent Megaman). For crystallization, UbcH5B (residues 2–147) with the mutations S22R (to prevent backside ubiquitin binding31) and C85K (to enable covalent ubiquitin linkage21) was cloned into the pET-NKI-6×His-3C-LIC vector. UbcH5B without S22R and C85K mutations (used for enzymatic assays), Cdc34, UbcH13 and Uev1a were cloned into the same vector. Untagged mono-ubiquitin with native N and C termini, used for crystallization and linear ubiquitination assays, was cloned into the pET29 vector (Novagen) using NdeI/XhoI restriction sites. N-terminally blocked mono-ubiquitin used for thioester assays was cloned in the pET-NKI-6×His-3C-LIC vector. Untagged linear di-ubiquitin was cloned with overlap extension PCR and ligated into the pET29 vector (Novagen) using NdeI/XhoI restriction sites. N- and C-terminally blocked di-ubiquitin with a N-terminal His tag and a C-terminal Ala–Ser sequence was cloned into the pET-NKI-6×His-3C-LIC vector. Human ubiquitin-activating enzyme E1 (Ube1) was cloned into a pET28 vector resulting in an N-terminal His tag. For NF-κB assays full-length HOIP with an N-terminal Flag tag and HOIL-1L with an N-terminal myc tag were cloned into pcDNA3.1(+) (Invitrogen) using EcoRI/NotI restriction sites. Mutations in UbcH5B, ubiquitin and HOIP were introduced using standard site-directed mutagenesis techniques. All proteins were expressed in BL21(DE3) E. coli after induction with 0.5 mM IPTG overnight at 20 °C. For expression of HOIP and HOIL-1L constructs, 0.5 mM ZnCl was added to the cultures before induction. Bacteria were harvested by centrifugation, lysed by addition of lysozyme and sonication in the presence of protease inhibitors (PMSF and leupeptin) and DNase. Lysates were cleared by centrifugation and His-tagged proteins were initially purified using Ni-NTA agarose (Qiagen). For HOIP RBR used for crystallization, and UbcH5B, Cdc34, UbcH13, Uev1a, wild-type ubiquitin to generate K48-linked di-ubiquitin and HOIL-1L His tags were removed by addition of 3C protease overnight at 4 °C. HOIP RBR and HOIL-1L were further purified using Superdex 200 10/300 GL or HiLoad 16/600 Superdex 200 pg size-exclusion chromatography columns (GE Healthcare) equilibrated in protein buffer (10 mM HEPES pH 7.9, 100 mM NaCl). UbcH5B used for biochemical assays was further purified on a Superdex 75 10/300 GL size-exclusion chromatography column (GE Healthcare) equilibrated in protein buffer. HOIP mutants for activity assays, and Cdc34, UbcH13 and Uev1a were desalted into protein buffer directly after Ni-NTA purification using PD MidiTrap G-25 desalting columns (GE Healthcare). Ube1 for biochemical assays was further purified using ion-exchange chromatography (Source Q) in 10 mM HEPES pH 7.9, 10 mM NaCl and eluted with a gradient from 10–500 mM NaCl. N-terminally His-tagged (di-)ubiquitin was purified using Ni-NTA as described above followed by size-exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in protein buffer or buffer exchange into protein buffer using PD MidiTrap G-25 desalting columns. To purify untagged mono- or di-ubiquitin, 0.5 mM EDTA and 100 mM sodium acetate pH 4.5 were added to the bacterial lysates and lysates were cleared by centrifugation, diluted sevenfold with 50 mM sodium acetate pH 4.5 and applied to a Source S 10/100 ion exchange column (GE Healthcare) equilibrated in 50 mM sodium acetate pH 4.5. Ubiquitin was eluted with a 0–500 mM NaCl gradient and further purified by size-exclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in protein buffer. His-eGFP-HOIP was purified using size-exclusion chromatography as described for HOIP RBR, followed by 3C cleavage and removal of His-eGFP via a second round of size-exclusion chromatography. All proteins were generally flash frozen in liquid nitrogen in small aliquots and stored at −80 °C. UbcH5B~ubiquitin linkage was performed based on published methods21. Briefly, Ube1, UbcH5B(S22R/C85K) and ubiquitin were mixed and buffer exchanged into 50 mM Tris pH 10, 150 mM NaCl using PD-10 desalting columns (GE Healthcare). 10 mM MgCl , 5 mM ATP and 1 mM TCEP were added and the protein solution was incubated at 37 °C for 16 h. The completeness of the reaction was monitored using SDS–PAGE and covalently linked UbcH5B~ubiquitin was purified from unreacted proteins and Ube1 using a Superdex 75 10/300 GL size-exclusion chromatography column (GE Healthcare) equilibrated in protein buffer. HOIP RBR was mixed with a 1.3-fold molar excess of UbcH5B~ubiquitin and applied to a Superdex 200 10/300 GL size-exclusion chromatography column equilibrated in protein buffer. Complex formation and purity was confirmed using SDS–PAGE, and complex containing fractions were pooled and concentrated to ~12 mg ml−1 for crystallization. Crystallization was performed using the vapour diffusion technique in sitting drop MRC 96-well plates (Molecular Dimensions). Initial crystals were obtained mixing HOIP/UbcH5B~ubiquitin complex solution with an equimolar amount of free ubiquitin in the Morpheus Screen (Molecular Dimensions). Subsequently, 2 μl of the protein complex were mixed with 0.6 μl reservoir solution (0.1 M Morpheus Buffer 3 pH 8.5 (Tris/Bicine), 0.12 M Morpheus Alcohols Mix (0.02 M each of 1,6-hexanediol; 1-butanol; 1,2-propanediol (racemic); 2-propanol; 1,4-butanediol; 1,3-propanediol), 30% Morpheus P550MME_P20K mix (20% PEG550MME, 10% PEG20K) and 8% glycerol) in MRC 48-well plates (Molecular Dimensions). Crystals appeared after about one week at 12 °C and were cryo-cooled, and evaluated on a rotating anode X-ray generator (Rigaku FR-E superbright). Seeding and dehydration of the crystals was performed to improve crystal diffraction. For successful dehydration, reservoir was slowly added to the protein drop (3 × 0.5 μl within ~2 h) and subsequently equilibrated overnight at 12 °C against a reservoir solution with increased P550MME_P20K concentration by adding 11 μl 60% Morpheus P550MME_P20K stock solution to 50 μl reservoir solution. The new reservoir solution was then slowly added to the protein drop (3 × 0.5 μl, followed by 2 × 1 μl with removal of 1 μl each in the last steps). After further overnight equilibration, crystals were harvested from the drop and directly cryo-cooled in a cryogenic nitrogen stream at 100 K. Crystals diffracted in-house to 4–6 Å. Complete diffraction data were measured at 100 K at beamline 23ID-D of the General Medical Sciences and Cancer Institutes Structural Biology Facility at the Advanced Photon Source (GM/CA @ APS), Argonne National Laboratory. Despite their size (common dimensions of ~200 × 140 × 100 μm3) crystals exhibited substantial inhomogeneity resulting in split and smeared diffraction spots. Using raster scans32, a suitable region for data collection could be identified at the edge of the crystal. Using a small (20 μm diameter) beam, split spots could be separated to allow reliable indexing and integration. Utilization of a small beam necessitated higher flux to retain reliable diffraction. To mitigate the radiation damage, the total dose was distributed over a 100-μm stretch of the crystal by using the ‘helical’ mode of ‘vector’ data collection as implemented in JBluIce33. Data were measured at 1.282 Å wavelength with a Pilatus3 6M pixel array detector with a 1-mm-thick sensor (Dectris). Data were collected from a single crystal and indexed, integrated and scaled in XDS/XSCALE34. Data were further processed using AIMLESS35 from the CCP4 suite36 with a resolution cut-off of 3.48 Å, resulting in an and CC1/2 = 0.648 in the highest resolution shell. Phasing was carried out in Phaser37 using an MR-SAD protocol as implemented in PHENIX38. For this, independent molecular replacement searches were initially performed for the RING2L domain of HOIP (from PDB: 4LJP (ref. 14)), UbcH5B (from PDB: 3A33 (ref. 39)), and ubiquitin (from PDB: 4LJP (ref. 14)) with the four C-terminal residues deleted. Various ambiguous solutions were identified that could not be separated, and Zn2+ sites could not be identified using MR-SAD due to incompleteness of resultant models. However, manual inspection revealed that some MR solutions contained ubiquitin oriented near identically to the symmetry-related donor ubiquitin observed in the HOIP RING2L/ubiquitin-ubiquitin transfer complex (PDB: 4LJP (ref. 14)). Based on this observation, a trimmed search model was created that contained a complex of the core of HOIP RING2L (with residues 906–924 and 949–999 removed) and C-terminally truncated ubiquitin. An MR search using this model found a single solution for two copies of the complex. After successful iterative searches for two UbcH5B molecules and two further ubiquitin molecules, MR-SAD using Phaser identified 15 distinct Zn2+ sites including the known Zn2+ sites in the RING2L domain of HOIP. Further molecular replacement in Phaser using a single unit of the initial HOIP RING2L/UbcH5B~ubiquitin complex (without the additional second ubiquitin), and the NMR structure of HOIP IBR (zinc atoms removed, deposited in Protein Data Bank40 under PDB accession number 2CT7, unpublished) correctly placed a single HOIP IBR domain, which was then manually copied to the other NCS-related HOIP in the asymmetric unit. For molecular replacement of the HOIP RING1, Sculptor41 was used to generate a search model based on the structure of the RING1 domain of HHARI (PDB: 4KBL (ref. 11)). However, Phaser was not able to correctly place this domain, probably owing to the low sequence conservation of only 27% identity. However, since mutational analysis of HOIP suggested that the RING/E2 interaction is preserved between RING-type E3 ligases and RBR-type E3 ligases5, we overlaid the E2 of the published RNF4–RING/UbcH5A~ubiquitin structure (PDB: 4AP4 (ref. 21)) with the E2 in our structure and then used this overlay to add the RING1 model generated by Sculptor. This overlay placed the HOIP RING1 Zn2+-coordinating residues near the last remaining free Zn2+ ions found earlier by Phaser MR-SAD, indicating correct placement of the RING1 domain. In the final round of molecular replacement, the two additional ubiquitin (Ub ) molecules were reinstated at the RING1–IBR interface. At this stage, Refmac42 was used for refinement using settings optimized for low-resolution refinement43 including ‘jelly body refinement’ and Babinet scaling. ProSMART44 was used to generate external restraints against high-resolution structures (PDB: 4LJO (ref. 14) for HOIP RING2L and ubiquitin, and PDB: 2ESK (ref. 45) for UbcH5B). After this, clear extra electron density became visible for the unmodelled helical linker regions of the RING1–IBR and IBR–RING2L transitions and for other regions omitted in the initial search models. Further model building and refinement was manually performed in Coot46 and Refmac. During refinement additional clear positive difference map electron density became visible and Phaser was used to place one additional UbcH5B molecule (UbcH5B ) into this density. TLS restraints were generated using the TLSMD server47 and NCS restraints were used throughout refinement. One overall B-factor was refined in Refmac. In later rounds of refinement the PDB_REDO server48 was used for refinement optimization and MolProbity49 was used for structure validation. Data processing and refinement statistics are summarized in Extended Data Fig. 2b. Ramachandran statistics were calculated using MolProbity and 94.8% of all residues are in favoured regions, 4.9% in allowed regions and 0.3% are outliers. The final structure has a MolProbity score of 1.75 (100th percentile). In the final structure the two HOIP RBR molecules (see also Extended Data Fig. 3) are defined by electron density from residues 699 to 707, 711 to 948, 969 to 991, and 996 to 1,011 (chain A) and 699 to 754, 760 to 957, 967 to 1,015, 1,019 to 1,035 and 1,054 to 1,066 (chain B). The catalytic UbcH5B~ubiquitin conjugates are defined from UbcH5B residues 3 to 147 and ubiquitin residues 1 to 76 (chains C and E), and UbcH5B residues 2 to 147 and ubiquitin residues 1 to 76 (chains D and F). The allosteric ubiquitin chains (chains G and H) are defined from residues 1 to 76 and the additional UbcH5B (chain I) is defined from residues 2 to 146. PHENIX was used to calculate simulated annealing (SA) composite omit maps and feature enhanced maps (FEM). All molecular figures were prepared in PyMOL (Schrödinger, LLC). K48-linked and K63-linked ubiquitin chains were formed through a linkage-specific enzymatic reaction using Cdc34 and UbcH13/Uev1a E2 ubiquitin-conjugating enzymes, respectively, as described in the literature50. Ubiquitin chains were separated using ion-exchange chromatography as described above for purification of mono-ubiquitin. Purified K48-linked di-ubiquitin was directly desalted into protein buffer using PD-10 desalting columns, whereas K63-linked di-ubiquitin was further purified on a Superdex 75 10/300 GL size-exclusion chromatography column equilibrated in protein buffer. Native ubiquitin without additional residues was used to generate di-ubiquitin chains for ITC experiments, whereas N-terminally blocked ubiquitin was used to form K48-linked di-ubiquitin for testing allosteric activation of HOIP RBR. Linear ubiquitin formation assays were performed in 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM MgCl and 0.6 mM DTT using 200 nM E1, 1 μM UbcH5B, 1 μM HOIP RBR or HOIP RING2L and 40 μM untagged ubiquitin. Reactions were started by addition of 10 mM ATP and were incubated at 30 °C for 2 h. Samples were taken at the indicated time points and treated with 50 mM sodium acetate pH 4.5 as described previously6, mixed with SDS sample buffer and analysed by SDS–PAGE using 12% Bolt Bis-Tris gels (Life Technologies). Proteins were visualized with Coomassie Brilliant blue dye. To test the activating effect of linear di-ubiquitin on auto-inhibited HOIP UBA–RBR, 5 μM HOIP UBA–RBR was pre-incubated with N- and C-terminally blocked linear di-ubiquitin or HOIL-1L at the indicated concentrations before addition of the remaining assay components. Samples were taken after 60 min and subsequently treated as described above. To monitor HOIP~ubiquitin thioester ubiquitin transfer from UbcH5B to HOIP, Ube1 (100 nM), UbcH5B (4 μM) and N-terminally blocked ubiquitin (32 μM) were mixed in 50 mM HEPES pH 7.9, 100 mM NaCl, 10 mM MgCl and 5 mM ATP and incubated at 25 °C for 5 min when 2 μM HOIP RBR was added. Samples were taken 10 s after HOIP addition, quenched by addition of pre-heated SDS protein-loading buffer without DTT, and run on a 12% SDS–PAGE gel (Life Technologies). The 10-s time point used was empirically determined with a time-course experiment (Extended Data Fig. 9g). Gels were stained with Coomassie Brilliant blue dye and scanned on a Li-COR Odyssey scanner using the 700 nm (red) channel. For the thioester transfer assay shown in Fig. 3d, 200 nM Ube1, 2 μM UbcH5B, 8 μM HOIP RBR, 8 μM N-terminally blocked ubiquitin and 10 mM ATP were used and samples taken after 30 s. Furthermore, proteins were transferred to a PVDF membrane and ubiquitin was visualized on a LI-COR Odyssey scanner at 800 nm using an anti-ubiquitin antibody (P4D1, Santa Cruz, 1:200 dilution in TBST (50 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween-20)) followed by an IRDye 800CW secondary antibody (LI-COR, 1:10,000 dilution in TBST). All quantitative experiments shown in graphs were performed in triplicates and band intensities were quantified using the ImageStudio software (LI-COR). HOIP thioester transfer activity was calculated as the fraction of HOIP~ubiquitin to total HOIP for each mutant and normalized against thioester transfer activity of wild-type HOIP. Data were analysed in GraphPad Prism using two-tailed unpaired Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. To test the allosteric activation of HOIP RBR by linear di-ubiquitin, a modified ubiquitin transfer assay was performed. HOIP RBR was pre-incubated with N- and C-terminally blocked linear di-ubiquitin at the indicated final concentrations for 5 min at 25 °C. At the same time, Ube1, UbcH5B, ubiquitin and ATP were premixed and incubated for 5 min at 25 °C, resulting in fully loaded UbcH5B~ubiquitin. Both mixtures were subsequently mixed together, resulting in final concentrations of 100 nM Ube1, 2 μM UbcH5B, 8 μM N-terminally blocked ubiquitin and 2 μM HOIP RBR in the final buffer described for the standard ubiquitin transfer assay. Samples were taken after 30 s and further treated as described for the standard transfer assay. A 30-s time point was determined to give the best results in this assay, in which lower E2 and mono-ubiquitin concentrations were used, resulting in an overall slower reaction rate. The experiments comparing the effects of linear versus K48-linked di-ubiquitin (Extended Data Fig. 9e) were performed similarly, with the difference that all samples were incubated with apyrase (Sigma) for 5 min to deplete ATP before addition of HOIP/di-ubiquitin and prevent E2-loading of K48-linked di-ubiquitin, which features a free C terminus on one of the ubiquitin units. Sedimentation equilibrium experiments were performed in a ProteomeLab XL-I (Beckman Coulter) analytical ultracentrifuge. HOIP RBR/UbcH5B~ubiquitin as used for crystallization was loaded into a 6-channel equilibrium cell at 5.0, 2.5 and 1.25 μM concentration and centrifuged at 10,000 r.p.m., 20 °C in an An-50 Ti 8-place rotor until equilibrium was achieved. Data were analysed using HeteroAnalysis software (J. L. Cole and J. W. Lary, University of Connecticut; http://www.biotech.uconn.edu/auf/). ITC experiments were performed on an ITC200 calorimeter (Microcal). Aliquots (2 μl each) of 500–650 μM UbcH5B~ubiquitin or di-ubiquitin solution were injected into the cell containing 40–50 μM HOIP RBR or HOIP RBR/di-ubiquitin complexes. The experiments were performed at 23 °C in buffer containing 10 mM HEPES pH 7.9, 100 mM NaCl. For titrations of UbcH5B~ubiquitin into HOIP RBR/di-ubiquitin complexes, HOIP RBR was pre-incubated with an equimolar amount of di-ubiquitin before the ITC experiments. Data were analysed using the Origin software (Microcal). Human embryonic kidney (HEK) 293T cells (ATCC) were co-transfected with NF-κB-luc reporter plasmid that contains an NF-κB response element upstream of the promoter driving the luciferase reporter gene, pGL4.74[hRluc/TK] control vector (Promega) and epitope tagged Flag-HOIP or myc-HOIL-1L pcDNA3.1(+) plasmids in 6-well plates in triplicates using Lipofectamine 2000 transfection reagent. Since this assay could be carried out in a variety of cellular contexts, HEK293T cells were used because they are easy to transfect and suitable for the assay. The cells tested negative for mycoplasma contamination. Empty pcDNA3.1(+) vector was used as control. After 36 h, cells were lysed and 20 μl cell lysates were used to measure firefly luciferase and Renilla luciferase (transfection control) signals using the dual luciferase reporter assay system according to the manufacturer’s protocol (Promega). Data were analysed in GraphPad Prism and one-way ANOVA followed by Tukey’s post hoc tests were used for statistical analysis. Immunoblotting was performed with anti-Flag (clone M2, Sigma-Aldrich) and anti-myc (clone 9E10, Sigma-Aldrich) antibodies, to confirm equivalent wild-type and mutant protein expression levels.
News Article | November 4, 2016
DOWNERS GROVE, Ill., Nov. 04, 2016 (GLOBE NEWSWIRE) -- FTD Companies, Inc. (Nasdaq:FTD) (“FTD” or the “Company”), a premier floral and gifting company, today announced that Christopher W. Shean, one of the Company’s directors, has been appointed interim President and Chief Executive Officer, effective immediately. Mr. Shean succeeds Robert S. Apatoff, who has stepped down from these positions and from FTD’s board of directors. Mr. Apatoff will continue in a transitional advisory role to the Company through December 31, 2016. In conjunction with this leadership change, the Company has also created an Office of the Chief Executive Officer comprised of Becky A. Sheehan, Executive Vice President and Chief Financial Officer, Helen Quinn, Executive Vice President, U.S. Consumer Floral, and Scott D. Levin, Executive Vice President, General Counsel and Secretary, all of whom will report directly to Mr. Shean. The Board will conduct a formal executive search for the position of Chief Executive Officer. “FTD is poised for future success and we have tremendous confidence in the leadership team to execute on our strategic growth initiatives which we believe will enhance value for our stockholders,” said Robert Berglass, Chairman of FTD’s Board of Directors. “On behalf of our employees and the Board, we would like to thank Rob for his commitment to FTD and numerous contributions over the last eight years, particularly through the spin-off from United Online and the acquisition of Provide Commerce. We wish him well in his future endeavors.” Mr. Berglass continued, “Chris is a strong executive and proven leader with significant e-commerce industry experience. He has the Board’s full support to lead the Company as we transition into our next phase of growth.” “I believe that FTD has tremendous opportunities to aggressively pursue growth initiatives,” commented Mr. Shean. “We have a talented management team, world class brands, and now that we have nearly finalized the integration of Provide Commerce, we have a strong foundation from which to build.” Mr. Shean has served as one of FTD’s directors since December 2014. Mr. Shean is Chief Executive Officer of Liberty Expedia Holdings. Mr. Shean has been a Senior Advisor of Liberty Interactive Corporation (“LIC”) and Liberty Media Corporation (together with LIC, “Liberty”), since October 2016. Prior to this, he served as Liberty’s Chief Financial Officer from November 2011. Prior to being named the Chief Financial Officer Mr. Shean served as Liberty’s Controller for eleven years. Prior to joining Liberty, Mr. Shean was an audit partner with KPMG focusing mainly on clients operating in the media and entertainment industry. Mr. Shean serves on the board of directors of Expedia, Inc. and the Atlanta Braves. From February 2013 to December 2015, Mr. Shean served on the board of directors of TripAdvisor, Inc. Mr. Shean also serves on the advisory committee for the Pamplin School of Business at Virginia Tech. Mr. Shean received a Bachelor of Science degree in accounting from Virginia Tech in 1987. FTD will report results for the third quarter ended September 30, 2016 on Monday, November 7, 2016 after market close. The Company will host a conference call to discuss these results with additional comments and details provided at that time. Participating on the call will be Christopher W. Shean, interim President and Chief Executive Officer, and Becky A. Sheehan, Executive Vice President and Chief Financial Officer. The conference call is scheduled to begin at 5:00 p.m. ET on Monday, November 7, 2016. Live audio of the call will be webcast and archived on the investor relations section of the Company's website at http://www.ftdcompanies.com. In addition, you may dial 877-407-0784 to listen to the live broadcast. A telephonic playback and archived webcast will be available from November 7, 2016, through November 21, 2016. Participants can dial 877-870-5176 to hear the playback. The passcode is 13647858. FTD Companies, Inc. is a premier floral and gifting company. Through our diversified family of brands, we provide floral, specialty foods, gifts and related products to consumers primarily in the United States, Canada, the United Kingdom and the Republic of Ireland. We also provide floral products and services to retail florists and other retail locations throughout these same geographies. FTD has been delivering flowers since 1910 and the highly-recognized FTD® and Interflora® brands are supported by the iconic Mercury Man logo®, which is displayed in approximately 40,000 floral shops in nearly 150 countries. In addition to FTD and Interflora, our diversified portfolio of brands includes the following trademarks: ProFlowers®, ProPlants®, Shari's Berries®, Personal Creations®, RedEnvelope®, Flying Flowers®, Flowers Direct™, Ink Cards™, Postagram™ and Gifts.com™. FTD Companies, Inc. is headquartered in Downers Grove, Ill. For more information, please visit www.ftdcompanies.com. This release contains certain forward-looking statements within the meaning of the “safe harbor” provisions of the Private Securities Litigation Reform Act of 1995, as amended, based on our current expectations, estimates and projections about our operations, industry, financial condition, performance, results of operations, and liquidity. Statements containing words such as “may,” “believe,” “anticipate,” “expect,” “intend,” “plan,” “project,” “projections,” “business outlook,” “estimate,” or similar expressions constitute forward-looking statements. These forward-looking statements include, but are not limited to, statements about the Company’s strategies and future financial performance; statements regarding expected synergies and benefits of the Company’s acquisition of Provide Commerce; expectations about future business plans, prospective performance and opportunities, including potential acquisitions; revenues; segment metrics; operating expenses; market trends, including those in the markets in which the Company competes; liquidity; cash flows and uses of cash; capital expenditures; depreciation and amortization; tax payments; foreign currency exchange rates; hedging arrangements; the Company’s ability to repay indebtedness and invest in initiatives; the Company’s products and services; pricing; marketing plans; competition; settlement of legal matters; and the impact of accounting changes and other pronouncements. Potential factors that could affect these forward-looking statements include, among others, the factors disclosed in the Company’s most recent Annual Report on Form 10-K and the Company’s other filings with the Securities and Exchange Commission (www.sec.gov), including, without limitation, information under the captions “Management’s Discussion and Analysis of Financial Condition and Results of Operations” and “Risk Factors.” Readers are cautioned not to place undue reliance on these forward-looking statements, which reflect management’s analysis only as of the date hereof. Such forward-looking statements are not guarantees of future performance or results and involve risks and uncertainties that may cause actual performance and results to differ materially from those predicted. Reported results should not be considered an indication of future performance. Except as required by law, we undertake no obligation to publicly release the results of any revision or update to these forward-looking statements that may be made to reflect events or circumstances after the date hereof or to reflect the occurrence of unanticipated events.
News Article | November 10, 2016
— Segments The market for global organic baby food market is segmented on the basis of by Systems and by Purpose, by Geography; On basis of Systems: • AEWC • Sensor • Electronic Warfare • Maritime Patrol On basis of Purpose: • Intelligence, • Surveillance, • Reconnaissance Market Synopsis of Airborne ISR Market The global Airborne ISR market is expected to grow at a CAGR of around 4% during 2016-2021. This growth is driven due to growing demand of unmanned aerial vehicle, investments on ISR payloads, and procurement of multirole UAV remote sensors. Key Players The leading market players in the global Airborne ISR market primarily include • BAE Systems • L-3 Communication • Lockheed Martin • Northrop Grumman • UTC Aerospace Systems • Boeing • General Dynamics • Raytheon • Rockwell Collins Global Airborne ISR Market ($ billion), 2016-2021 The need for UAV remote sensors emerged from the changes in military security missions, such as, shift to Net-centric warfare (NCW), increasing Low intensity conflicts (LIC), need for persistence, and reduced tolerance to casualties. The Americas is the dominant player with 55% of the market share and is likely to be the leading region in the forecast period. Whereas, with the growing defense spending and procurement of airborne systems, APAC region will grow significantly at a CAGR of around 9% during the forecast period. Regional and Country Analysis of Airborne ISR Market As per the MRFR analysis, the Americas Airborne ISR market is poised to reach $XX Billion in 2021, to grow at a CAGR of around 2% during the forecasted period. Whereas, EMEA and Asia Pacific will grow at a CAGR of around XX% and XX% respectively. “Ask for your specific company profile and country level customization on reports.” Table of content 1. INTRODUCTION 1.1 REPORT DESCRIPTION 1.2 RESEARCH OBJECTIVE 2. EXECUTIVE SUMMARY 2.1 KEY FINDINGS / HIGHLIGHTS 2.1.1 INVESTMENT OPPORTUNITIES 2.1.2 MARKET STARTEGIES 2.1.3 LATEST DEVELOPMENTS 3. SCOPE OF THE STUDY 3.1 MARKETS COVERED 3.2 YEARS CONSIDERED FOR THE STUDY (2016-2021) 3.2 GEOGRAPHIC SCOPE 3.3 KEY STAKEHOLDERS 4. ASSUMPTIONS AND LIMITATIONS 5. RESEARCH METHODOLOGY 5.1 PRIMARY RESEARCH 5.2 SECONDARY RESEARCH 5.3 ECONOMETRIC AND FORECASTING MODEL 6. MARKET SIZE ESTIMATION 6.1 TOP DOWN APPROACH 6.2 BOTTOM UP APPROACH 7. MARKET FACTOR ANALYSIS 7.1 VALUE CHAIN ANALYSIS 7.2 SUPPLY CHAIN ANALYSIS 7.3 PORTER'S FIVE FORCES ANALYSIS 8. MARKET DYNAMICS 8.1 DRIVERS 8.2 RESTRAINTS 8.3 OPPORTUNITIES 8.4 TRENDS 9. MARKET SEGMENTATION 9.1 BY SYSTEMS 9.2 BY PURPOSE 9.3 BY REGION 10. GLOBAL AIRBORNE ISR MARKET BY SYSTEMS, 2016-2021 10.1 MARKET SIZE BY SYSTEMS ($ BILLIONS) 10.1.1 AIRBORNE EARLY WARNING AND CONTROL (AEWC) 10.1.2 MARITIME PATROL 10.1.3 SENSOR 10.1.4 ELECTRONIC WARFARE 11. GLOBAL AIRBORNE ISR MARKET BY PURPOSE, 2016-2021 11.1 MARKET SIZE BY PURPOSE ($ BILLIONS) 11.1.1 INTELLIGENCE 11.2.2 SURVELLIENCE 11.2.3 RECONNAISSANCE 12. GLOBAL AIRBORNE ISR MARKET BY REGION, 2016-2021 12.1 MARKET SIZE BY REGION ($ BILLIONS) 12.1.1 AMERICAS (NORTH & LATIN) Continued….. Reasons to Purchase this report: From an insight perspective, this research report has focused on various levels of analyses—industry analysis (industry trends), market share analysis of top players, supply chain analysis, and company profiles, which together comprise and discuss the basic views on the competitive landscape, emerging and high-growth segments of the Global Airborne ISR Market, high-growth regions, and market drivers, restraints, and opportunities. Related Reports Global Aerospace Composites Market Research Report- Forecast to 2021 The Global Aerospace Composites market is expected to grow at a CAGR of around 8% during 2016-2021. This growth is driven due to significant demand for aircraft weight reduction and fuel efficiency and high corrosion resistance. Know more about this report @ https://www.marketresearchfuture.com/reports/global-aerospace-composites-market-research-report-forecast-to-2021 About Market Research Future: At Market Research Future (MRFR), we enable our customers to unravel the complexity of various industries through our Cooked Research Report (CRR), Half-Cooked Research Reports (HCRR), Raw Research Reports (3R), Continuous-Feed Research (CFR), and Market Research & Consulting Services. MRFR team have supreme objective to provide the optimum quality market research and intelligence services to our clients. Our market research studies by products, services, technologies, applications, end users, and market players for global, regional, and country level market segments, enable our clients to see more, know more, and do more, which help to answer all their most important questions. For more information, please visit https://www.marketresearchfuture.com
News Article | December 2, 2016
Market Research Report Provides Manufacturers Profiles/Analysis: Ajinomoto Co., Inc., Amy's Kitchen, Inc., Ardo N.V., Arena Agroindustrie Alimentari SPA, Bellisio Foods, Inc., Bonduelle SCA, Conagra Foods, Inc., Findus Group., General Mills, Inc., Goya Foods, Inc., H.J. Heinz Company, Iceland Foods Ltd., Kraft Food, Inc., Mccain Foods Limited, Nature's Peak, LIC., Nestl SA & more -with detail like Company Basic Information, Manufacturing Base and Competitors. The report provides a basic overview of Frozen Food industry including definitions, applications and industry chain structure. United States market analysis and Chinese domestic market analysis are provided with a focus on history, developments, trends and competitive landscape of the market. A comparison between the international and Chinese situation is also offered. United States Frozen Food Industry Research Report 2016 also focuses on development policies and plans for the industry as well as a consideration of a cost structure analysis. Capacity production, market share analysis, import and export consumption and price cost production value gross margins are discussed. A key feature of this report is it focus on major industry players, providing an overview, product specification, product capacity, production price and contact information for United States Top15 companies. This enables end users to gain a comprehensive insight into the structure of the international and Chinese Frozen Food industry. Development proposals and the feasibility of new investments are also analyzed. Companies and individuals interested in the structure and value of the Frozen Food industry should consult this report for guidance and direction. The report begins with a brief overview of the United States Frozen Food market and then moves on to evaluate the key trends of the market. The key trends shaping the dynamics of the United States Frozen Food market have been scrutinized along with the related current events, which is impacting the market. Drivers, restraints, opportunities, and threats of the United States Frozen Food market have been analyzed in the report. Moreover, the key segments and the sub-segments that constitutes the market is also explained in the report.
News Article | November 14, 2016
This report studies Frozen Food in Global market, especially in North America, Europe, China, Japan, Southeast Asia and India, focuses on top manufacturers in global market, with production, price, revenue and market share for each manufacturer, covering Ajinomoto Co., Inc. Amy's Kitchen, Inc. Ardo N.V. Arena Agroindustrie Alimentari SPA Bellisio Foods, Inc. Bonduelle SCA Conagra Foods, Inc. Findus Group. General Mills, Inc. Goya Foods, Inc. H.J. Heinz Company Iceland Foods Ltd. Kraft Food, Inc. Mccain Foods Limited Nature's Peak, LIC. Nestlé SA Market Segment by Regions, this report splits Global into several key Regions, with production, consumption, revenue, market share and growth rate of Frozen Food in these regions, from 2011 to 2021 (forecast), like North America Europe China Japan Southeast Asia India Split by product type, with production, revenue, price, market share and growth rate of each type, can be divided into Frozen Fruits & Vegetables Frozen Ready Meals Frozen Meat Frozen Fish Others Split by application, this report focuses on consumption, market share and growth rate of Frozen Food in each application, can be divided into Residential Commercial Application 3 Global Frozen Food Market Research Report 2016 1 Frozen Food Market Overview 1.1 Product Overview and Scope of Frozen Food 1.2 Frozen Food Segment by Type 1.2.1 Global Production Market Share of Frozen Food by Type in 2015 1.2.2 Frozen Fruits & Vegetables 1.2.3 Frozen Ready Meals 1.2.4 Frozen Meat 1.2.5 Frozen Fish 1.2.6 Others 1.3 Frozen Food Segment by Application 1.3.1 Frozen Food Consumption Market Share by Application in 2015 1.3.2 Residential 1.3.3 Commercial 1.3.4 Application 3 1.4 Frozen Food Market by Region 1.4.1 North America Status and Prospect (2011-2021) 1.4.2 Europe Status and Prospect (2011-2021) 1.4.3 China Status and Prospect (2011-2021) 1.4.4 Japan Status and Prospect (2011-2021) 1.4.5 Southeast Asia Status and Prospect (2011-2021) 1.4.6 India Status and Prospect (2011-2021) 1.5 Global Market Size (Value) of Frozen Food (2011-2021) 7 Global Frozen Food Manufacturers Profiles/Analysis 7.1 Ajinomoto Co., Inc. 7.1.1 Company Basic Information, Manufacturing Base and Its Competitors 7.1.2 Frozen Food Product Type, Application and Specification 18.104.22.168 Type I 22.214.171.124 Type II 7.1.3 Ajinomoto Co., Inc. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.1.4 Main Business/Business Overview 7.2 Amy's Kitchen, Inc. 7.2.1 Company Basic Information, Manufacturing Base and Its Competitors 7.2.2 Frozen Food Product Type, Application and Specification 126.96.36.199 Type I 188.8.131.52 Type II 7.2.3 Amy's Kitchen, Inc. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.2.4 Main Business/Business Overview 7.3 Ardo N.V. 7.3.1 Company Basic Information, Manufacturing Base and Its Competitors 7.3.2 Frozen Food Product Type, Application and Specification 184.108.40.206 Type I 220.127.116.11 Type II 7.3.3 Ardo N.V. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.3.4 Main Business/Business Overview 7.4 Arena Agroindustrie Alimentari SPA 7.4.1 Company Basic Information, Manufacturing Base and Its Competitors 7.4.2 Frozen Food Product Type, Application and Specification 18.104.22.168 Type I 22.214.171.124 Type II 7.4.3 Arena Agroindustrie Alimentari SPA Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.4.4 Main Business/Business Overview 7.5 Bellisio Foods, Inc. 7.5.1 Company Basic Information, Manufacturing Base and Its Competitors 7.5.2 Frozen Food Product Type, Application and Specification 126.96.36.199 Type I 188.8.131.52 Type II 7.5.3 Bellisio Foods, Inc. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.5.4 Main Business/Business Overview 7.6 Bonduelle SCA 7.6.1 Company Basic Information, Manufacturing Base and Its Competitors 7.6.2 Frozen Food Product Type, Application and Specification 184.108.40.206 Type I 220.127.116.11 Type II 7.6.3 Bonduelle SCA Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.6.4 Main Business/Business Overview 7.7 Conagra Foods, Inc. 7.7.1 Company Basic Information, Manufacturing Base and Its Competitors 7.7.2 Frozen Food Product Type, Application and Specification 18.104.22.168 Type I 22.214.171.124 Type II 7.7.3 Conagra Foods, Inc. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.7.4 Main Business/Business Overview 7.8 Findus Group. 7.8.1 Company Basic Information, Manufacturing Base and Its Competitors 7.8.2 Frozen Food Product Type, Application and Specification 126.96.36.199 Type I 188.8.131.52 Type II 7.8.3 Findus Group. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.8.4 Main Business/Business Overview 7.9 General Mills, Inc. 7.9.1 Company Basic Information, Manufacturing Base and Its Competitors 7.9.2 Frozen Food Product Type, Application and Specification 184.108.40.206 Type I 220.127.116.11 Type II 7.9.3 General Mills, Inc. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.9.4 Main Business/Business Overview 7.10 Goya Foods, Inc. 7.10.1 Company Basic Information, Manufacturing Base and Its Competitors 7.10.2 Frozen Food Product Type, Application and Specification 18.104.22.168 Type I 22.214.171.124 Type II 7.10.3 Goya Foods, Inc. Frozen Food Production, Revenue, Price and Gross Margin (2015 and 2016) 7.10.4 Main Business/Business Overview 7.11 H.J. Heinz Company 7.12 Iceland Foods Ltd. 7.13 Kraft Food, Inc. 7.14 Mccain Foods Limited 7.15 Nature's Peak, LIC. 7.16 Nestlé SA
News Article | December 7, 2016
VANCOUVER, British Columbia, December 7, 2016 /PRNewswire/ -- LiCo Energy Metals Inc. "The Company" or "LiCo" (OTCQB: WCTXF), (TSX-V: LIC) is pleased to report that it has commenced a Phase One exploration program on the Teledyne Property located close to Cobalt, Ontario. The Cobalt...
News Article | December 5, 2016
Wearable Electronics are minute electronics devices worn by the consumer which enable wireless networking and mobile computing. The word “wearable technology” refers to any electronic device or product which can be worn by a person to add computing in his action or work and utilize technology to benefit advanced features and characteristics. Wearable electronics may consist of glasses, jewelry, headgear, belts, arm wear, wrist wear, leg wear, footwear, skin patches, and e-textiles. In recent years, there has been lots of research and development going in the field of wearable electronics attributed to their popularity and wide acceptance in global market. North America is the largest market for wearable electronics followed by Europe and Asia Pacific. In the last few years, North America has been witnessing the fastest growth rate; however Asia Pacific region is expected to take over as the fastest growing market in upcoming years. Some of the major drivers of the industry include increasing demand of consumers towards communication, networking, positioning and recognition technologies in compact and portable forms, developments in material science, augmented reality and chip evolution and low power inter device connectivity (Bluetooth 4.0, infra red and NFC). Some of the key challenges for the industry are thermal consideration, negative effect of radiation on human health, and device protection. With the growth of sensors, particularly in the health and medical space, the potential uses of wearable computing technologies are quite extensive. Wearable electronics are expected to increase their adoption levels in different sectors such as health and fitness, gaming, fashion, mobile money, education and transportation. Rising average life expectancy, baby boomers population and larger proportion of patients requiring long term treatment are some of the key drivers in healthcare and medicine segment whereas demands from professional athletes, recreational fitness consumers, and corporate wellness programs are propelling the market of wearable technology in fitness and wellness sector. The ongoing military up gradation programs around the globe such as, the U.S.’s ‘Future Force Warrior (FFW)’, Australia’s ‘Project Wundurra’, Israel’s ‘Integrated Advanced Soldier (IAS)’, and the U.K.’s ‘Future Infantry Soldier Technology’ are expected to increase the overall market share of wearable electronics market in military and defense. Request for Sample Report and Table of content @: http://www.persistencemarketresearch.com/toc/3035 Some of the key players in the wearable electronics market include Adidas AG, Recon Instruments, Inc., Fibretronic Ltd., Jawbone, Inc., Fitbit, Inc., Nike, Inc. (U.S.), Olympus Corporation, and Weartech s.l, Vuzix Corporation , Google, Inc., Apple, Inc.,By-Wire.Net, Imprint Energy, Inc, Jawbone, Inc., O’neill Wetsuits LIC, Plastic Logic Ltd., Texas Instruments, Inc., Zoog Technologies, Inc., Weartech S.L, Shimmer Research, Inc., Vancive Medical Technologies, Infineon Technologies Ag, Glassup SRL, Eurotech S.P.A, and AT&T, Inc.
News Article | November 10, 2016
The market for global organic baby food market is segmented on the basis of by Systems and by Purpose, by Geography; The global Airborne ISR market is expected to grow at a CAGR of around 4% during 2016-2021. This growth is driven due to growing demand of unmanned aerial vehicle, investments on ISR payloads, and procurement of multirole UAV remote sensors. The leading market players in the global Airborne ISR market primarily include The need for UAV remote sensors emerged from the changes in military security missions, such as, shift to Net-centric warfare (NCW), increasing Low intensity conflicts (LIC), need for persistence, and reduced tolerance to casualties. The Americas is the dominant player with 55% of the market share and is likely to be the leading region in the forecast period. Whereas, with the growing defense spending and procurement of airborne systems, APAC region will grow significantly at a CAGR of around 9% during the forecast period. As per the MRFR analysis, the Americas Airborne ISR market is poised to reach $XX Billion in 2021, to grow at a CAGR of around 2% during the forecasted period. Whereas, EMEA and Asia Pacific will grow at a CAGR of around XX% and XX% respectively. “Ask for your specific company profile and country level customization on reports.” From an insight perspective, this research report has focused on various levels of analyses—industry analysis (industry trends), market share analysis of top players, supply chain analysis, and company profiles, which together comprise and discuss the basic views on the competitive landscape, emerging and high-growth segments of the Global Airborne ISR Market, high-growth regions, and market drivers, restraints, and opportunities. The Global Aerospace Composites market is expected to grow at a CAGR of around 8% during 2016-2021. This growth is driven due to significant demand for aircraft weight reduction and fuel efficiency and high corrosion resistance. Know more about this report @ https://www.marketresearchfuture.com/reports/global-aerospace-composites-market-research-report-forecast-to-2021 At Market Research Future (MRFR), we enable our customers to unravel the complexity of various industries through our Cooked Research Report (CRR), Half-Cooked Research Reports (HCRR), Raw Research Reports (3R), Continuous-Feed Research (CFR), and Market Research & Consulting Services. MRFR team have supreme objective to provide the optimum quality market research and intelligence services to our clients. Our market research studies by products, services, technologies, applications, end users, and market players for global, regional, and country level market segments, enable our clients to see more, know more, and do more, which help to answer all their most important questions.