News Article | April 6, 2016
No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were carried out using the TR146 buccal epithelial squamous cell carcinoma line32 obtained from the European Collection of Authenticated Cell Cultures (ECACC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were routinely tested for mycoplasma contamination using mycoplasma-specific primers and were found to be negative. Prior to stimulation, confluent TR146 cells were serum-starved overnight, and all experiments were carried out in serum-free DMEM. C. albicans wild-type strains included the autotrophic strain BWP17 + CIp30 (ref. 33) and the parental strain SC5314 (ref. 34). Other C. albicans strains used and their sources are listed in Extended Data Tables 1 and 2. C. albicans cultures were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30 °C overnight. Cultures were washed in sterile PBS and adjusted to the required cell density. Antibodies to phospho-MKP1 and c-Fos were from Cell Signalling Technologies (New England Biolabs UK), mouse anti-human α-actin was from Millipore (UK), and goat anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were from Jackson Immunologicals (Stratech Scientific, UK). Ece1p peptides were synthesized commercially (Proteogenix (France) or Peptide Synthetics (UK). ECE1 deletion was performed as previously described35. Deletion cassettes were generated by PCR36. Primers ECE1-FG and ECE1-RG were used to amplify pFA-HIS1 and pFA-ARG4 -based markers. C. albicans BWP17 (ref. 37), was sequentially transformed38 with the ECE1-HIS1 and ECE1-ARG4 deletion cassettes and then transformed with CIp10 (ref. 39), yielding the ece1∆/Δ deletion strain. For complementation, the ECE1 gene plus upstream and downstream intergenic regions were amplified with primers ECE1-RecF3k and ECE1-RecR and cloned into plasmid CIp10 at MluI and SalI sites. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1∆/Δ + ECE1 complemented strain. For generation of the ece1Δ/Δ + ECE1 strain, the CIp10-ECE1 was amplified with primers Pep3-F1 and Pep3-R1, digested with ClaI and re-ligated, yielding the CIp10 + ECE1 plasmid. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1Δ/Δ + ECE1 strain. All integrations were confirmed by PCR/sequencing and at least two independent isogenic transformants were created to confirm results. KEX1 deletion was performed exactly as the ECE1 deletion but using primers KEX1-FG and KEX1-RG for creating the deletion cassette. Fluorescent strains of ece1Δ/Δ and BWP17 were constructed as previously described40. Briefly, the ece1Δ/Δ and BWP17 strains were transformed with the pENO1-dTom-NATr plasmid. Primers used to clone and construct the ECE1 genes and intragenic regions are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. ECE1 promoter (primers 5′ECE1prom–NarI / 3′ECE1prom–XhoI) and terminator (5′ECE1term–SacII / 5′ECE1term–SacI) were amplified and cloned into pADH1-GFP. Resulting pSK-pECE1-GFP was verified by sequencing. C. albicans SC5314 was transformed with the pECE1-GFP transformation cassette38. Resistance to nourseothricin was used as selective marker and correct integration of GFP into the ECE1 locus was verified by PCR. Primers for cloning and validation are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 μl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65 °C) before subjected to 2× freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at −20 °C. Precipitated pellets were washed (2× 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at −80 °C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicentre) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used for qPCR with EVAgreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for actin, ECE1-F and ECE1-R for ECE1 Extended Data Table 4) were used at a final concentration of 500 nM. qPCR amplifications were performed using a Biorad CFX96 thermocycler. Data was evaluated using Bio-Rad CFX Manager 3.1 (Bio-Rad) with ACT1 as the reference gene and t as the control sample. TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors41, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 μg of total protein was separated on 12% SDS–PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1,000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-ray film (Fuji film). Human α-actin was used as a loading control. DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described41, 42. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Active Motif) according to the manufacturer’s protocol. Protein concentration was determined (BCA protein quantitation kit (Perbio Science)) and 5 μg of nuclear extract was assayed in the TransAM system according to the manufacturer’s protocol. Data was expressed as fold-change in A relative to resting cells. Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analysed using Bioplex Manager 6.1 software to determine analyte concentrations. Following incubation, culture supernatant was collected and assayed for lactate dehydrogenase (LDH) activity using the Cytox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer’s instructions. Recombinant porcine LDH (Sigma-Aldrich) was used to generate a standard curve. Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously43. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 105) were added into 1 ml serum-free DMEM, incubated for 60 min (37 °C/5% CO ) and non-adherent C. albicans cells removed by aspiration. Following washing (3× 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high-magnification fields of 200 μm × 200 μm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip. C. albicans invasion of epithelial cells was determined as described previously43. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 105), for 3 h in a humidified incubator (37 °C/5% CO ). Following washing (3× PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti-Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalized cells by the total number of adherent cells. At least 100 fungal cells were counted on each coverslip. TR146 cells (105 per ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 104 cfu per ml) in DMEM and incubated for 6 h (37 °C/5% CO ). Cells were washed with PBS, fixed overnight (4 °C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 μg ml−1) for 45 min at room temperature in the dark with gentle shaking (70 r.p.m.) to stain the fungal cell wall. Epithelial cells were permeabilized with 0.1% Triton X-100 for 15 min at 37 °C in the dark, then washed and stained with 10 μg ml−1 Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 μl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase images were taken per picture. For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4 °C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in high vacuum mode. Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on two-tailed t-tests in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 – beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33 °C in E3 + PTU and used as previously described40. Briefly, 4 days post-fertilization (dpf) larvae were treated with 20 μg ml−1 dexamethasone dissolved in 0.1% DMSO 1 h before infection and thereafter. For tissue damage and neutrophil recruitment, individual AB or mpo:GFP fish (respectively) were injected into the swimbladder with 4 nl of PBS with or without 25–40 C. albicans yeast cells of ece1Δ/Δ-dTomato, ece1Δ/Δ + ECE1 + dTomato, ece1Δ/Δ + ECE1 + dTomato or BWP17-dTomato. For tissue damage, 1 nl of Sytox green (0.05 mM in 1% DMSO) was injected at 20 h post-infection into the swimbladder and fish were imaged by confocal microscopy at 24 h post-infection. For neutrophil recruitment, fish were imaged at 24 h post-injection. For synthetic peptide damage, AB or α-catenin:citrine44 fish were injected with 2 nl of peptide (9 ng or 1.25 ng per fish) or vehicle (40% DMSO or 5% DMSO) + SytoxGreen (0.05 mM in 1% DMSO) or SytoxOrange (0.5 mM in 10% DMSO) and the fish imaged by confocal microscopy 4 h later. Numbers of neutrophils and damaged cells observed were counted and tabulated for each fish. Live zebrafish imaging was carried out as previously described40. Briefly, fish were anaesthetized in Tris-buffered Tricaine (200 μg ml−1, Western Chemicals) and further immobilized in a solution of 0.4% low-melting-point agarose (LMA, Lonza) in E3 + Tricaine in a 96-well plate glass-bottom imaging dish (Greiner Bio-On). Confocal imaging was carried out using an Olympus IX-81 inverted microscope with an FV-1000 laser scanning confocal system (Olympus). Images were collected and processed using Fluoview (Olympus) and Photoshop (Adobe Systems). Panels are either a single slice for the differential interference contrast channel (DIC) with maximum projection overlays of fluorescence image channels (red-green), or maximum projection overlays of fluorescence channels. The number of slices for each maximum projection is specified in the legend of individual figures. Murine infections were performed under UK Home Office Project Licence PPL 70/7598 in dedicated animal facilities at King’s College London. No statistical method was used to pre-determine sample size. No method of randomization was used to allocate animals to experimental groups. Mice in the same cage were part of the same treatment. The investigators were not blinded during outcome assessment. A previously described murine model of oropharyngeal candidiasis using female BALB/c mice45 was modified to use for investigating early infection events. Briefly, mice were treated subcutaneously with 3 mg per mouse (in 200 μl PBS with 0.5% Tween 80) of cortisone acetate on days −1 and +1 post-infection. On day 0, mice were sedated for ~75 min with an intra-peritoneal injection of 110 mg per kg ketamine and 8 mg per kg xylazine, and a swab soaked in a 107 cfu per ml of C. albicans yeast culture in sterile saline was placed sublingually for 75 min. After 2 days, mice were euthanized, the tongue excised and divided longitudinally in half. One half was weighed, homogenized and cultured to derive quantitative Candida counts. The other half was processed for histopathology and immunohistochemistry. C. albicans-infected murine tongues were fixed in 10% (v/v) formal-saline before being embedded and processed in paraffin wax using standard protocols. For each tongue, 5-μm sections were prepared using a Leica RM2055 microtome and silane coated slides. Sections were dewaxed using xylene, before C. albicans and infiltrating inflammatory cells were visualized by staining using Periodic Acid-Schiff (PAS) stain and counterstaining with haematoxylin. Sections were then examined by light microscopy. Histological quantification of infection was undertaken by measuring the area of infected epithelium and expressed as a percentage relative to the entire epithelial area. TR146 epithelial cells were grown in 35-mm Petri dishes (Nunc) for 48 h before recordings at low cell density (10–30% confluence). Cells were superfused with a modified Krebs solution (120 mM NaCl, 3 mM KCl, 2.5 mM CaCl , 1.2 mM MgCl , 22.6 mM NaHCO , 11.1 mM glucose, 5 mM HEPES pH 7.4). Isolated cells were recorded at room temperature (21–23 °C) in whole cell mode using microelectrodes (5–7 MΩ) containing 90 mM potassium acetate, 20 mM KCl, 40 mM HEPES, 3 mM EGTA, 3 mM MgCl , 1 mM CaCl (free Ca2+ 40 nM), pH 7.4. Cells were voltage clamped at −60 mV using an Axopatch 200A amplifier (Axon Instruments) and current/voltage curves were generated by 1 s steps between −100 to +50 mV. Treatments were applied to the superfusate to produce the final required concentration, with vehicle controls similarly applied. Data was recorded using Clampex software (PClamp 6, Axon Instrument) and analysed with Clampfit 10. TR146 cells were grown in a 96-well plate overnight until confluent. The medium was removed and 50 μl of a Fura-2 solution (5 μl Fura-2 (Life Technologies) (2.5 mM in 50% Pluronic F-127 (Life Technologies):50% DMSO), 5 μl probenecid (Sigma) in 5 ml saline solution (NaCl (140 mM), KCl (5 mM), MgCl (1 mM), CaCl (2 mM), glucose (10 mM) and HEPES (10 mM), adjusted to pH 7.4)) was added and the plate incubated for 1 h at 37 °C/5% CO . The Fura-2 solution was replaced with 50 μl saline solution and baseline fluorescence readings (excitation 340 nm/emission 520 nm) taken for 10 min using a FlexStation 3 (Molecular Devices). Ece1 peptides were added at different concentrations and readings immediately taken for up to 3 h. The data was analysed using Softmax Pro software to determine calcium present in the cell cytosol and expressed as the ratio between excitation and emission spectra. tBLMs with 10% tethering lipids and 90% spacer lipids (T10 slides) were formed using the solvent exchange technique46, 47 according to the manufacturer’s instructions (SDx Tethered Membranes Pty Ltd, Sydney, Australia). Briefly, 8 μl of 3 mM lipid solutions in ethanol were added, incubated for 2 min and then 93.4 μl buffer (100 mM KCl, 5 mM HEPES, pH 7.0) was added. After rinsing 3× with 100 μl buffer the conductance and capacitance of the membranes were measured for 20 min before injection of Ece1 peptides at different concentrations. All experiments were performed at room temperature. Signals were measured using the tethaPod (SDx Tethered Membranes Pty, Sydney, Australia). Intercalation of Ece1 peptides into phospholipid liposomes was determined by FRET spectroscopy applied as a probe-dilution assay48. Phospholipids mixed with each 1% (mol/mol) of the donor dye NBD-phosphatidylethanolamine (NBD-PE) and of the acceptor dye rhodamine-PE, were dissolved in chloroform, dried, solubilized in 1 ml buffer (100 mM KCl, 5 mM HEPES, pH 7.0) by vortexing, sonicated with a titan tip (30 W, Branson sonifier, cell disruptor B15), and subjected to three cycles of heating to 60 °C and cooling down to 4 °C, each for 30 min. Lipid samples were stored at 4 °C for at least 12 h before use. Ece1 peptide was added to liposomes and intercalation was monitored as the increase of the quotient between the donor fluorescence intensity I at 531 nm and the acceptor intensity I at 593 nm (FRET signal) independent of time. CD measurements were performed using a Jasco J-720 spectropolarimeter (Japan Spectroscopic Co., Japan), calibrated as described previously49. CD spectra represent the average of four scans obtained by collecting data at 1 nm intervals with a bandwidth of 2 nm. The measurements were performed in 100 mM KCl, 5 mM HEPES, pH 7.0 at 25 °C and 40 °C in a 1.0 mm quartz cuvette. The Ece1-III concentration was 15 μM. Planar lipid bilayers were prepared using the Montal-Mueller technique50 as described previously51. All measurements were performed in 5 mM HEPES, 100 mM KCl, pH 7.0 (specific electrical conductivity 17.2 mS per cm) at 37 °C. Candida strains were cultured for 18 h in hyphae inducing conditions (YNB medium containing 2% sucrose, 75 mM MOPSO buffer pH 7.2, 5 mM N-acetyl-d-glucosamine, 37 °C). Hyphal supernatants were collected by filtering through a 0.2 μm PES filter, and peptides were enriched by solid phase extraction (SPE) using first C4 and subsequently C18 columns on the C4 flowthrough. After drying in a vacuum centrifuge, samples were resolubilized in loading solution (0.2% formic acid in 71:27:2 ACN/H O/DMSO (v/v/v)) and filtered through a 10 kDa MWCO filter. The filtrate was transferred into HPLC vials and injected into the LC-MS/MS system. LC-MS/MS analysis was carried out on an Ultimate 3000 nano RSLC system coupled to a QExactive Plus mass spectrometer (ThermoFisher Scientific). Peptide separation was performed based on a direct injection setup without peptide trapping using an Accucore C4 column as stationary phase and a column oven temperature of 50 °C. The binary mobile phase consisting of A) 0.2% (v/v) formic acid in 95:5 H O/DMSO (v/v) and B) 0.2% (v/v) formic acid in 85:10:5 ACN/H O/DMSO (v/v/v) was applied for a 60 min gradient elution: 0–1.5 min at 60% B, 35–45 min at 96% B, 45.1–60 min at 60% B. The Nanospray Flex Ion Source (ThermoFisher Scientific) provided with a stainless steel emitter was used to generate positively charged ions at 2.2 kV spray voltage. Precursor ions were measured in full scan mode within a mass range of m/z 300–1600 at a resolution of 70k FWHM using a maximum injection time of 120 ms and an automatic gain control target of 1e6. For data-dependent acquisition, up to 10 most abundant precursor ions per scan cycle with an assigned charge state of z = 2–6 were selected in the quadrupole for further fragmentation using an isolation width of m/z 2.0. Fragment ions were generated in the HCD cell at a normalized collision energy of 30 V using nitrogen gas. Dynamic exclusion of precursor ions was set to 20 s. Fragment ions were monitored at a resolution of 17.5k (FWHM) using a maximum injection time of 120 ms and an AGC target of 2e5. Thermo raw files were processed by the Proteome Discoverer (PD) software v188.8.131.528 (Thermo). Tandem mass spectra were searched against the Candida Genome Database (http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz; status: 2015/05/03) using the Sequest HT search algorithm. Mass spectra were searched for both unspecific cleavages (no enzyme) and tryptic peptides with up to 4 missed cleavages. The precursor mass tolerance was set to 10 p.p.m. and the fragment mass tolerance to 0.02 Da. Target Decoy PSM Validator node and a reverse decoy database was used for (q value) validation of the peptide spectral matches (PSMs) using a strict target false discovery (FDR) rate of <1%. Furthermore, we used the score versus charge state function of the Sequest engine to filter out insignificant peptide hits (xcorr of 2.0 for z = 2, 2.25 for z = 3, 2.5 for z = 4, 2.75 for z = 5, 3.0 for z = 6). At least two unique peptides per protein were required for positive protein hits. TransAM and patch clamp data were analysed using a paired t-test while cytokines, LDH and calcium influx data were analysed using one-way ANOVA with all compared groups passing an equal variance test. Murine in vivo data was analysed using the Mann–Whitney test. Zebrafish data was analysed using the Kruskal–Wallis test with Dunn’s multiple comparison correction. In all cases, P < 0.05 was taken to be significant.
News Article | February 16, 2017
« Honda R&D partners with new smart mobility innovation center DRIVE in Israel | Main | SEAT Componentes to manufacture new 6-speed manual for Volkswagen Group MQB platform; 450K units/year » GKN Driveline’s Chinese Joint Venture partner SDS has commenced production of its complete all-wheel drive (AWD) Disconnect system for small- to medium-sized vehicles in China. The system will initially feature on the Jeep Renegade built for the Chinese market by GAC Fiat Chrysler Automobiles Co., Ltd. (GAC FCA) in Guangzhou City, southern Guangdong Province. GKN’s Disconnect function enables vehicles to combine the enhanced traction, dynamics and stability of all-wheel drive with improved on-highway fuel efficiency. The system reacts to driver inputs and road conditions to seamlessly switch between two-wheel drive and all-wheel drive. During steady state cruising, the clutch system disengages the rear section of the driveline, eliminating rotating losses and improving highway fuel economy by up to 4%—compared to standard AWD. If the driver or conditions require more traction, the system reconnects within just 300 milliseconds. GKN’s Chinese joint venture, Shanghai GKN HUAYU Driveline Systems (SDS), will supply GAC FCA from its production facility in Shanghai. GKN manufactures the complete all-wheel drive system, including the power transfer unit (PTU), rear drive module (RDM), propshaft and sideshafts, as well as supplying software controls. The move marks another milestone in the growth of GKN’s AWD business in China. SDS successfully localized production of PTUs and RDMs for Jaguar Land Rover models in 2015, including the Range Rover Evoque and Land Rover Discovery Sport, as well as localizing production for two further automakers last year. More localization programs are due to start production for a range of manufacturers over the next two years. SDS added six new AWD assembly lines and a new hypoid gear production line across two of its facilities in Shanghai during the past 18 months, as well as testing and other equipment, to support growing levels of business. The investment significantly expanded the company’s AWD production capacity in China. GKN has capabilities to manufacture sophisticated all-wheel drive systems on a global basis and that’s why automakers choose to partner with us on their mega-platforms. The demand for all-wheel drive vehicles is growing at a rapid rate in China and GKN and SDS have a number of localization and domestic programmes in the pipeline for the coming years. GKN’s scalable AWD Disconnect system was first launched on the Jeep Renegade and Fiat 500X subcompact crossover SUVs. As well as China, GKN builds the system at facilities in Europe and North America, and supplies the system in India and South America.
Grewal J.S.,Guru Nanak Institutions |
Sidhu B.S.,PTU |
Prakash S.,Indian Institute of Technology Roorkee
Proceedings of the International Thermal Spray Conference | Year: 2015
Titanium aluminium based nitride (Ti, A1)N coatings possess excellent tribological behaviour with respect to metal cutting and polymer forming contacts. In the present work TiAIN coatings were deposited by plasma spray process. Three coatings of TiAIN were deposited on AISI-347 grade boiler steel substrate out of which two were thin nano coatings deposited at different temperatures of 500°C and 200°C and one conventional coating was deposited by plasma spraying. The as sprayed coatings were characterized with relative to coating thickness, microhardness, porosity and microstructure. The optical microscopy (OM), the XRD analysis and field mission scanning electron microscope (FESEM with EDAX attachment) techniques have been used to identify various phases formed after coating deposited on the surface of the substrate. Subsequently the sliding wear behaviour of uncoated, PVD sprayed nanostructured thin TiAIN coatings deposited at 500°C and 200°C and plasma sprayed conventional coated AISI-347 grade boiler steel were investigated according to ASTM standard G99-03 using pin on disk wear test rig. Cumulative wear volume loss and coefficient of friction, p were calculated for the coated as well as uncoated specimens for 10, 15 and 20 N normal loads at a constant sliding velocity of 1 m/sec. The worn out samples were analysed with SEM/EDAX. Wear rates in terms of volumetric loss (mm3/g) for uncoated and coated alloys were compared. The nanostructured TiAIN coatings deposited at 500°C and 200°C has shown minimum wear rate as compared to conventional TiAIN coating and uncoated AISI-347 grade boiler steel. Nanostructured TiAIN coatings were found to be successful in retaining surface contact with the substrate after the wear tests. © (2015) by ASM International All rights reserved.
News Article | November 24, 2016
TORONTO, ON--(Marketwired - November 24, 2016) - Purepoint Uranium Group Inc. ( the "Company" or "Purepoint") (TSX VENTURE: PTU) is pleased to report on last week's Hook Lake Preliminary Technical Committee meeting, a project owned jointly by Cameco Corp. (39.5%), AREVA Resources Canada Inc. (39.5%) and Purepoint Uranium Group Inc. (21%). The Hook Lake JV project resides within the Patterson Uranium District that hosts Fission Uranium's high-grade PLS uranium discovery, NexGen's Arrow discovery and the Hook Lake JV Spitfire discovery. Mobilization of camp and drill equipment has commenced, utilizing the remaining $500,000 from the 2016 budget. Initial drilling will follow-up the Spitfire high-grade intercept returned earlier this year by hole HK16-53 with 10.3% U3O8 over 10.0 metres. The proposed 2017 Hook Lake JV exploration program plans for twenty-five diamond drill holes, approximately 10,000 metres of drilling, at a budgeted cost of $4,000,000. Drilling is planned to further delineate the Spitfire Discovery and to follow the associated mineralized structure towards the northeast. The Hook Lake JV project technical and management meetings will be held within the next two weeks. The Hook Lake JV project is owned jointly by Cameco Corp. (39.5%), AREVA Resources Canada Inc. (39.5%) and Purepoint Uranium Group Inc. (21%) and consists of nine claims totaling 28,683 hectares situated in the southwestern Athabasca Basin. The Hook Lake JV is considered one of the highest quality uranium exploration projects in the Athabasca Basin due to its location along the prospective Patterson Lake trend and the relatively shallow depth to the unconformity. Current exploration is targeting the Patterson Uranium District that hosts Fission's Triple R Deposit (indicated mineral resource 79,610,000 lbs U3O8 at an average grade of 1.58% U3O8), NexGen Energy's Arrow Deposit (inferred mineral resource 201,900,000 lbs U3O8 at an average grade of 2.63% U3O8) and the Spitfire Discovery (10.0 metres of 10.3% U3O8) by the Hook Lake JV. Purepoint Uranium Group Inc. is focused on the precision exploration of its seven projects in the Canadian Athabasca Basin. Purepoint proudly maintains project ventures in the Basin with two of the largest uranium producers in the world, Cameco Corporation and AREVA Resources Canada Inc. Established in the Athabasca Basin well before the initial resurgence in uranium earlier last decade. Purepoint is actively advancing a large portfolio of multiple drill targets in the world's richest uranium region. Scott Frostad BSc, MASc, PGeo, Purepoint's Vice President, Exploration, is the Qualified Person responsible for technical content of this release. Mr. Frostad has supervised the preparation of, and approved the scientific and technical disclosures in, this news release. THE TSX VENTURE EXCHANGE HAS NOT REVIEWED AND DOES NOT ACCEPT RESPONSIBILITY FOR THE ADEQUACY OR ACCURACY OF THIS RELEASE.
News Article | November 21, 2016
CLEARWATER, Fla., Nov. 21, 2016 (GLOBE NEWSWIRE) -- Tech Data Corporation (Nasdaq:TECD) today announced the appointment of Linda Rendleman to vice president of Product Marketing, Client and Mobile Solutions. In this role, Rendleman will be responsible for the strategic direction and go-to-market execution of the company’s Client and Mobile Solutions business in the United States, including development of growth strategies and program implementation. She will report to Brian Davis, the company’s senior vice president, U.S. Marketing and Purchasing. “We are excited to welcome Linda back to Tech Data in this new role,” said Davis. “As Tech Data remains strategically focused on growing our partners’ client and mobile businesses, we look forward to the innovative ideas and experience that Linda brings to our team, as well as the leadership that she’s demonstrated both at Microsoft and at Tech Data.” Rendleman rejoins Tech Data following more than seven years with Microsoft, where she most recently served as Director, Partner Team Unit (PTU) Lead. In this role, she led a team responsible for the growth of Microsoft’s business with Tech Data in the United States. Previously, Rendleman worked for Microsoft in the United Kingdom (UK) for more than two years, serving as UK Partner Business and Development Director and UK Distribution and Scale Reseller Director. She began her career in sales and customer service at Tech Data in 1992, quickly rising through the organization and holding multiple sales leadership positions. Prior to joining Microsoft in 2009, Rendleman served as Director of Sales, National Account at Tech Data. She holds a B.A. in Business Administration from Eckerd College. About Tech Data Tech Data Corporation is one of the world’s largest wholesale distributors of technology products, services and solutions. Its advanced logistics capabilities and value added services enable 105,000 resellers to efficiently and cost effectively support the diverse technology needs of end users in more than 100 countries. Tech Data generated $26.4 billion in net sales for the fiscal year ended January 31, 2016. It is ranked No. 108 on the Fortune 500® and one of Fortune’s “World’s Most Admired Companies.” To learn more, visit www.techdata.com, or follow us on Facebook and Twitter.
News Article | October 31, 2016
TORONTO, ONTARIO--(Marketwired - Oct. 31, 2016) - Purepoint Uranium Group Inc. (TSX VENTURE:PTU) ("Purepoint" or the "Company") is pleased to announce that it will be hosting a public presentation to discuss the history, recent findings and future potential of the Patterson Lake Structural Corridor in Saskatchewan's Athabasca Basin - host to an emerging world class uranium district. Utilizing results from all publically available data and dismissing arbitrary claim lines, the Company's objective is to knit together a single data set producing a consistent, macro interpretation of the Patterson Lake Structural Corridor and better understand the relationships between the discoveries of the past few years and the potential for more. What: Webinar on the Patterson Uranium District When: Thursday November 3, 2016 at 12:00 pm ET/ 9:00 am PT Spots are limited. Interested parties should register as soon as possible. The Patterson Uranium District is located on the south western edge of Canada's Athabasca Basin, home to the world's richest uranium deposits. Since 2012, eight related deposits and showings have been discovered along the Patterson Lake Structural Corridor including: Purepoint Uranium Group Inc. is focused on the precision exploration of its seven projects in the Canadian Athabasca Basin. Purepoint proudly maintains project ventures in the Basin with two of the largest uranium producers in the world, Cameco Corporation and AREVA Resources Canada Inc. Established in the Athabasca Basin well before the initial resurgence in uranium earlier last decade, Purepoint is actively advancing a large portfolio of multiple drill targets in the world's richest uranium region. Scott Frostad BSc, MASc, PGeo, Purepoint's Vice President, Exploration, is the Qualified Person responsible for technical content of this release and the presentation. THE TSX VENTURE EXCHANGE HAS NOT REVIEWED AND DOES NOT ACCEPT RESPONSIBILITY FOR THE ADEQUACY OR ACCURACY OF THIS RELEASE.
News Article | December 14, 2016
TORONTO, ONTARIO--(Marketwired - Dec. 14, 2016) - Purepoint Uranium Group Inc. (the "Company" or "Purepoint") (TSX VENTURE:PTU) today reported that a 2017 exploration budget of $5,000,000 has been approved by the Hook Lake Joint Venture partners (AREVA Resources Canada Inc. and Cameco Corp.) for the diamond drill program that is being operated by Purepoint. The Hook Lake JV project resides within the Patterson Uranium District on the southwest edge of the Athabasca Basin, Saskatchewan. The proposed 2017 Hook Lake JV exploration program plan for 25 diamond drill holes at a budgeted cost of $4-million has now been revised for 30 drill holes and an increased budget of $5-million has been approved. The total exploration budget for the 2016/2017 winter program is $5,500,000, with the $500,000 coming from the remaining 2016 budget. Further to the Company's previous announcement (Purepoint PR of November 24th, 2016), mobilization of camp and drill equipment is now complete with initial drilling commencing shortly to follow-up the Spitfire high-grade intercept by hole HK16-53 which returned 10.3% U O over 10.0 metres earlier this year. Drilling is planned to further delineate the Spitfire Discovery and to follow the associated mineralized structure towards the northeast. The 2017 drill program will be focused on the Patterson Lake Corridor, an emerging, world class uranium district that is attracting significant exploration investment. The prospective Patterson structural trend currently hosts the Spitfire discovery and two high grade uranium deposits, Fission's Triple R deposit and NexGen Energy's Arrow deposit, over a 14 kilometre strike length and remains virtually untested on the Hook JV project for an additional 12 kilometres. Purepoint will be hosting a public presentation to discuss the Hook Lake Project within the context of the Patterson Uranium District, the most recent work completed and the details of the current program. When: Monday December 19, 2016 at 12:00 pm ET/ 9:00 am PT Spots are limited. Interested parties should register as soon as possible. The Hook Lake JV project is owned jointly by Cameco Corp. (39.5%), AREVA Resources Canada Inc. (39.5%) and Purepoint Uranium Group Inc. (21%) and consists of nine claims totaling 28,683 hectares situated in the southwestern Athabasca Basin. The Hook Lake JV is considered one of the highest quality uranium exploration projects in the Athabasca Basin due to its location along the prospective Patterson Lake trend and the relatively shallow depth to the unconformity. Current exploration is focused on the Patterson Uranium District that hosts Fission's Triple R Deposit (indicated mineral resource 79,610,000 lbs U O at an average grade of 1.58% U O ), NexGen Energy's Arrow Deposit (inferred mineral resource 201,900,000 lbs U O at an average grade of 2.63% U O ) and the Spitfire Discovery (10.0 metres of 10.3% U O ) by the Hook Lake JV. Purepoint Uranium Group Inc. continues to conduct precision exploration of its seven projects in the Canadian Athabasca Basin. Purepoint proudly maintains project ventures in the Basin with two of the largest uranium producers in the world, Cameco Corporation and AREVA Resources Canada Inc. Established in the Athabasca Basin well before the initial resurgence in uranium earlier last decade, Purepoint is actively advancing a large portfolio of multiple drill targets in the world's richest uranium region. Scott Frostad BSc, MASc, PGeo, Purepoint's Vice President, Exploration, is the Qualified Person responsible for technical content of this release. Mr. Frostad THE TSX VENTURE EXCHANGE HAS NOT REVIEWED AND DOES NOT ACCEPT RESPONSIBILITY FOR THE ADEQUACY OR ACCURACY OF THIS RELEASE.
Kaushik B.,P.T.U. |
Kaur N.,C.G.C. |
Kohli A.K.,Thapar University
Applied Soft Computing Journal | Year: 2013
The objective of this paper is to present a novel method to achieve maximum reliability for fault tolerant optimal network design when network has variable size. Reliability calculation is most important and critical component when fault tolerant optimal network design is required. A network must be supplied with certain parameters that guarantee proper functionality and maintainability under worse situations. Many alternative methods for measuring reliability have been stated in literature for optimal network design. Most of these methods mentioned in literature for evaluating reliability may be analytical and simulation based. These methods provide significant way to compute reliability when network has limited size. Also, significant computational effort is required for growing variable sized networks. Therefore, a novel neural network method is presented to achieve significant high reliability for fault tolerant optimal network design in highly growing variable networks. This paper computes reliability with improved learning rate gradient descent based neural network method. The result shows that improved optimal network design with maximum reliability is achievable by novel neural network at manageable computational cost. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
Kang H.S.,PTU |
Smart Innovation, Systems and Technologies | Year: 2015
The innovative features of information system, known as, Radiology Information System (RIS), for electronic medical records has shown a good impact in the hospital. The interoperability of RIS with the other Intra-hospital Information Systems that interacts with, dealing with the compatibility and open architecture issues, are accomplished by two novel mechanisms. The first one is the particular message handling system that is applied for the exchange of information, according to the Health Level Seven (HL7) protocol’s specifications and serves the transfer of medical and administrative data among the RIS applications and data store unit. The same mechanism allows the secure and HL7-compatible interactions with the Hospital Information System (HIS) too. The second one implements the translation of information between the formats that HL7 and Digital Imaging and Communication in Medicine (DICOM) protocols specify, providing the communication between RIS and Picture and Archive Communication System (PACS). © Springer India 2015.
Chhabra A.,PTU |
Conference Proceeding - 2015 International Conference on Advances in Computer Engineering and Applications, ICACEA 2015 | Year: 2015
Supporting quality of service (QoS) in multimedia application like Voice over IP is a key requirement. Wireless Mesh Networking is envisioned as a solution for next networks generation and a key technology for supporting VoIP application. VoIP has become a killer application and is gradually being tested over emerging areas like Wireless mesh networks. There are various challenges for VoIP in WMN. This Paper discusses E-Model which is the most reliable method for evaluating quality of voice recommended by the International Telecommunication Union-Telecommunication. The paper assesses quality of voice in form of Mean Opinion Score and R-Score in Wireless Mesh Scenario. The Paper gives a review of various codec's used in voice transmission. This paper also analyzes affect of variation of number of mesh routers over transmission of voice signal in wireless mesh scenario. © 2015 IEEE.