News Article | May 18, 2017
84 PoE Industrial Switches complete phase three of an industrial portfolio launch. More and more companies are using PoE technology to increase installation flexibility, lower costs and make their networks efficient, comments John Feeney, Chief Operating Officer at Perle Systems. Adding PoE technology to Managed Ethernet Switches to meet the unique needs of Industrial-grade environments just makes sense. Nashville, TN, May 18, 2017 --( “More and more companies are using PoE technology to increase installation flexibility, lower costs and make their networks efficient,” comments John Feeney, Chief Operating Officer at Perle Systems. “Adding PoE technology to Managed Ethernet Switches to meet the unique needs of Industrial-grade environments just makes sense.” Industrial-grade Managed Ethernet Switches with PoE are designed to withstand extreme temperatures, surges, vibrations, and shocks found in industrial automation, government, military, oil, gas, mining and outdoor applications. Classified as Power Sourcing Equipment (PSE), they use standard UTP cables that carry Ethernet data to also provide up to 30 watts of power to Powered Devices (PDs) such as wireless access points, Voice over IP phones and IP cameras. Perle IDS-500 PoE Industrial Managed Switches come with a PRO Feature Set to meet the needs enterprise-grade level environments where additional security and network integration functionality is required: TACACS+ and RADIUS authentication, authorization and accounting (AAA) security services Secure management sessionsvia SSH, SNMPv3, Telnet and HTTPS Management Access Lists (ACL) by IP address and IP Port number Password Strength Checking IEEE 802.1x Authentication and Port Security for protection of User Access Ports Advanced protocols to optimize the performance and intelligence of the network: LLDP, GVRP, Voice LANs, MSTP, GMRP, IPv4 IGMP Snooping and IPv6 MLD Snooping IEEE 1588 Precision Time Protocol (PTP) Boundary clock capabilities These rugged fan-less switches feature a corrosion resistant IP20 aluminum case, a voltage range of 44 to 57 volts DC and operate in temperatures from -40 to 75°C. In addition, Perle DIN Rail Switches will have numerous certifications for harsh environments including UL 61010-1, ANSI Class1/Div2 and ATEX Class1/Zone2. With this product launch, Perle now offers over 720 Industrial Managed Ethernet Switches. Since entering the Industrial Switch Market, Perle has sold to numerous high profile customers such as the FAA, NBCUniveral, SolarCity, Dupont, Rolls Royce and Siemens. Nashville, TN, May 18, 2017 --( PR.com )-- Perle Systems, a global manufacturer of secure device networking hardware, today announced the launch of fully compliant IEEE802.3 af/at Power over Ethernet (PoE) Industrial Managed Switches. This third phase of an Industrial Managed Switch portfolio launch includes 84 models of the IDS-500 PoE Switches with variable 10/100/1000Base-T, SFP, Fiber and combo ports.“More and more companies are using PoE technology to increase installation flexibility, lower costs and make their networks efficient,” comments John Feeney, Chief Operating Officer at Perle Systems. “Adding PoE technology to Managed Ethernet Switches to meet the unique needs of Industrial-grade environments just makes sense.”Industrial-grade Managed Ethernet Switches with PoE are designed to withstand extreme temperatures, surges, vibrations, and shocks found in industrial automation, government, military, oil, gas, mining and outdoor applications. Classified as Power Sourcing Equipment (PSE), they use standard UTP cables that carry Ethernet data to also provide up to 30 watts of power to Powered Devices (PDs) such as wireless access points, Voice over IP phones and IP cameras.Perle IDS-500 PoE Industrial Managed Switches come with a PRO Feature Set to meet the needs enterprise-grade level environments where additional security and network integration functionality is required:TACACS+ and RADIUS authentication, authorization and accounting (AAA) security servicesSecure management sessionsvia SSH, SNMPv3, Telnet and HTTPSManagement Access Lists (ACL) by IP address and IP Port numberPassword Strength CheckingIEEE 802.1x Authentication and Port Security for protection of User Access PortsAdvanced protocols to optimize the performance and intelligence of the network: LLDP, GVRP, Voice LANs, MSTP, GMRP, IPv4 IGMP Snooping and IPv6 MLD SnoopingIEEE 1588 Precision Time Protocol (PTP) Boundary clock capabilitiesThese rugged fan-less switches feature a corrosion resistant IP20 aluminum case, a voltage range of 44 to 57 volts DC and operate in temperatures from -40 to 75°C. In addition, Perle DIN Rail Switches will have numerous certifications for harsh environments including UL 61010-1, ANSI Class1/Div2 and ATEX Class1/Zone2. With this product launch, Perle now offers over 720 Industrial Managed Ethernet Switches.Since entering the Industrial Switch Market, Perle has sold to numerous high profile customers such as the FAA, NBCUniveral, SolarCity, Dupont, Rolls Royce and Siemens. Click here to view the list of recent Press Releases from Perle Systems
News Article | May 17, 2017
Ntn1βgeo(ref. 7) and Dcc (ref. 18) knockout lines have previously been described and genotyped by PCR. The Ntn1 conditional knockout was created (Genoway) by inserting two loxP sites flanking the coding sequences containing both the principal ATG (based on Ntn1 cDNA sequence NM_008744) and the cryptic ATG (based on Ntn1 cDNA: BC141294) and the alternative promoter described in intron 3 (ref. 28). The targeting vector was constructed as follows: three fragments of 2.1 kb, 3.4 kb and 4.6 kb (respectively, the 5′, floxed and 3′ arms) were amplified by PCR using 129Sv/Pas ES DNA as a template and sequentially subcloned into the pCR4-TOPO vector (Invitrogen). These fragments were used for the construction of the targeting vector in which a FRT-flanked neomycin cassette was inserted in 5′ of the loxP-flanked region. The linearized construct was electroporated into 129Sv/Pas mouse embryonic stem (ES) cells. After selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blot analysis both with a neomycin and a 5′ external probe. The positive ES cell clones were injected into C57BL/6J blastocysts and gave rise to male chimaeras with a significant ES cell contribution. Breeding was established with C57BL/6 mice expressing the Flp-recombinase, to produce the heterozygous Ntn1 conditional knockout line devoid of the neomycin cassette. To generate a null allele of Ntn1, Ntn1fl/fl mice were crossed to Krox20:cre mice, which express Cre recombinase in the male and female germline after sexual maturity29. To ablate netrin-1 expression in the floor plate we used the Shh:cre line30 (Jackson laboratories). In this line, the eGFP reporter was also inserted in the Shh locus. Lastly, we crossed Ntn1fl/fl mice to Foxg1:cre mice31 and Nes:cre mice22 (Jackson laboratories). The Ai9 RosatdTomato reporter line (RosaTom; Jackson Laboratories) was used to monitor Cre expression. Developing inferior olivary neurons were visualized by crossing the RosaTom line with the Ptf1a:creERT2 line15. They were also further crossed to Ntn1βgeo mice. All mice are kept in C57BL/6 background. The day of the vaginal plug was counted as embryonic day 0.5 (E0.5). Mice were anaesthetized with intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg). All animal procedures were carried out in accordance to institutional guidelines and approved by the UPMC Charles Darwin Ethics Committee. Embryos of either sex were used. Ptf1a:creERT2;RosaTom pregnant mice were intraperitoneally injected at E10 with 1 mg of tamoxifen (Sigma-Aldrich, T-5648) dissolved in corn oil (Sigma-Aldrich, C-8267). The embryos were collected at E11. Pregnant females were injected intraperitoneally with EdU (1 mg per 10 g body weight) and killed three hours later. The proliferating cells were visualized after immunohistochemistry using the Alexa Fluor 647 Click-iT EdU Imaging Kit (Invitrogen). Antisense riboprobes were labelled with digoxigenin-11-d-UTP (Roche Diagnostics) as described elsewhere12, by in vitro transcription of cDNA of mouse Ntn1 (ref. 7) or mouse Ntn1 exon 3. E12-E13 hindbrains fixed in 4% PFA in an open book configuration were injected using a glass micropipette with DiI crystals or small drops of DiI (Invitrogen) diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich). Samples were kept for 24 h at 37 °C in 4% PFA. Embryos were fixed by immersion in 4% PFA in 0.12 M phosphate buffer, pH 7.4 (PFA) overnight at 4 °C. Samples were cryoprotected in a solution of 10% sucrose in 0.12 M phosphate buffer (pH 7.2), frozen in isopentane at 50 °C and then cut at 20 μm with a cryostat (Leica Microsystems). Immunohistochemistry was performed on cryostat sections after blocking in 0.2% gelatin in PBS containing 0.5% Triton-X 100 (Sigma-Aldrich). Sections were then incubated overnight with the following primary antibodies: goat anti-human Robo3 (1:250, R&D Systems AF3076), goat anti-Dcc (1:500, Santa Cruz sc-6535), goat anti-Robo1 (1:500, R&D Systems AF1749), rat anti-mouse netrin-1 (1:500, R&D Systems MAB1109), mouse anti-nestin–Alexa488 (1:1000, Abcam ab197495), mouse anti-neurofilament (1:300, DSHB 2H3), goat anti-Foxp2 (1:1000, Santa Cruz sc-21069), rabbit anti-Foxp2 (1:1000, Abcam ab16046), rabbit anti-Sox2 (1:500, Abcam ab97959), rabbit anti-βgal (1:500, Cappel 55976), goat anti-human ALCAM (1:500, R&D Systems AF656), rabbit anti-GFP (1:800, Life Technologies A11122), rabbit anti-DsRed (1:500, Clontech 632496) followed by 2 h incubation in species-specific secondary antibodies directly conjugated to fluorophores (Cy-5, Cy-3, Alexa Fluor 647 from Jackson ImmunoResearch, or from Invitrogen). For netrin-1 immunostaining, an antigen retrieval treatment was performed on the sections before to process them for immunochemistry. The sections were boiled in citrate buffer (pH 6) during 9 min. Sections were counterstained with DAPI (1:1,000, Sigma-Aldrich). In the case of netrin-1 immunostaining on non-permeabilized tissue, the Triton was removed from all the steps. Slides were scanned with a Nanozoomer (Hamamatsu) and laser scanning confocal microscope (FV1000, Olympus). Brightness and contrast were adjusted using Adobe Photoshop. Whole-mount immunostaining and 3DISCO optical clearing procedure has been described previously32. 3D imaging was performed with an ultramicroscope using Inspector Pro software (LaVision BioTec). HEK-293T cells (from ATCC, not authenticated, tested for mycoplasma contamination with a negative result) were transfected with pCDNA3, pCDNA3-human NTN3, pCDNA3-human NTN1 or pCDNA3-mouse Ntn1 plasmids using Fugene HD transfection reagent (Promega) according to the manufacturer’s instructions. Cells were harvested and lysed 36 h after transfection. Cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail (Roche Diagnostics)) and incubated for 1 h at 4 °C. Floor plates were micro-dissected from hindbrains and spinal cords from Ntn1fl/fl, Shh:cre;Ntn1fl/fl and Ntn1−/− E11 embryos. Floor plates were lysed in RIPA buffer and incubated for 20 min at 4 °C. Protein content was determined by a BCA assay. 25 μg of total protein was loaded on a 10% Mini Protean TGX precast gel (Biorad) and blotted onto a nitrocellulose membrane (Biorad). Membranes were blocked with 5% dried milk and 3% of BSA in TBS-0.1% Tween (TBS-T) for 1 h at room temperature and incubated for 90 min at room temperature with primary antibodies: anti-actin (Sigma-Aldrich, A5060, rabbit polyclonal, 1:1,500), anti-HPRT (Abcam, ab109021, rabbit monoclonal EPR5299, 1:10,000), anti-Ntn1 (R&D Systems, MAB1109, rat monoclonal, 1:500), anti-NTN3 (Abcam, ab185200, rabbit polyclonal, 1:1,000) and anti-Slit2 (Abcam, ab134166, rabbit monoclonal, 1:400). After three washes in TBS-T, membranes were incubated with the appropriate HRP-conjugated secondary antibody (1:5,000, Jackson ImmunoResearch). Detection was performed using Pierce ECL Western Blotting Substrate (ThermoScientific). All data quantification was done by an observer blinded to the experimental conditions. We did not perform randomization into groups. No statistical methods were used to predetermine sample size. Data are presented as mean values ± s.e.m. Statistical significance was calculated using one-sided unpaired tests for non-parametric tendencies (Kruskal–Wallis and Mann–Whitney). For western blot, at least three independent cases were quantified from independent experiments using densitometric analysis (ImageJ) by normalizing phosphorylation signals to total protein levels. The control cases were normalized to 1 and for the mutants, data were presented as mean values ± s.e.m. (0.1133 ± 0.05 for Shh:cre; Ntn1fl/fl 1 for Ntn1fl/fl and 0 for Ntn1−/−). Differences were considered significant when P < 0.05. The thickness of hindbrain commissural bundles was quantified for each embryo on nine coronal sections. The sections were representative of three different hindbrain antero-posterior levels (three sections for each level). To minimize the developmental variations, mutant embryos and littermate controls were compared (except for Ntn1−/− embryos which were compared to wild-type embryos). The ratio of the commissural axon bundle size was normalized to controls. Six embryos of each genotype were quantified, from at least two different litters. Data are presented as mean ± s.e.m.: wild type: Dcc, 0.994 ± 0.052; Robo3, 1 ± 0.061; neurofilament, 1 ± 0.048; Ntn1βgeo/βgeo: Dcc, 0.416 ± 0.013; Robo3, 0.402 ± 0.007; neurofilament, 0.416 ± 0.01; Ntn1−/−: Dcc, 0.091 ± 0.008; Robo3, 0.061 ± 0.008; neurofilament, 0.104 ± 0.007; Shh:cre;Ntn1fl/fl: Dcc, 1.051 ± 0.011; Robo3, 1.084 ± 0.028; neurofilament, 1.063 ± 0.019; and Foxg1:cre;Ntn1fl/fl: Dcc, 0.411 ± 0.053; Robo3, 0.369 ± 0.056; neurofilament, 0.416 ± 0.050. Differences were considered significant when P < 0.05. Statistical analyses of the mean and variance were performed with Prism 7 (GraphPad Software). The data that support the findings of this study are available from the corresponding author upon reasonable request.
News Article | May 24, 2017
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. To construct the 35S:uORFs –LUC reporter, the 35S promoter and the TBF1 exon1 (including the R-motif, uORF1-uORF2, and the coding sequence of the first 73 amino acids of TBF1) were amplified from p35S:uORF1-uORF2-GUS1 using Reporter-F/R primers, and ligated into pGWB235 (ref. 22) via gateway recombination. The 35S:ccdB cassette–LUC-NOS construct was generated by fusing PCR fragments of the 35S promoter from pMDC140 (ref. 23), the ccdB cassette, and the NOS terminator from pRNAi-LIC24 and LUC from pGWB235 (ref. 22). The 35S:ccdB cassette–LUC-NOS was then inserted into pCAMBIA1300 via PstI and EcoRI and designated as pGX301 for cloning 5′ leader sequences through replacement of the ApaI-flanked ccdB cassette24. Similarly, the 35S:RLUC-HA-rbs terminator construct was made through fusion of PCR fragments of 35S from pMDC140 (ref. 23), RLUC from pmirGLO (Promega, E1330), and rbs terminator from pCRG3301 (ref. 25). The 35S:RLUC-HA-rbs fragment flanked with EcoRI was inserted into pTZ-57rt (Thermo Fisher, K1213) via TA cloning to generate pGX125. The 5′ leader sequences were amplified from the Arabidopsis (Col-0) genomic DNA or synthesized by Bio Basics (New York, USA) and inserted into pGX301 followed by transferring 35S:RLUC-HA-rbs from pGX125 via EcoRI. EFR, PAB2, PAB4, and PAB8 were amplified from U21686, C104970, U10212, and U15101 (from the Arabidopsis Biological Resource Center), respectively, and fused with the amino (N) terminus of enhanced green fluorescent protein (EGFP) by PCR. Fusion fragments were then inserted between the 35S promoter and the rbs terminator to generate 35S:EFR–EGFP (pGX664), 35S:EFR (pGX665), and 35S:PAB2–EGFP (pGX694). Information on all plasmids and primers in this study can be found in Supplementary Table 6. Plants were grown on soil (Metro Mix 360) at 22 °C under 12/12-h light/dark cycles with 55% relative humidity. Mutants efr-1 (ref. 6), ers1-10 (a weak gain-of-function mutant; ERS, ethylene receptor-related gene family member)26, ein4-1 (a gain-of-function mutant; EIN4, ethylene receptor-related gene family member)27, wei7-4 (a loss-of-function mutant; WEI7, involved in ethylene-mediated auxin increase)28, eicbp.b (camta 1-3; SALK_108806; EICBP.B, an ethylene-induced calmodulin-binding protein)29, and pab2/4 (ref. 18) and pab2/8 (ref. 18) were previously described; erf7 (SALK_205018; ERF7, a homologue of the ethylene responsive transcription factor gene ERF1) and gcn2 (GABI_862B02) were from the Arabidopsis Biological Resource Center. Transgenic plants were generated using the floral dip method30. Leaves from ~24 3-week-old plants (two leaves per plant; ~1.0 g) were collected. Tissue was fast frozen and ground in liquid nitrogen. Five millilitres of cold polysome extraction buffer (PEB; 200 mM Tris pH 9.0, 200 mM KCl, 35 mM MgCl , 25 mM EGTA, 5 mM DTT, 1 mM phenylmethanesulfonylfluoride (PMSF), 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol, 1% (v/v) Brij-35, 1% (v/v) Igepal CA630, 1% (v/v) Tween 20, 1% (v/v) Triton X-100, 1% sodium deoxycholate (DOC), 1% (v/v) polyoxyethylene 10 tridecyl ether (PTE)) was added. After thawing on ice for 10 min, lysate was centrifuged at 4 °C/16,000g for 2 min. Supernatant was transferred to 40 μm filter falcon tube and centrifuged at 4 °C/7,000g for 1 min. Supernatant was then transferred into a 2-ml tube and centrifuged at 4 °C/16,000g for 15 min and this step was repeated once. Lysate (0.25 ml) was saved for total RNA extraction for making the RNA-seq library. Another 1 ml of lysate was layered on top of 0.9 ml sucrose cushion (400 mM Tris·HCl pH 9.0, 200 mM KCl, 35 mM MgCl , 1.75 M sucrose, 5 mM DTT, 50 μg ml−1 chloramphenicol, 50 μg ml−1 cycloheximide) in an ultracentrifuge tube (349623, Beckman). The samples were then centrifuged at 4 °C/70,000 r.p.m. for 4 h in a TLA100.1 rotor. The pellet was washed twice with cold water, resuspended in 300 μl RNase I digestion buffer (20 mM Tris·HCl pH 7.4, 140 mM KCl, 35 mM MgCl , 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol)10 and then transferred to a new tube for brief centrifugation. The supernatant was then transferred to another new tube where 10 μl RNase I (100 U μl−1) was added before 60 min incubation at 25 °C. 15 μl SUPERase-In (20 U μl−1) was then added to stop the reaction. The subsequent steps including ribosome recovery, footprint fragment purification, PNK treatment, and linker ligation were performed as previously reported31. Two and a half microlitres of 5′ deadenylase (NEB) were then added to the ligation system and incubated at 30 °C for 1 h. Two and a half microlitres of RecJ exonuclease (NEB) was subsequently added for 1 h incubation at 37 °C. The enzymes were inactivated at 70 °C for 20 min and 10 μl of the samples were taken as template for reverse transcription (Extended Data Fig. 2). The rest of the steps for the library construction were performed as in the reported protocol31, with the exception of using biotinylated oligos, rRNA1 and rRNA2, for Arabidopsis according to another reported method10. TRIzol LS (0.75 ml; Ambion) was added to the 0.25 ml lysate saved from the Ribo-seq library construction, from which total RNA was extracted, quantified, and qualified using Nanodrop (Thermo Fisher Scientific). Total RNA (50-75 μg) was used for mRNA purification with Dynabeads Oligo (dT) (Invitrogen). Twenty microlitres of the purified poly (A) mRNA was mixed with 20 μl 2× fragmentation buffer (2 mM EDTA, 10 mM Na CO , 90 mM NaHCO ) and incubated for 40 min at 95 °C before cooling on ice. Five hundred microlitres of cold water, 1.5 μl of GlycoBlue, and 60 μl of cold 3 M sodium acetate were then added to the samples and mixed. Subsequently, 600 μl isopropanol was added before precipitation at −80 °C for at least 30 min. Samples were then centrifuged at 4°C/15,000g for 30 min to remove all liquid and air dried for 10 min before resuspension in 5 μl of 10 mM Tris pH 8. The rest of the steps were the same as Ribo-seq library preparation with quality control data shown in Extended Data Fig. 3. To record the 35S:uORFs –LUC reporter activity, 3-week-old Arabidopsis plants were sprayed with 1 mM luciferin 12 h before infiltration with either 10 μM elf18 (synthesized by GenScript) or 10 mM MgCl as Mock. Luciferase activity was recorded in a CCD (charge-coupled device) camera-equipped box (Lightshade Company) with each exposure time of 20 min. For the dual-luciferase assay, Nicotiana benthamiana plants were grown at 22 °C under 12/12-h light/dark cycles. Dual-luciferase constructs were transformed into the Agrobacterium strain GV3101, which was cultured overnight at 28 °C in Luria-Bertani broth supplied with kanamycin (50 mg l−1), gentamycin (50 mg l−1), and rifampicin (25 mg l−1). Cells were then spun down at 2,600g for 5 min, resuspended in infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid (MES), 10 mM MgCl , 200 μM acetosyringone), adjusted to an opitcal density at 600 nm (OD ) = 0.1, and incubated at room temperature for an additional 4 h before infiltration using 1 ml needleless syringes. For elf18 induction, 10 mM MgCl (Mock) solution or 10 μM elf18 were infiltrated 20 h after the dual-luciferase construct and EFR–EGFP had been co-infiltrated at the ratio of 1:1, and samples were collected 2 h after treatment. For the PAB2–EGFP co-expression assay, Agrobacterium containing a dual-luciferase construct was mixed with Agrobacterium containing the PAB2–EGFP construct at a ratio of 1:5. Leaf discs were collected, ground in liquid nitrogen, and lysed with the PLB buffer (Promega, E1910). Lysate was spun down at 15,000g for 1 min, from which 10 μl was used for measuring LUC and RLUC activity using a Victor3 plate reader (PerkinElmer). At 25 °C, substrates for LUC and RLUC were added using the automatic injector and after 3 s shaking and 3 s delay, the signals were captured for 3 s and recorded as counts per second. For elf18-induced growth inhibition assay, seeds were sterilized in a 2% PPM solution (Plant Cell Technology) at 4 °C for 3 days and sown on MS media (1/2 MS basal salts, 1% sucrose, and 0.8% agar) with or without 100 nM elf18. Ten-day-old seedlings were weighed with ten seedlings per sample. For elf18-induced resistance to Psm ES4326, 1 μM elf18 or Mock (10 mM MgCl ) was infiltrated into 3-week-old soil-grown plants 1 day before Psm ES4326 (OD = 0.001) infection of the same leaf. Bacterial growth was scored 3 days after infection. For elf18-induced resistance to Psm ES4326 in primary transformants overexpressing PAB2 in the pab2/8 mutant (OE-PAB2), transgenic plants expressing yellow fluorescent protein (YFP) in the WT background were used as control, and both control and OE-PAB2 were selected for basta-resistance and further confirmed by PCR. For MAPK activation, 12-day-old seedlings grown on MS media were flooded with 1 μM elf18 solution and 25 seedlings were collected at the indicated time points. Protein was extracted with co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100, 0.2% (v/v) Nonidet P-40, protease inhibitor cocktail (Roche), phos-stop phosphatase inhibitor cocktail (Roche)). Antibody information and conditions can be found in Supplementary Table 6. For callose deposition, 3-week-old soil-grown plants were infiltrated with 1 μM elf18. After 20 h of incubation, leaves were collected, decolorized in 100% ethanol with gentle shaking for 4 h, and rehydrated in water for 30 min before stained in 0.01% (w/v) aniline blue in 0.01 M K PO pH 12 covered with aluminium foil for 24 h with gentle shaking. Callose deposition was observed with a Zeiss-510 inverted confocal microscope using a 405 nm laser for excitation and 420–480 nm filter for emission. PAB2–EGFP was amplified from pGX694. GA, G(A) , and G(A) were synthesized using Bio Basics (New York, USA) while poly(A) and G(A) were synthesized by IDT (https://www.idtdna.com/site). The sequences used for in vitro biotin-RNA synthesis can be found in Supplementary Table 6. In vitro transcription and translation were performed using the wheat germ translation system according to the manufacturer’s instructions (BioSieg, Japan). To make biotin-labelled RNA probes, 2 μl of 10 mM biotin-16-UTP (11388908910, Roche) was added into the transcription system. DNase I was then used to remove the DNA template. Biotin-labelled RNA (0.2 nmol) was conjugated to 50 μl streptavidin magnetic beads (65001, Thermo Fisher) according to the manufacturer’s instructions. In vitro synthesized PAB2–EGFP was incubated with biotin-labelled RNA in the glycerol-co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, 10% (v/v) glycerol, 1 mM PMSF, 20 U ml−1 Super-In RNase inhibitor, protease inhibitor cocktail (Roche)). To perform in vivo pull-down experiment, PAB2–EGFP was co-expressed with the elf18 receptor EFR (pGX665) for 40 h in N. benthamiana, which was then treated with Mock or elf18 for 2 h. Protein was extracted with glycerol-co-IP buffer and used in the pull-down assay at 4 °C for 4 h. YFP was expressed as a control. Antibody information and assay conditions can be found in Supplementary Table 6. Arabidopsis tissue (0.6 g) was ground in liquid nitrogen with 2 ml cold PEB buffer. One millilitre of crude lysate was loaded to 10.8 ml 15–60% sucrose gradient and centrifuged at 4 °C for 10 h (35,000 r.p.m., SW 41 Ti rotor). A absorbance recording and fractionation were performed as described previously32. Polysomal RNA was isolated by pelleting polysomes, and polysomal/total mRNA ratio was calculated as described previously8. About 50 mg of leaf tissue was used for total RNA extraction using TRIzol following the manufacturer’s instructions (Ambion). After DNase I (Ambion) treatment, reverse transcription was performed following the instruction of SuperScript III Reverse Transcriptase (Invitrogen) using oligo (dT). Real-time reverse-transcription polymerase chain reaction (RT–PCR) was done using FastStart Universal SYBR Green Master (Roche). Primer sequences can be found in Supplementary Table 6. Read processing and statistical methods were conducted following the criteria illustrated in Extended Data Fig. 4. Generally, Bowtie2 (ref. 33) was used to align reads to the Arabidopsis TAIR10 genome. Read assignment was achieved using HT-Seq34. Transcriptome and translatome changes were calculated using DESeq2 (ref. 35). Transcriptome fold changes (RS ) for protein-coding genes were determined using reads assigned to exon by gene. Translatome fold changes (RF ) for protein-coding genes were measured using reads assigned to CDS by gene. Translational efficiency was calculated by combining reads for all genes that passed reads per kilobase of transcript per million mapped reads (RPKM) ≥ 1 in CDS threshold in two biological replicates and normalizing Ribo-seq RPKM to RNA-seq RPKM as reported12. The criteria used for uORF prediction are shown in Extended Data Fig. 6 and were performed using systemPipeR (https://github.com/tgirke/systemPipeR). The MEME online tool36 was used to search strand-specific 5′ leader sequences for enriched consensuses compared with whole-genome 5′ leader sequences with default parameters. The density plot was presented using IGB37. The nucleotide resolution of the coverage around start and stop codons was performed using the 15th nucleotide of 30-nucleotide reads of Ribo-seq, similar as reported previously10, 38. Whole-transcriptome R-motif search was performed using the FIMO tool in the MEME suite36. LUC/RLUC ratio was first tested for normal distribution using a Shapiro–Wilk test. A two-sided Student’s t-test was used for comparison between two samples. Two-sided one-way or two-way analysis of variance was used for more than two samples, and Tukey’s test was used for multiple comparisons. GraphPad Prism 6 was used for all the statistical analyses. Unless specifically stated, sample size n means the biological replicate and experiment was performed three times with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate significant increases; NS, no significance; †††P < 0.001 indicates a significant decrease. The authors declare that the main data supporting the findings of this study are available within the article and its Source Data files. Extra data are available from the corresponding author upon request. The RNA-seq and Ribo-seq data have been deposited in Gene Expression Omnibus under accession number GSE86581.
News Article | February 15, 2017
Today Euronext (Paris:ENX) (Amsterdam:ENX) (Brussels:ENX) announced its results for the full year 2016. “The Euronext team is proud of the results we announce today for 2016. In a year marked by lower volumes on our markets that induced, as guided with our Q3 2016 results, a 4.3% decrease in revenue, we have managed to maintain our EBITDA in absolute terms stable compared to last year. Our cost base has been significantly reduced, and is now fully covered by our non-volume related revenue only. As a result, our profitability and EPS increased significantly and we are in a position to propose for the approval of our Annual General Meeting on 19 May 2017, based on our consistent dividend policy, the payment of a dividend of €1.42 per share, increased by 14.5% compared to last year. These results demonstrate Euronext’s continuous capability to service our shareholders and customers, and to deliver value against a tough trading environment. 2017 will be a critical year for our industry landscape. We will remain focused on executing our Agility for Growth strategy and maximizing opportunities that may arise, as we did with the agreement to potentially acquire LCH.Clearnet S.A.,” said Stéphane Boujnah, Chairman and CEO of the Managing Board of Euronext NV. The year 2016 was marked by a down-trend in volatility, both for cash and derivatives markets, with some volatility spikes around the UK referendum in June and the US elections in November. Ongoing uncertainty also resulted in low activity on primary markets. As a result, revenue for the full-year decreased by -4.3% to €496.4 million (2015: €518.5 million). As a reminder, 2015 was the strongest year since 2008, both in terms of trading volumes and in terms of primary markets activity. This performance results from the strict execution of our strategic plan with €15.6 million of cost savings since Q2 2016. The level of operational expenses has been reduced by some reversal of accruals for a total amount of €3.3 million. The onboarding of costs, linked to the execution of the growth initiatives, ramped up after the summer break, impacting the cost base of the Company by €2.1 million for the full year 2016 and generating €0.8 million of revenue. The relocation of our IT operations from Belfast to Porto is nearly complete, with 88 people hired in Porto, while the closure of the Belfast office will be effective at the end of Q1 2017. This strong focus on costs has allowed us to contain the impact of the reduced revenue on EBITDA, which remained stable in 2016, to €283.9 million, representing a margin of 57.2% (2015: 54.7% or €283.8 million). Depreciation and Amortization decreased by -11.6% in 2016, to €15.1 million (2015: €17.1 million). The impact of the end of the accelerated depreciation of assets, resulting from relocations of our offices in Belgium and France, and of the roll-out of a portion of UTP amortization were partially offset by the start of Interbolsa T2S project depreciation in Q3 2016. Operating profit before exceptional items was €268.8 million, an increase of +0.8% compared to 2015 (€266.8 million). €10.0 million of exceptional items were booked in 2016 compared to €28.7 million in 2015. These costs consisted mainly of M&A related costs, for €3.3 million, and of restructuring costs for €7.1 million in 2016. In 2015 some reversal of provision related to Cannon Bridge House premises for €14.4 million had partially compensated the €35.0 million of restructuring costs. Results from equity investment increased by 29.8% in 2016, to €6.0 million (2015: €4.6 million), due to the increase in dividends paid by Euroclear (both direct and indirect stakes), and to €0.7 million of dividend paid this year by LCH Group (2015: zero). Income tax for the full year 2016 was €67.0 million, representing an effective tax rate for the year of 25.4% (2015: 27.6%), both years being impacted by the release of tax provisions (€16.3 million in 2016 and €13.9 million in 2015). As a result, the net profit for 2016 increased by 14.1%, to €197.0 million (2015: €172.7 million). This represents an EPS (basic) of € 2.83 (€2.82 fully diluted) in 2016 compared to €2.47 (€2.46 fully diluted) in 2015. The number of shares used for the basic calculation was 69,526,615 for 2016, compared to 69,851,603 in 2015. As of 31 December 2016, the Company had cash and cash equivalents of €174.5 million, and total debt of €69.1 million, after the partial debt repayment of €40 million in Q3 2016 and the €86.2 million dividend payment in Q2 2016. Revenue was €68.7 million in 2016, a decrease of -2.6% compared to the €70.5 million achieved in 2015. This decrease was driven by the fall in IPO fees and reduced listing fees for ETPs in comparison with 2015. In 2015, large transactions such as Lafarge-Holcim, Altice, Amundi and ABN Amro were key contributors to the listing revenue performance. In 2016 much uncertainty and volatility induced low activity on primary markets even though two benchmark transactions took place on our market (Coca Cola European Partners and AB Inbev). 28 new listings took place in 2016, raising €3.7 billion, compared to 52 listings for €12.4 billion during 2015, due to the uncertainty created by various global factors throughout the year (UK referendum in June, US elections in November, etc.). Euronext continued to be the venue of choice for Tech SMEs. Although the number of new SME listings decreased from 36 in 2015 to 23 in 2016, these companies raised €1,430 million in 2016 compared to €1,337 million in 2015. Amongst these listings, Euronext markets registered interesting small and mid cap deals in 2016, confirming the relevance of its strategy and its willingness to develop specific offers for these kinds of companies: Maisons du Monde (a regional based mid-cap furniture company), Nextstage (the first listing of a private equity fund on Euronext Paris) as well as Geneuro and Noxxon Pharma (listing of a Swiss and a German Biotech). In 2016, Euronext saw an all time high on secondary issues, to €56.4 billion, up 72% compared to €32.9 billion in 2015. Debt capital markets issuance also increased in 2016, with €80.6 billion raised by corporates on our markets in 2016 vs €66.4 billion in 2015. In total €140.7 billion in equity and debt was raised on our markets in 2016, compared to €111.7 billion in 2015. Full-year 2016 volumes in Cash Trading were down due to the macro factors mentioned above, translating into reduced investor confidence post Brexit and lower volatility, particularly in Q3. Average daily volumes were € 7.0 billion (-15.3% compared to 2015). However, efficient yield management enabled us to partially de-correlate revenue from volumes, thus minimising the impact on our top line. Yield averaged 0.50 bps in 2016, compared to 0.47 bps in 2015, a +7.8% increase. Revenue for the full-year is down by -8.4% to €180.7 million (2015: €197.2 million). Average market share for the year was 61.0%. The year ended strongly with December market share at 65.2%, following lows in October at 58.6% as a result of the launch of several initiatives (notably new fee scheme for non-member proprietary flow and a new best execution service for retail investors). Average market share in 2015 was 63.5%, in a context of buoyant market activity. Activity on ETFs followed a similar trend, with an average daily transaction value at €554 million, down 9.7% compared to €613 million in 2015. However, we recorded a record number of new ETFs listed on our markets, at 137, bringing the total number of ETFs listed on our markets at 790 by year end (up +13.5% versus end of 2015: 696). Our client alignment is demonstrated by Euronext winning four awards during 2016, being consistently recognised as the Best ETF Exchange in Europe. A new issuer, UBS AG has been onboarded in January 2016. Derivatives trading revenue decreased by -9.8% in 2016 compared to 2015, amounting to €40.1 million (2015: €44.5 million) as a result of the lower volatility . Index product trading volumes declined by -9.8% in 2016 compared to 2015 (average daily volumes of 214,520 lots in 2016 vs 237,881 in 2015). Trading activity on our individual equity options franchise decreased by -5.2% during 2016 compared to 2015 as market volatility was higher last year (average daily volumes was 222,942 lots in 2016 vs 235,135 in 2015). A materially sub-standard 2016 French wheat harvest have impacted volumes in commodity products that declined by -4.1% in 2016 compared to 2015 (average daily volumes of 53,536 lots in 2016 vs 55,843 lots in 2015). Market data and indices revenue delivered a strong performance in 2016 with revenue up + 6.0% compared to 2015, to €105.7 million (2015: €99.8 million), benefiting from the positive impact of the new products and services launched during the course of 2015 as well as from some fee adjustments. Consistent with the trend in derivatives trading mentioned above, clearing revenue decreased by -7.6%, from €51.9 million in 2015 to €48.0 million in 2016. Revenue from Interbolsa in Portugal in 2016 was €19.6 million; stable compared to the €19.7 million achieved in 2015. Revenue from market solutions decreased by -3.3% in 2016, to €33.0 million (2015: €34.1 million). The decrease in software solution revenue during transition to Optiq platform was partially offset by the introduction of a new Market Abuse Regulation compliance service in July. For comparative purposes, the company provides unaudited non-IFRS measures including: We define the non-IFRS measures as follows: Non-IFRS financial measures are not meant to be considered in isolation or as a substitute for comparable IFRS measures and should be read only in conjunction with the consolidated financial statements. Update on the Agility for Growth plan Since the publication of its strategic plan in May 2016, Euronext has started the deployment of the Agility for Growth plan and has achieved a number of significant milestones. 2016 has been a year of intense focus on our technology capabilities. The relocation of IT operations from Belfast to Porto is being delivered on time and within budget. As of 31 December 2016, 88 people had been onboarded in Porto and the Belfast office will be closed, as planned, at the end of Q1 2017. Over time this will enable us to operate more efficiently at lower cost. The development of Optiq, our new trading platform, is taking shape. The customer test platform opened as scheduled in November 2016 for market data. The launch of the new market data infrastructure for cash and derivatives products is scheduled for May 2017. The migration to Optiq for the cash businesses will be completed in October 2017 and will include MiFID II compliance. By the end of 2016, Euronext already achieved 70% of the gross cost reduction programme (based on 2016 cost base that was also reduced by €3.3 million of non-recurring reversals of accruals), thanks to strong cost discipline and due to fact that the onboarding of costs related to the growth initiatives started only after the summer break. Euronext has begun enhancing its agility, demonstrated by a number of product launches leveraging the regulatory environment, to help clients comply with major changes implied by MiFID II or MAR. Thanks to reinforced client centricity, Euronext also identified selected opportunities that translate into innovative projects such as the involvement in a Blockchain consortium and new areas of business, evidenced by the expansion of our fixed income offering. In addition Euronext announced several milestones that will strengthen its core business: The Agility for Growth strategic plan is already bearing its first fruits and delivering enhanced shareholder value. If approved during the Annual General Meeting on 19 May 2017, the dividend paid of €1.42 per share will represent an increase of 14.5% compared to the dividend paid last year. Stronger profitability combined with consistent dividend policy translates into increased shareholder remuneration while Euronext maintains significant flexibility to deploy capital. 2016 has been the first year of capital deployment since IPO; Euronext will pursue the implementation of its strategy in 2017 where organic milestones and various products and services launches will be combined with additional acquisitions and partnerships. Euronext is the leading pan-European exchange in the Eurozone with more than 1,300 listed issuers worth close to €3.3 trillion in market capitalisation as of end December 2016, an unmatched blue chip franchise consisting of 25 issuers in the EURO STOXX 50® benchmark and a strong diverse domestic and international client base. Euronext operates regulated and transparent equity and derivatives markets. Its total product offering includes Equities, Exchange Traded Funds, Warrants & Certificates, Bonds, Derivatives, Commodities and Indices. Euronext also leverages its expertise in running markets by providing technology and managed services to third parties. Euronext operates regulated markets, Alternext and the Free Market; in addition it offers EnterNext, which facilitates SMEs’ access to capital markets. For the latest news, find us on Twitter (twitter.com/euronext) and LinkedIn (linkedin.com/euronext). This press release is for information purposes only and is not a recommendation to engage in investment activities. This press release is provided “as is” without representation or warranty of any kind. While all reasonable care has been taken to ensure the accuracy of the content, Euronext does not guarantee its accuracy or completeness. Euronext will not be held liable for any loss or damages of any nature ensuing from using, trusting or acting on information provided. No information set out or referred to in this publication may be regarded as creating any right or obligation. The creation of rights and obligations in respect of financial products that are traded on the exchanges operated by Euronext’s subsidiaries shall depend solely on the applicable rules of the market operator. All proprietary rights and interest in or connected with this publication shall vest in Euronext. This press release speaks only as of this date. Euronext refers to Euronext N.V. and its affiliates. Information regarding trademarks and intellectual property rights of Euronext is located at www.euronext.com/terms-use.
News Article | November 2, 2016
Geneva, Switzerland, Nov. 02, 2016 (GLOBE NEWSWIRE) -- HDBaseT Automotive technology enables simultaneous transmissions of up to 6Gbps of high-definition video & audio, USB, data, and power over a single twisted-pair cable Hod Hasharon, Israel; Geneva, Switzerland - November 2, 2016 - Valens and STMicroelectronics announced today their collaboration to bring HDBaseT Automotive into the next-generation of connected cars. The highly efficient technology optimizes in-vehicle connectivity by enabling the transmission of reliable 6Gbps high-throughput infotainment, road safety, and automotive-control content over a low-cost infrastructure with near-zero latency. Valens, as the inventor of HDBaseT and founder of the HDBaseT Alliance, brings the technology and expertise to accomplish the goal of commercializing HDBaseT-enabled vehicles. STMicroelectronics will contribute its extensive design and manufacturing experience and know-how in compliance with the strict automotive quality and reliability requirements. "As Valens meets significant milestones in our vision to optimize in-vehicle connectivity, we are excited to welcome ST as our major partner in pursuing this goal. ST will help us accelerate the introduction of HDBaseT Automotive to the market," said Dror Jerushalmi, CEO, Valens. "With long leadership in serving the Automotive industry and Smart Driving as one of our key focus areas, we can see the potential of HDBaseT Automotive as a high-throughput, low-latency, low-cost technology. That's why we are excited to be part of this project to help Valens bring the HDBaseT Automotive technology and devices to the market, with the highest levels of quality and reliability expected," said Fabio Marchio, Vice President and General Manager, Automotive Digital Division, STMicroelectronics. Learn more about HDBaseT Automotive at the Electronica 2016 show in Munich, on November 8th-11th, at the HDBaseT Alliance booth (Hall A6, Booth 264). To schedule an appointment, contact us at firstname.lastname@example.org. HDBaseT Automotive is the only technology today that enables the tunneling of up to 6Gbps of video, audio, data, USB and more, with native networking capabilities over a single unshielded twisted-pair (UTP) cable for up to 15m (50ft). HDBaseT Automotive also allows for daisy-chaining and multistreaming, to simplify and optimize in-vehicle connectivity. For more information about Valens' HDBaseT Automotive solution, click here. About Valens Established in 2006, Valens provides semiconductor products for the distribution of uncompressed ultra-high-definition (HD) multimedia content. The company's HDBaseT technology enables long-reach connectivity of devices over a single cable and is a global standard for advanced digital media distribution. Valens is a private company headquartered in Israel. For more information, visit www.valens.com. About STMicroelectronics ST is a global semiconductor leader delivering intelligent and energy-efficient products and solutions that power the electronics at the heart of everyday life. ST's products are found everywhere today, and together with our customers, we are enabling smarter driving and smarter factories, cities and homes, along with the next generation of mobile and Internet of Things devices. By getting more from technology to get more from life, ST stands for life.augmented. In 2015, the Company's net revenues were $6.9 billion, serving more than 100,000 customers worldwide. Further information can be found at www.st.com. A photo accompanying this announcement is available at http://www.globenewswire.com/NewsRoom/AttachmentNg/67933ea3-2204-491d-b1cc-5d113757aa51
News Article | January 27, 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. Bovine Pol II was prepared as described11 with modifications. Unless otherwise noted, all steps were completed at 4 °C. Protease inhibitors included 1 mM PMSF, 1 mM benzamidine, 60 μM leupeptin, and 200 μM pepstatin. Calf thymus was homogenized for 3 min in buffer A (50 mM Tris, pH 7.9 at 4 °C, 1 mM EDTA, 10 μM ZnCl , 10% glycerol, 1 mM DTT, protease inhibitors) using a 2 l blender (Waring). The homogenized material was centrifuged and the supernatant filtered through two layers of Miracloth. A 5% solution of polyethyleneimine, pH 7.9 at 25 °C, was added to a final concentration of 0.02%, and the material was stirred for 10 min then centrifuged. The resulting pellets were washed with buffer A before resuspension in buffer A (0.15 M ammonium sulfate). After centrifugation, the conductivity of the supernatant was adjusted to that of buffer A (0.2 M ammonium sulfate), and the resulting material was loaded on a 225-ml MacroPrepQ column equilibrated in buffer A (0.2 M ammonium sulfate). The column was washed with two column volumes of buffer A (0.2 M ammonium sulfate), followed by Pol II elution with buffer A (0.4 M ammonium sulfate). The eluate was precipitated by addition of finely ground ammonium sulfate added to 50% saturation, and pellets were collected by centrifugation. The pellets were resuspended in buffer A, and the conductivity was adjusted to that of buffer A (0.15 M ammonium sulfate). The material was clarified by centrifugation, and further purified using a 5-ml gravity flow column of 8WG16 (αRPB1 CTD) antibody-coupled sepharose equilibrated in buffer A (0.15 M ammonium sulfate). After application of the input material, the antibody column was washed with five column volumes of buffer A (0.5 M ammonium sulfate), sealed, and allowed to equilibrate to room temperature (20–25 °C) for 15 min. Pol II was eluted using buffer A (0.5 M ammonium sulfate, 50% (v/v) glycerol), and Pol-II-containing fractions were immediately mixed with buffer A (2 mM DTT, lacking glycerol and protease inhibitors). The diluted material was centrifuged and subjected to anion exchange chromatography using a UNO-Q column equilibrated in buffer A (0.1 M ammonium sulfate, 2 mM DTT, lacking protease inhibitors). Pol II was eluted using a linear gradient from 0.1 M to 0.5 M ammonium sulfate in buffer A (2 mM DTT, lacking protease inhibitors). For the purification of 12-subunit bovine Pol II, the Gdown1-free Pol II fraction was applied to a Sephacryl S-300 HiLoad sizing column equilibrated in buffer B (150 mM NaCl, 5 mM HEPES pH 7.25 at 25 °C, 10 μM ZnCl , 10 mM DTT). For the purification of bovine Pol II containing Gdown1, the Gdown1-free Pol II fraction was incubated with a 3× molar excess of human Gdown1 for 1 h at 4 °C before application to the Sephacryl S-300 HiLoad sizing column. Pol-II-containing fractions were concentrated using a 100-kDa cutoff Amicon concentrator to a final concentration of 2–4 mg ml−1. Gene-optimized human Gdown1 (Life Technologies) was cloned into pOPINB (N-terminal His tag and 3C protease site). After transformation, Escherichia coli BL21(DE3)RIL cells were grown at 37 °C in Lysogeny broth (LB) medium to an absorbance at 600 nm, A , of 0.5 before protein expression with 0.5 mM IPTG for 3–4 h at 37 °C. Subsequent steps were completed at 4 °C unless otherwise noted. Cells were lysed by sonication in buffer C (50 mM HEPES pH 7.5 (25 °C), 300 mM NaCl, 1 mM CaCl , 10% glycerol) supplemented with 10 mM imidazole, 1 mM PMSF, 1 mM benzamidine, 1 mM sodium metabisulfite, 1 mM DTT, and 2 μg ml−1 DNase I. Cleared lysate was subjected to affinity chromatography using Ni-NTA agarose (Qiagen), and excess chaperone was removed by washing the resin with a 5 mM ATP and 2 mg ml−1 denatured E. coli protein wash at room temperature in buffer C supplemented as above containing 30 mM imizdazole. Protein was eluted with buffer C supplemented as above, but lacking DNase I and containing 250 mM imidazole. Elutions were exchanged into buffer C supplemented with 10 mM imidazole and 1 mM DTT via a PD10 desalting column, followed by 3C protease cleavage at 4 °C overnight. Cleaved Gdown1 was subjected to reverse chromatography (Ni-NTA agarose) followed by dilution with buffer D (50 mM HEPES pH 7.5 (25 °C), 1 mM CaCl , 10% glycerol, 2 mM DTT) to a conductivity of buffer D containing 0.05 M NaCl. Diluted protein was subjected to cation exchange chromatography (MonoS 5/50) to remove additional chaperone, and eluted with a linear gradient from 0.05 M to 0.5 M NaCl in buffer D. The conductivity of the Gdown1-containing fractions was again adjusted to that of buffer D containing 0.05 M NaCl, and applied to a MonoQ 5/50 anion exchange column. Gdown1 was eluted using a linear gradient from 0.05 M to 0.5 M NaCl in buffer D. Fractions containing purified Gdown1 were pooled, resulting in a final concentration of 1–1.5 mg ml−1. Yield was approximately 2.5 mg per 2 l of E. coli culture. Purification of human SPT4 and SPT5 was as described31, with adaptations. Gene-optimized human SPT5 (pMK vector, no tag) and SPT4 were purchased from Life Technologies, and SPT4 was recloned into pOPINJ (N-terminal HIS6 and GST tags followed by a 3C protease cleavage site). SPT4 and SPT5 vectors were co-transformed into E. coli BL21(DE3)RIL cells, which were then grown at 37 °C in LB medium supplemented with 10 μM ZnCl to A = 0.6. Expression was induced with 1 mM IPTG for 18 h at 18 °C. Cells were lysed by sonication in buffer E (25 mM Tris pH 7.4 (4 °C), 500 mM NaCl, 10 μM ZnCl , 5 mM DTT) supplemented with 5 mM imidazole and protease inhibitors (1 mM PMSF, 1 mM benzamidine, 60 μM leupeptin, and 200 μM pepstatin). Soluble material was passed over a Ni-NTA agarose column and washed with ten column volumes each of buffer E supplemented with 20 mM or 40 mM imidazole before elution in buffer E supplemented with 300 mM imidazole. Eluted protein was cleaved with 3C protease during overnight dialysis (4 °C) against buffer E, then subjected to reverse chromatography. Protein was passed over a HiTrap Q HP anion exchange column to remove DNA, and the flow-through fraction containing SPT4/5 was concentrated using a 50-kDa cutoff Amicon concentrator to 1–4 mg ml−1. The nucleic-acid scaffold (Metabion) was only slightly modified from what was used for a yeast Pol II EC crystal structure20 by mutation of the DNA templating base from C to A to generate a fully mismatched open bubble and by removing the upstream and downstream CC overhangs. A 1.5× molar excess of pre-annealed template DNA (sequence 5′-AAGCTCAAGTACTTAAGCCTGGTCATTACTAGTACTGCC-3′), non-template DNA (sequence 5′-GGCAGTACTAGTAAACTAGTATTGAAAGTACTTGAGCTT-3′), and RNA (sequence 5′-UAUAUGCAUAAAGACCAGGC-3′) were incubated with Pol II–Gdown1 at 4 °C for 10 min, then 20 °C for 15 min. To increase the randomness of Pol II EC particle orientations, the resulting complex (0.85 μM) was crosslinked with 3 mM BS3 (Thermo) for 30 min at 30 °C, then quenched with 50 mM ammonium bicarbonate. Crosslinked complex was applied to a Superdex 200 increase 10/300 GL column equilibrated in buffer B (150 mM NaCl, 5 mM HEPES pH 7.25 at 25 °C, 10 μM ZnCl , 10 mM DTT). The nucleic-acid-containing peak was concentrated to ~0.3 mg ml−1 as described above and used immediately for cryo-EM grid preparation. Four microlitres of sample were applied to glow-discharged Quantifoil R 3.5/1 holey carbon grids, which were then blotted and plunge-frozen in liquid ethane using a Vitrobot (FEI). Data were acquired using an FEI Titan Krios operated in energy-filtered transmission electron microscopy (EFTEM) mode at 300 kV equipped with a Gatan K2 Summit direct detector. Automated data collection was performed using the TOM toolbox32. Movie images were collected at a nominal magnification of ×37,000 (1.35 Å per pixel) in ‘super-resolution mode’ (0.675 Å per pixel) at a dose rate of about nine electrons per pixel per second. Two movies were acquired per hole, and each movie encompassed a total dose of ~43 electrons per square ångström over 8 s fractionated into 40 frames (0.2 s each). Defocus values ranged from −0.6 μm to −3.1 μm. Movies were aligned and binned as previously described15, 33, except that images were not partitioned into quadrants. Unless otherwise noted, processing was performed using RELION 1.3 (ref. 34). Contrast transfer function (CTF) parameters were estimated using CTFFIND4 (ref. 35). Initial 2D classes were generated after semi-automated picking of ~10,000 particles (box size 204) using e2boxer.py (EMAN2)36. Sixteen distinct classes were low-pass filtered to 25 Å resolution and used as templates for autopicking37, resulting in 476,100 particles selected from 1,172 micrographs. The autopicked particles were subjected to manual screening followed by screening by 2D classification, yielding an input data set of 409,401 particles. A previously published 22-Å-resolution cryo-negative stain reconstruction of human Pol II (EMD-1282)4 filtered to 50 Å was used as an initial reference for 3D refinement. Before any 3D classification, data were subjected to the particle polishing movie-processing algorithm of RELION 1.3 (ref. 38), resulting in an improvement in resolution from 3.7 Å to 3.4 Å. Three-dimensional classification was performed without image alignment as outlined in Extended Data Fig. 2. Masks were chosen to include either the entire Pol II EC or a smaller region of interest. The full data set was used as input for the classification of heterogeneity in the region of upstream DNA density. Only classes displaying strong clamp density were used as input for classification of conformations of the RPB4–RPB7 stalk. After classification, data were again subjected to 3D refinement with a 50 Å filtered reference volume. B-factors were automatically estimated in RELION39 and resolutions were reported on the basis of the gold-standard Fourier shell correlation (FSC) (0.143 criterion)40 as described41. The Pol II EC1 reconstruction was calculated from 264,134 particles to 3.4 Å resolution and sharpened with a B-factor of −137 Å2. The Pol II EC2 (improved RPB4–RPB7 stalk density) reconstruction was calculated from 219,265 particles to 3.6 Å and sharpened with a B-factor of −128 Å2. The Pol II EC3 (improved upstream DNA density) reconstruction was calculated from 184,122 particles to 3.7 Å and sharpened with a B-factor of −123 Å2. Focused refinements were achieved by continuing a refinement of the full data set from the iteration at which local searches began, but replacing the mask encompassing the entire Pol II EC density with a soft mask around the region of interest and allowing the refinement to continue to convergence. Local resolution was calculated using a sliding window method as described15, 42, except that a single pair of half maps was used per estimation and local resolution was not capped at the nominal value. Figures were generated using UCSF Chimera43. Alignments for each Pol II subunit were generated using the Homo sapiens, Bos taurus, Drosophila melanogaster, Schizosaccharomyces pombe, and S. cerevisiae sequences followed by alignment using Clustal Omega44. The Pol II crystal structure PDB 4BBS45 was chosen as a reference, as it displayed good stereochemistry and included the most complete model of the RPB2 protrusion domain. A starting model of ten-subunit Pol II was generated using the CCP4 (ref. 46) program chainsaw47 along with the alignment of the B. taurus and S. cerevisiae sequences. Conserved amino acids were retained, and non-conserved amino acids were pruned back to the gamma atom. The starting model was placed in the Pol II EC density by fitting in UCSF Chimera43, followed by fitting of rigid body groups in COOT48. Groups for rigid body refinement were chosen on the basis of observations from Pol II X-ray studies and visual inspection of the initial fit to the density. In regions of sufficient density quality, the model was manually adjusted to include complete side chains, and missing or divergent regions were built in COOT using B-factor sharpened maps. Multiple maps were used during model building, including the densities for the refinement using all data, Pol II EC1, Pol II EC2, Pol II EC3, and focused refinements. To generate a complete EC model, one molecule (chains A and B) of the crystal structure of human RPB4–RPB7 (PDB 2C35)49 was docked into the Pol II EC2 map. Regions near the ten-subunit core were adjusted manually to fit the density. Amino-acid side chains (previously stubbed) were added to the model if side chain density was visible. Ideal B-form DNA was manually fitted into the Pol II EC3 upstream DNA density. To improve backbone geometry, the EC model was subjected to PHENIX real space refinement (global minimization and ADP refinement) into one of the three unsharpened EC maps using Ramachandran, rotamer, and nucleic-acid restraints50. EC3 was used for refinement of the upstream DNA (chain N residues 1–13 and chain T residues 27–39). Because of the lower local resolution of the distal end of the upstream DNA, residues 1–10 of the non-template strand and residues 29–39 of the template strand were replaced with ideal B-form DNA that had been aligned to the refined upstream nucleic acids. EC2 was used for refinement of RPB4 and RPB7, and EC1 was used for the remaining model (EC body). The EC body was additionally refined as described above using the sharpened EC1 map to better position well-resolved side chains and nucleic acids within the density50. The final model was validated using Molprobity51, EMRinger52, and the FSC of the final model versus the EC1 map (Extended Data Fig. 3b). For model versus map FSC calculations, the EC1 map was masked using the RELION-generated soft automask used in postprocessing. Nucleic-acid scaffold used to assemble Pol II EC complexes was modified to include a 50 nucleotide RNA (sequence 5′-GAACGAGAUCAUAACAUUUGAACAAGAAUAUAUAUACAUAAAGACCAGGC-3′), as previous data have shown that DSIF affinity for ECs is increased as RNA length increases53. RNA was produced and purified as previously described54. A twofold molar excess of pre-annealed RNA and template DNA was incubated with Pol II for 20 min at 25 °C, followed by incubation with fourfold excess of non-template DNA for an additional 20 min at 25 °C. A fivefold molar excess of DSIF was incubated with the resulting Pol II EC for 20 min at 25 °C. Pol II–DSIF EC sample was applied to two consecutive Superdex 200 10/300 size-exclusion columns equilibrated in buffer B (150 mM NaCl, 5 mM HEPES pH 7.25 at 25 °C, 10 μM ZnCl , 10 mM DTT). Purified Pol II–DSIF EC was concentrated to ~0.5 mg ml−1 (~0.74 μM) and crosslinked with 3 mM BS3 (BS3-d0/d12, Creative Molecules) as described above. Crosslinked sample was again applied to two Superdex 200 10/300 columns, resulting in ~ 25 μg material. Sample was digested with trypsin, and analysed as previously described15, 55. Pol II–DSIF EC complexes were prepared as described above. Sample (~30 μg ml−1) was applied to glow-discharged 400 mesh copper grids coated with continuous carbon (Plano EM) for 1 min, washed on 500 μl water for 30 s, floated for 20 s on three consecutive 20-μl drops of 2% uranyl formate stain, and blotted to remove excess stain. Data were collected using an FEI Tecnai Spirit operated at 120 kV and a magnification of ×90,600. Micrographs were collected using a defocus range from −1.0 to −1.5 μm with an FEI Eagle CCD (charge-coupled device) camera binned 2× (image dimensions 2,048 pixels × 2,048 pixels) at a pixel size of 3.31 Å. Semi-automatic picking using e2boxer.py (EMAN2) yielded 11,531 particles from 120 micrographs. Data were subjected to 3D classification in RELION (eight classes, no CTF correction) using the cryo-negative stain reconstruction of human Pol II (EMD-1282)4 low-pass filtered to 60 Å as an initial reference. The two highest populated classes (comprising 85% of the data) were further classified into two classes each, for a total of four classes in which Pol II features beyond 60 Å were recognizable. Two classes did not have discernible additional density compared with Pol II (42% of data). A third class (28% of the data) displayed additional density near the RPB4–RPB7 stalk. A final class (15% of the data) showed additional density over the Pol II DNA binding cleft, as well as additional density near the RPB4–RPB7 stalk. Refinement of this subset of data (1,630 particles) resulted in a 3D reconstruction at 26 Å resolution, revealing extra density consistent with results from crosslinking coupled to mass spectrometry, previous DSIF–RNA crosslinking56, and the published interaction between SPT5 and the Pol II clamp coiled-coil motif26. Activity assays were performed as described57, with modifications. For reactions using fully complementary template and non-template DNA sequences, Pol II ECs were assembled stepwise beginning with either 12-subunit bovine Pol II or 12-subunit bovine Pol II in complex with human Gdown1, as indicated. Per reaction, Pol II was pre-assembled for 20 min at 28 °C with a 0.5 molar ratio of 5′ 6-FAM-labelled 20-nucleotide RNA annealed to template DNA, followed by incubation with a 1.0 molar ratio of fully complementary non-template DNA. The RNA and DNA sequences were the same as for the Pol II EC, except for an additional 46 nucleotides of downstream DNA. The template DNA sequence was 5′-ACAAATTACTGGGAAGTCGACTATGCAATACAGGCATCATTTGATCAAGCTCAAGTACTTAAGCCTGGTCATTACTAGTACTGCC-3′; the non-template DNA sequence was 5′-GGCAGTACTAGTAATGACCAGGCTTAAGTACTTGAGCTTGATCAAATGATGCCTGTATTGCATAGTCGACTTCCCAGTAATTTGT-3′, and RNA sequence was 5′-UAUAUGCAUAAAGACCAGGC-3′. Transcription was allowed to proceed for 10 min at 30 °C in the presence of 1–100 μM nucleoside triphosphates (NTPs) as indicated, and 0.2 pmol product per reaction was visualized on a 15% denaturing urea polyacrylamide gel. Transcription assays were also performed on the bubble scaffold used for structural studies. Pol II–EC complexes were prepared as described for cryo-EM, except that the samples were not crosslinked and the 20-nucleotide RNA used for assembly was 5′-labelled with 6-FAM. Assembled complex was incubated with 10–1,000 μM UTP at 30 °C for 10 min, allowing the extension of the RNA by two additional nucleotides. Product was visualized on a 20% denaturing urea polyacrylamide gel and imaged using a Typhoon FLA 9500.
News Article | March 16, 2016
No statistical methods were used to predetermine sample size for biochemical or cell-based assays, or for pharmacokinetic studies. Investigators were not blinded to outcome assessment during these investigations. For GS-5734 efficacy assessments in nonhuman primates, statistical power analysis was used to predetermine sample size, and subjects were randomly assigned to experimental group, stratified by sex and balanced by body weight. Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals and outcome.
GS-5734, Nuc, and NTP were synthesized at Gilead Sciences, Inc., and chemical identity and sample purity were established using NMR, HRMS, and HPLC analysis (Supplementary Information). The radiolabelled analogue [14C]GS-5734 (specific activity, 58.0 mCi mmol−1) was obtained from Moravek Biochemicals (Brea, California) and was prepared in a similar manner described for GS-5734 using [14C]trimethylsilylcyanide (Supplementary Information). Small molecule X-ray crystallographic coordinates and structure factor files have been deposited in the Cambridge Structural Database (http://www.ccdc.cam.ac.uk/) and accession numbers are supplied in the Supplementary Information.
RSV A2 was purchased from Advanced Biotechnologies, Inc. EBOV (Kikwit and Makona variants), Sudan virus (SUDV, Gulu), Marburg virus (MARV, Ci67), Junín virus (JUNV, Romero), Lassa virus (LASV, Josiah), Middle East respiratory syndrome virus (MERS, Jordan N3), Chikungunya virus (CHIV, AF 15561), and Venezuelan equine encephalitis virus (VEEV, SH3) were all prepared and characterized at the United States Army Medical Research Institute for infectious diseases (USAMRIID). EBOV containing a GFP reporter gene (EBOV–GFP), EBOV Makona (Liberia, 2014), and MARV containing a GFP reporter gene (MARV–GFP) were prepared and characterized at the Centers for Disease Control and Prevention26, 27.
HEp-2 (CCL-23), PC-3 (CCL-1435), HeLa (CCL-2), U2OS (HTB-96), Vero (CCL-81), HFF-1 (SCRC-1041), and HepG2 (HB-8065) cell lines were purchased from the American Type Culture Collection. Cell lines were not authenticated and were not tested for mycoplasma as part of routine use in assays. HEp-2 cells were cultured in Eagle’s Minimum Essential Media (MEM) with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 100 U ml−1 penicillin and streptomycin. PC-3 cells were cultured in Kaighn’s F12 media supplemented with 10% FBS and 100 U ml−1 penicillin and streptomycin. HeLa, U2OS, and Vero cells were cultured in MEM supplemented with 10% FBS, 1% l-glutamine, 10 mM HEPES, 1% non-essential amino acids, and 1% penicillin/streptomycin. HFF-1 cells were cultured in MEM supplemented with 10% FBS and 0.5 mM sodium pyruvate. HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and 0.1 mM non-essential amino acids. The MT-4 cell line was obtained from the NIH AIDS Research and Reference Reagent Program and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and 2 mM l-glutamine. The Huh-7 cell line was obtained from C. M. Rice (Rockefeller University) and cultured in DMEM supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and non-essential amino acids.
Primary human hepatocytes were purchased from Invitrogen and cultured in William’s Medium E medium containing cell maintenance supplement. Donor profiles were limited to 18- to 65-year-old nonsmokers with limited alcohol consumption. Upon delivery, the cells were allowed to recover for 24 h in complete medium with supplement provided by the vendor at 37 °C. Human PBMCs were isolated from human buffy coats obtained from healthy volunteers (Stanford Medical School Blood Center, Palo Alto, California) and maintained in RPMI-1640 with GlutaMAX supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin. Rhesus fresh whole blood was obtained from Valley Biosystems. PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation. Briefly, blood was overlaid on 15 ml Ficoll-Paque (GE Healthcare Bio-Sciences AB), and centrifuged at 500g for 20 min. The top layer containing platelets and plasma was removed, and the middle layer containing PBMCs was transferred to a fresh tube, diluted with Tris buffered saline up to 50 ml, and centrifuged at 500g for 5 min. The supernatant was removed and the cell pellet was resuspended in 5 ml red blood cell lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.5). To generate stimulated PBMCs, freshly isolated quiescent PBMCs were seeded into a T-150 (150 cm2) tissue culture flask containing fresh medium supplemented with 10 U ml−1 of recombinant human interleukin-2 (IL-2) and 1 μg ml−1 phytohaemagglutinin-P at a density of 2 × 106 cells ml−1 and incubated for 72 h at 37 °C. Human macrophage cultures were isolated from PBMCs that were purified by Ficoll gradient centrifugation from 50 ml of blood from healthy human volunteers. PBMCs were cultured for 7 to 8 days in in RPMI cell culture media supplemented with 10% FBS, 5 to 50 ng ml−1 granulocyte-macrophage colony-stimulating factor and 50 μM β-mercaptoethanol to induce macrophage differentiation. The cryopreserved human primary renal proximal tubule epithelial cells were obtained from LifeLine Cell Technology and isolated from the tissue of human kidney. The cells were cultured at 90% confluency with RenaLife complete medium in a T-75 flask for 3 to 4 days before seeding into 96-well assay plates. Immortalized human microvascular endothelial cells (HMVEC-TERT) were obtained from R. Shao at the Pioneer Valley Life Sciences Institute28. HMVEC-TERT cells were cultured in endothelial basal media supplemented with 10% FBS, 5 μg of epithelial growth factor, 0.5 mg hydrocortisone, and gentamycin/amphotericin-B.
RNA POLII was purchased as part of the HeLaScribe Nuclear Extract in vitro Transcription System kit from Promega. The recombinant human POLRMT and transcription factors mitochondrial transcription factors A (mtTFA or TFAM) and B2 (mtTFB2 or TFB2M) were purchased from Enzymax. RSV ribonucleoprotein (RNP) complexes were prepared according to a method modified from ref. 29.
The intracellular metabolism of GS-5734 was assessed in different cell types (HMVEC and HeLa cell lines, and primary human and rhesus PBMCs, monocytes and monocyte-derived macrophages) following 2-h pulse or 72-h continuous incubations with 10 μM GS-5734. For comparison, intracellular metabolism during a 72-h incubation with 10 μM of Nuc was completed in human monocyte-derived macrophages. For pulse incubations, monocyte-derived macrophages isolated from rhesus monkeys or humans were incubated for 2 h in compound-containing media followed by removal, washing with 37 °C drug-free media, and incubated for an additional 22 h in media which did not contain GS-5734. Human monocyte-derived macrophages, HeLa and HMVEC were grown to confluence (approximately 0.5, 0.2, and 1.2 × 106 cells per well, respectively) in 500 μl of media in 12-well tissue culture plates. Monocyte and PBMCs were incubated in suspension (approximately 1 × 106 cells ml−1) in 1 ml of media in micro centrifuge tubes.
For adherent cells (HMVEC, HeLa, and monocyte-derived macrophages), media was removed at select time points from duplicate wells, cells washed twice with 2 ml of ice-cold 0.9% normal saline. For non-adherent cells (monocytes and PBMCs), duplicate incubations were centrifuged at 2,500g for 30 s to remove media. The cell pellets were re-suspended with 500 μl cell culture media (RPMI with 10% FBS) and layered on top of a 500 μl oil layer (Nyosil M25; Nye Lubricants) in a microcentrifuge tube. Samples were then centrifuged at room temperature at 13,000 r.p.m. for 45 s. The media layer was removed and the oil layer was washed twice with 500 μl water. The oil layer was then carefully removed using a Pasteur pipet attached to vacuum. A volume of 0.5 ml of 70% methanol containing 100 nM of the analytical internal standard 2-chloro-adenosine-5′-triphosphate (Sigma-Aldrich) was added to isolated cells. Samples were stored overnight at −20 °C to facilitate extraction, centrifuged at 15,000g for 15 min and then supernatant was transferred to clean tubes for drying in a MiVac Duo concentrator (Genevac). Dried samples were then reconstituted in mobile phase A containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine (DMH) in water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS).
LC-MS/MS was performed using low-flow ion-pairing chromatography, similar to methods described previously30. Briefly, analytes were separated using a 50 × 2 mm × 2.5 μm Luna C18(2) HST column (Phenomenex) connected to a LC-20ADXR (Shimadzu) ternary pump system and HTS PAL autosampler (LEAP Technologies). A multi-stage linear gradient from 10% to 50% acetonitrile in a mobile phase containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine over 8 min at a flow rate of 150 μl min−1 was used to separate analytes. Detection was performed on an API 4000 (Applied Biosystems) MS/MS operating in positive ion and multiple reaction monitoring modes. Intracellular metabolites alanine metabolite, Nuc, nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate were quantified using 7-point standard curves ranging from 0.274 to 200 pmol (approximately 0.5 to 400 μM) prepared in cell extract from untreated cells. Levels of adenosine nucleotides were also quantified to assure dephosphorylation had not taken place during sample collection and preparation. In order to calculate intracellular concentration of metabolites, the total number of cells per sample were counted using a Countess automated cell counter (Invitrogen).
Antiviral assays were conducted in biosafety level 4 containment (BSL-4) at the Centers for Disease Control and Prevention. EBOV antiviral assays were conducted in primary HMVEC-TERT and in Huh-7 cells. Huh-7 cells were not authenticated and were not tested for mycoplasma. Ten concentrations of compound were diluted in fourfold serial dilution increments in media, and 100 μl per well of each dilution was transferred in duplicate (Huh-7) or quadruplicate (HMVEC-TERT) onto 96-well assay plates containing cell monolayers. The plates were transferred to BSL-4 containment, and the appropriate dilution of virus stock was added to test plates containing cells and serially diluted compounds. Each plate included four wells of infected untreated cells and four wells of uninfected cells that served as 0% and 100% virus inhibition controls, respectively. After the infection, assay plates were incubated for 3 days (Huh-7) or 5 days (HMVEC-TERT) in a tissue culture incubator. Virus replication was measured by direct fluorescence using a Biotek HTSynergy plate reader. For virus yield assays, Huh-7 cells were infected with wild-type EBOV for 1 h at 0.1 plaque-forming units (PFU) per cell. The virus inoculum was removed and replaced with 100 μl per well of media containing the appropriate dilution of compound. At 3 days post-infection, supernatants were collected, and the amount of virus was quantified by endpoint dilution assay. The endpoint dilution assay was conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% cytopathic effects (TCID ). To measure levels of viral RNA from infected cells, total RNA was extracted using the MagMAX-96 Total RNA Isolation Kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT–PCR) assay with primers and probes specific for the EBOV nucleoprotein gene.
Antiviral assays were conducted in BSL-4 at USAMRIID. HeLa or HFF-1 cells were seeded at 2,000 cells per well in 384-well plates. Ten serial dilutions of compound in triplicate were added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard) in twofold dilution increments starting at 10 μM at 2 h before infection. The DMSO concentration in each well was normalized to 1% using an HP D300 digital dispenser. The assay plates were transferred to the BSL-4 suite and infected with EBOV Kikwit at a multiplicity of infection of 0.5 PFU per cell for HeLa cells and with EBOV Makona at a multiplicity of infection of 5 PFU per cell for HFF-1 cells. The assay plates were incubated in a tissue culture incubator for 48 h. Infection was terminated by fixing the samples in 10% formalin solution for an additional 48 h before immune-staining, as described in Supplementary Table 1.
Antiviral assays were conducted in BSL-4 at USAMRIID. Primary human macrophage cells were seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate were added directly to the cell cultures using an HP D300 digital dispenser in threefold dilution increments 2 h before infection. The concentration of DMSO was normalized to 1% in all wells. The plates were transferred into the BSL-4 suite, and the cells were infected with 1 PFU per cell of EBOV in 100 μl of media and incubated for 1 h. The inoculum was removed, and the media was replaced with fresh media containing diluted compounds. At 48 h post-infection, virus replication was quantified by immuno-staining as described in Supplementary Table 1.
For antiviral tests, compounds were threefold serially diluted in source plates from which 100 nl of diluted compound was transferred to a 384-well cell culture plate using an Echo acoustic transfer apparatus. HEp-2 cells were added at a density of 5 × 105 cells per ml, then infected by adding RSV A2 at a titer of 1 × 104.5 tissue culture infectious doses (TCID ) per ml. Immediately following virus addition, 20 μl of the virus and cells mixture was added to the 384-well cell culture plates using a μFlow liquid dispenser and cultured for 4 days at 37 °C. After incubation, the cells were allowed to equilibrate to 25 °C for 30 min. The RSV-induced cytopathic effect was determined by adding 20 μl of CellTiter-Glo Viability Reagent. After a 10-min incubation at 25 °C, cell viability was determined by measuring luminescence using an Envision plate reader.
Antiviral assays were conducted in 384-or 96-well plates in BSL-4 at USAMRIID using a high-content imaging system to quantify virus antigen production as a measure of virus infection. A ‘no virus’ control and a ‘1% DMSO’ control were included to determine the 0% and 100% virus infection, respectively. The primary and secondary antibodies and dyes used for nuclear and cytoplasmic staining are listed in Supplementary Table 1. The primary antibody specific for a particular viral protein was diluted 1,000-fold in blocking buffer (1 × PBS with 3% BSA) and added to each well of the assay plate. The assay plates were incubated for 60 min at room temperature. The primary antibody was removed, and the cells were washed three times with 1 × PBS. The secondary detection antibody was an anti-mouse (or rabbit) IgG conjugated with Dylight488 (Thermo Fisher Scientific, catalogue number 405310). The secondary antibody was diluted 1,000-fold in blocking buffer and was added to each well in the assay plate. Assay plates were incubated for 60 min at room temperature. Nuclei were stained using Draq5 (Biostatus) or 33342 Hoechst (ThermoFisher Scientific) for Vero and HFF-1 cell lines. Both dyes were diluted in 1× PBS. The cytoplasm of HFF-1 (EBOV assay) and Vero E6 (MERS assay) cells were counter-stained with CellMask Deep Red (Thermo Fisher Scientific). Cell images were acquired using a Perkin Elmer Opera confocal plate reader (Perkin Elmer) using a ×10 air objective to collect five images per well. Virus-specific antigen was quantified by measuring fluorescence emission at a 488 nm wavelength and the stained nuclei were quantified by measuring fluorescence emission at a 640 nm wavelength. Acquired images were analysed using Harmony and Acapella PE software. The Draq5 signal was used to generate a nuclei mask to define each nuclei in the image for quantification of cell number. The CellMask Deep Red dye was used to demarcate the Vero and HFF-1 cell borders for cell-number quantitation. The viral-antigen signal was compartmentalized within the cell mask. Cells that exhibited antigen signal higher than the selected threshold were counted as positive for viral infection. The ratio of virus-positive cells to total number of analysed cells was used to determine the percentage of infection for each well on the assay plates. The effect of compounds on the viral infection was assessed as percentage of inhibition of infection in comparison to control wells. The resultant cell number and percentage of infection were normalized for each assay plate. Analysis of dose–response curve was performed using GeneData Screener software applying Levenberg–Marquardt algorithm for curve-fitting strategy. The curve-fitting process, including individual data point exclusion, was pre-specified by default software settings. R2 value quantified goodness of fit and fitting strategy was considered acceptable at R2 > 0.8.
All virus infections were quantified by immuno-staining using antibodies that recognized the relevant viral glycoproteins, as described in Supplementary Table 1.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 1 PFU per cell MARV, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.08 PFU SUDV per cell, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.3 PFU per cell JUNV, which resulted in ~50% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 PFU per cell LASV, which resulted in >60% of the cells expressing virus antigen in a 48-h period.
African green monkey (Chlorocebus sp.) kidney epithelial cells (Vero E6) were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 PFU per cell of MERS virus, which resulted in >70% of the cells expressing virus antigen in a 48-h period.
U2OS cells were seeded at 3,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 PFU per cell of CHIK, which resulted in >80% of the cells expressing virus antigen in a 48-h period.
HeLa cells were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 PFU per cell VEEV, which resulted in >60% of the cells expressing virus antigen in a 20-h period.
HEp-2 (1.5 × 103 cells per well) and MT-4 (2 × 103 cells per well) cells were plated in 384-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. PC-3 cells (2.5 × 103 cells per well), HepG2 cells (4 × 103 cells per well), hepatocytes (1 × 106 cells per well), quiescent PBMCs (1 × 106 cells per well), stimulated PBMCs (2 × 105 cells per well), and RPTEC cells (1 × 103 cells per well) were plated in 96-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. Cells were cultured for 4–5 days at 37 °C. Following the incubation, the cells were allowed to equilibrate to 25 °C, and cell viability was determined by adding Cell-Titer Glo viability reagent. The mixture was incubated for 10 min, and the luminescence signal was quantified using an Envision plate reader. Cell lines were not authenticated and were not tested for mycoplasma as part of routine use in cytotoxicity assays.
RNA synthesis by the RSV polymerase was reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1–14 of the RSV leader promoter31, 32, 33 (3′-UGCGCUUUUUUACG-5′). RNA synthesis reactions were performed as described previously, except that the reaction mixture contained 250 μM guanosine triphosphate (GTP), 10 μM uridine triphosphate (UTP), 10 μM cytidine triphosphate (CTP), supplemented with 10 μCi [α-32P]CTP, and either included 10 μM adenosine triphosphate (ATP) or no ATP. Under these conditions, the polymerase is able to initiate synthesis from the position 3 site of the promoter, but not the position 1 site. The NTP metabolite of GS-5734 was serially diluted in DMSO and included in each reaction mixture at concentrations of 10, 30, or 100 μM as specified in Fig. 1f. RNA products were analysed by electrophoresis on a 25% polyacrylamide gel, containing 7 M urea, in Tris–taurine–EDTA buffer, and radiolabelled RNA products were detected by autoradiography.
Transcription reactions contained 25 μg of crude RSV RNP complexes in 30 μL of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl , 3 mM DTT, 2 mM EGTA, 50 μg ml−1 BSA, 2.5 U RNasin, 20 μM ATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, and 1.5 μCi [α-32P]ATP (3,000 Ci mmol−1)). The radiolabelled nucleotide used in the transcription assay was selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription.
To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds were added using a six-step serial dilution in fivefold increments. After a 90-min incubation at 30 °C, the RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.
For a 25 μl reaction mixture, 7.5 μl 1 × transcription buffer (20 mM HEPES (pH 7.2–7.5), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol), 3 mM MgCl , 100 ng CMV positive or negative control DNA, and a mixture of ATP, GTP, CTP and UTP was pre-incubated with various concentrations (0–500 μM) of the inhibitor at 30 °C for 5 min. The mixture contained 5–25 μM (equal to K ) of the competing 33P-labelled ATP and 400 μM of GTP, UTP, and CTP. The reaction was started by addition of 3.5 μl of HeLa and extract. After 1 h of incubation at 30 °C, the polymerase reaction was stopped by addition of 10.6 μl proteinase K mixture that contained final concentrations of 2.5 μg μl−1 proteinase K, 5% SDS, and 25 mM EDTA. After incubation at 37 °C for 3–12 h, 10 μl of the reaction mixture was mixed with 10 μl of the loading dye (98% formamide, 0.1% xylene cyanol and 0.1% bromophenol blue), heated at 75 °C for 5 min, and loaded onto a 6% polyacrylamide gel (8 M urea). The gel was dried for 45 min at 70 °C and exposed to a phosphorimager screen. The full length product, 363 nucleotide runoff RNA, was quantified using a Typhoon Trio Imager and Image Quant TL Software.
Twenty nanomolar POLRMT was incubated with 20 nM template plasmid (pUC18-LSP) containing POLRMT light-strand promoter region and mitochondrial (mt) transcription factors TFA (100 nM) and mtTFB2 (20 nM) in buffer containing 10 mM HEPES (pH 7.5), 20 mM NaCl, 10 mM DTT, 0.1 mg ml−1 BSA, and 10 mM MgCl 34. The reaction mixture was pre-incubated to 32 °C, and the reactions were initiated by addition of 2.5 μM of each of the natural NTPs and 1.5 μCi of [32P]GTP. After incubation for 30 min at 32 °C, reactions were spotted on DE81 paper and quantified.
A homology model of RSV A2 and EBOV polymerases were built using the HIV reverse transcriptase X-ray crystal structure (PDB:1RTD). Schrödinger Release 2015-1: Prime, version 3.9 (Schrödinger, LLC), default settings with subsequent rigid body minimization and side-chain optimization. Loop insertions not in 1RTD of greater than 10 amino acids were not built.
For quantitative assessment of viral RNA nonhuman primate plasma samples, whole blood was collected using a K3 EDTA Greiner Vacuette tube (or equivalent) and sample centrifuged at 2500 (± 200) relative centrifugal force for 10 ± 2 min. To inactivate virus, plasma was treated with 3 parts (300 μl) TriReagent LS and samples were transferred to frozen storage (−60 °C to −90 °C), until removal for RNA extraction. Carrier RNA and QuantiFast High Concentration Internal Control (Qiagen) were spiked into the sample before extraction, conducted according to manufacturer’s instructions. The viral RNA was eluted in AVE buffer. Each extracted RNA sample was tested with the QuantiFast Internal Control RT–PCR RNA Assay (Qiagen) to evaluate the yield of the spiked-in QuantiFast High Concentration Internal Control. If the internal control amplified within manufacturer-designated ranges, further quantitative analysis of the viral target was performed. RT–PCR was conducted using an ABI 7500 Fast Dx using primers specific to EBOV glycoprotein. Samples were run in triplicate using a 5 μl template volume. For quantitative assessments, the average of the triplicate genomic equivalents (GE) per reaction were determined and multiplied by 800 to obtain GE ml−1 plasma. Standard curves were generated using synthetic RNA. The limits of quantification for this assay are 8.0 × 104 − 8.0 × 1010 GE ml−1 of plasma. Acceptance criteria for positive template control (PTC), negative template control (NTC), negative extraction control (NEC), and positive extraction control (PEC) are specified by standard operating procedure. For qualitative assessments, the limit of detection (LOD) was defined as C 38.07, based on method validation testing. An animal was considered to have tested positive for detection of EBOV RNA when a minimum of 2 of 3 replicates were designated as ‘positive’ and PTC, NTC, and NEC controls met specified method-acceptance criteria. A sample was designated as ‘positive’ when the C value was
News Article | December 2, 2016
Research and Markets has announced the addition of the "Global Automotive Connectors Market 2016-2020" report to their offering. The global automotive connectors market to grow at a CAGR of 8.12% during the period 2016-2020. The report covers the present scenario and the growth prospects of the global automotive connectors market for 2016-2020. To calculate the market size, the report considers the revenue generated from the sale of automotive connectors. The report covers the market landscape and its growth prospects over the coming years. The report also includes a discussion of the key vendors operating in this market. One of latest trends in the market is induction of high-end optical fibers into automobile sector. Traditional cars used the crimp and poke connector system owing to its simplified architecture for wiring harnesses. However, as the demand for driver support systems such as ADAS and ESC increased, traditional connector system could no longer provide the bandwidth. This gave rise to the development of other complex architecture for wiring harnesses such as UTP and STP. According to the report, one of the primary drivers in the market is rising demand for high-speed connectivity solutions in automobiles. Earlier, automobiles consisted of more mechanical components than electronic components. However, in the recent years, the electronic content per vehicle has increased to more than 50% in some vehicles. This is due to the increased adoption of advanced safety features and powertrain electrification. Modern-day vehicles have an array of sensors installed in them. This has led to the development of more complex safety systems. Further, the report states that one major challenge in the market is rising labor cost in low-cost manufacturing countries. One such means to optimize cost was to outsource manufacturing activities to countries, such as China, Taiwan, South Korea, and India, which have low-cost labor. These countries are among the largest manufacturers of automotive connectors in the world. The primary reason for the high concentration of automotive connectors manufacturers in these countries is attributed to the drastic shift of electronic manufacturing units to these countries from developed countries such as the US and the UK. For more information about this report visit http://www.researchandmarkets.com/research/6fr9kr/global_automotive
News Article | February 23, 2017
NAPERVILLE, Ill.--(BUSINESS WIRE)--Coriant, a global supplier of SDN-enabled end-to-end packet optical networking and DCI solutions, today announced that PT Telekomunikasi Indonesia, Tbk (Telkom) has deployed Coriant’s best-in-class coherent optical transport and universal switching technology as part of a nationwide upgrade to its fiber-based backbone network. Completed at the end of 2016, the network modernization initiative will improve the performance and increase the capacity of Telkom’s terrestrial and subsea optical transport infrastructure to meet growing demand for high-bandwidth mobile and fixed line services and applications. “We continue to transform our network infrastructure with cutting-edge technologies to provide the best digital experience for our customers,” said Agus Subandrio, Deputy EGM of Backbone and Core Network Planning and Deployment, Telkom. “With growing demand for data in Indonesia and across the Southeast Asia region, Coriant’s advanced traffic grooming, switching, and transport solution will play a key role in our ability to cost-efficiently scale backbone capacity and deliver an uninterrupted quality of service under any network condition.” The Coriant solution enables Telkom to seamlessly scale its DWDM infrastructure to 100G per channel without replacing existing transmission systems. In addition to terrestrial 100G backbone upgrades, the combination of the industry-leading Coriant® hiT 7300 Multi-Haul Transport Platform and Coriant CloudWave™ Optics coherent interface technology enables Telkom to further boost capacity on its JASUKA undersea cable, which provides high-speed connectivity amongst Java, Sumatera, and Kalimantan islands. Initial implementation includes operational deployment of sixteen 100G optical channels across an unrepeatered and DCM-free subsea transmission distance of over 350 kilometers between Dumai and Dangas. The Telkom nationwide upgrade project includes expanded deployment of the Coriant® mTera® Universal Transport Platform to optimize service traffic and maximize utilization of fiber assets as the network scales. The mTera® UTP is an extremely flexible multi-service transport solution that supports software-defined Universal Switching, including OTN, Carrier Ethernet, MPLS-TP and SONET/SDH in a single, power-efficient system architecture. To simplify capacity provisioning and efficiently manage service traffic across the multi-domain Coriant optical transport network, Telkom is using the Coriant® Transport Network Management System (TNMS), a robust management platform that helps service providers reduce OpEx and CapEx expenses through sophisticated end-to-end network control, automated provisioning features, and advanced planning capabilities. “Telkom remains a premier provider of fixed and mobile network services in the region, and we are pleased to help them address the challenges of backbone traffic growth with the latest innovations in our end-to-end packet optical transport solutions portfolio,” said Tarcisio Ribeiro, Executive Vice President and Chief Commercial and Marketing Officer, Coriant. Coriant will showcase the new 7300 OLS and its complete portfolio of SDN-enabled Smart Router and Packet Optical Transport Solutions at Mobile World Congress 2017 in Barcelona from February 27 – March 2 (Hall 2, Stand 2I30). From performance-based multi-layer network optimization to purpose-built metro and long haul DCI, Coriant will demonstrate a broad range of networking solutions that help operators realize the business value of intelligent, open, and highly-programmable networks optimized for the hyperscale generation. Coriant delivers innovative and dynamic networking solutions for a fast-changing and cloud-centric business world. The Coriant portfolio of SDN-enabled, edge-to-core packet optical networking and DCI solutions enables network operators to cost-efficiently scale network capacity, reduce operational complexity, and create the resilient foundation for a new generation of mobile, video, and cloud services. Coriant serves leading network operators around the world, including mobile and fixed line service providers, cloud and data center operators, Web 2.0 content providers, cable MSOs, government agencies, and large enterprises. With a distinguished heritage of technology innovation and service excellence, Coriant is helping its global customers maximize the value of their network infrastructure as demand for bandwidth explodes and the communications needs of businesses and consumers continue to evolve. Learn more at www.coriant.com and follow us on Twitter for the latest @Coriant news and information.
News Article | November 2, 2016
Hod Hasharon (Israel) et Genève (Suisse), le 2 novembre 2016 - Valens et STMicroelectronics annoncent leur collaboration en vue d'intégrer la technologie HDBaseT Automotive dans la prochaine génération de véhicules automobiles connectés. Cette technologie à haut rendement optimise la connectivité à bord des véhicules en transmettant des informations à haut débit d'infotainment, de sécurité routière et de contrôle automobile au débit de 6 Gbits/s sur une infrastructure à faible coût et avec un temps de latence quasiment nul. Valens, inventeur du HDBaseT et fondateur de l'Alliance HDBaseT, apporte sa technologie et son expertise pour atteindre la commercialisation de véhicules dotés de la technologie HDBaseT. STMicroelectronics apporte son savoir-faire et sa vaste expérience de la conception et de la fabrication conformément aux très strictes exigences de qualité et de fiabilité de l'industrie automobile. « Notre longue expérience au service de l'industrie automobile et notre stratégie en faveur du Smart Driving nous ont convaincus du potentiel du HDBaseT Automotive, une technologie à haut débit et faible temps de latence dotée pour un coût peu élevé. C'est pourquoi nous sommes heureux de nous associer à ce projet et d'aider Valens à lancer la technologie et les produits HDBaseT Automotive sur le marché avec les plus hauts niveaux de qualité et de fiabilité possibles », a déclaré Fabio Marchio, Directeur Général de la division Automotive Digital de STMicroelectronics. La technologie HDBaseT Automotive est actuellement la seule technologie capable d'acheminer des signaux vidéo, audio, données, USB, etc. à un débit pouvant atteindre 6 Gbits/s avec des capacités de connexion natives via un unique câble à paires torsadées non blindé (UTP) sur une distance pouvant atteindre 15 mètres. Cette technologie autorise également la connexion en cascade (daisy chaining) et la lecture en multistreaming, ce qui simplifie et optimise la connectivité à bord des véhicules. Pour de plus amples informations sur la solution HDBaseT Automotive de Valens, cliquez ici. Créée en 2006, la société Valens fournit des circuits intégrés conçus pour diffuser des contenus multimédias ultra-haute définition (HD) non compressés. Sa technologie HDBaseT permet de connecter des produits sur une longue distance via un unique câble et représente un standard mondial pour la distribution avancée de médias numériques. Valens est une entreprise privée dont le siège est situé en Israël. Pour plus d'informations, visitez le site www.valens.com. ST, un leader mondial sur le marché des semiconducteurs, fournit des produits et des solutions intelligents qui consomment peu d'énergie et sont au coeur de l'électronique que chacun utilise au quotidien. Les produits de ST sont présents partout, et avec nos clients, nous contribuons à rendre la conduite automobile, les usines, les villes et les habitations plus intelligentes et à développer les nouvelles générations d'appareils mobiles et de l'Internet des objets.