LitCovid-sample-sentences  

PMC:7253482 / 27038-36048 JSONTXT 25 Projects

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Id Subject Object Predicate Lexical cue
T150 0-7 Sentence denotes Methods
T151 9-71 Sentence denotes Expression and purification of coronavirus spike glycoproteins
T152 72-350 Sentence denotes Human embryonic kidney 293 Freestyle (HEK293F) cells were transfected with mammalian-codon-optimised genes encoding 2P-stabilised SARS MERS and HKU1 S proteins containing a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, an 8xHis-tag and a Twin-Strep-tag41.
T153 351-424 Sentence denotes H3N2 Victoria 2011 hemagglutinin was also expressed in the HEK293F cells.
T154 425-632 Sentence denotes The 200 ml cultures were harvested 6 days after transfection, filtered and purified by nickel-affinity chromatography and size exclusion chromatography using a SuperdexTM 16/600 75 pg column (GE Healthcare).
T155 634-675 Sentence denotes Release and labelling of N-linked glycans
T156 676-797 Sentence denotes Excised coronavirus S gel bands were washed alternately with acetonitrile and water before drying in a vacuum centrifuge.
T157 798-892 Sentence denotes The bands were rehydrated with 100 μL of water and incubated with PNGase F at 37 °C overnight.
T158 893-1155 Sentence denotes Aliquots of released N-linked glycans were also fluorescently labelled with procainamide, by adding 100 μL of labelling mixture (110 mg/mL procainamide and 60 mg/mL sodium cyanoborohydrate in 70% DMSO and 30% glacial acetic acid) and incubating for 4 h at 65 °C.
T159 1156-1251 Sentence denotes Procainamide labelled glycans were purified using Spe-ed Amide 2 columns (Applied Separations).
T160 1253-1282 Sentence denotes Glycan analysis by HILIC-UPLC
T161 1283-1495 Sentence denotes Labelled glycans were analysed using a 2.1 mm × 150 mm Acquity BEH Glycan column (Waters) on an Acquity H-Class UPLC instrument (Waters), with fluorescence measurements occurring at λex = 310 nm and λem = 370 nm.
T162 1496-1542 Sentence denotes The following gradient was used: time (t) = 0:
T163 1543-1591 Sentence denotes 22% A, 78% B (flow rate = 0.5 mL/min); t = 38.5:
T164 1592-1632 Sentence denotes 44.1% A, 55.9% B (0.5 mL/min); t = 39.5:
T165 1633-1670 Sentence denotes 100% A, 0% B (0.25 mL/min); t = 44.5:
T166 1671-1708 Sentence denotes 100% A, 0% B (0.25 mL/min); t = 46.5:
T167 1709-1811 Sentence denotes 22% A, 78% B (0.5 mL/min), where solvent A was 50 mM ammonium formate (pH 4.4) and B was acetonitrile.
T168 1812-1992 Sentence denotes Quantification of oligomannose-type glycans was achieved by digestion of fluorescently labelled glycans with Endo H, and clean-up using a PVDF protein-binding membrane (Millipore).
T169 1993-2050 Sentence denotes Empower 3 software (Waters) was used for data processing.
T170 2052-2080 Sentence denotes Mass spectrometry of glycans
T171 2081-2334 Sentence denotes Prior to ion-mobility electrospray ionisation MS and tandem MS analysis, PNGase F released N-linked glycans were purified on a Nafion® 117 membrane (Sigma-Aldrich) and a trace amount of ammonium phosphate was added to promote phosphate adduct formation.
T172 2335-2751 Sentence denotes Glycans were analyzed by direct infusion using a Synapt G2Si instrument (Waters) with the following settings: capillary voltage, 0.8–1.0 kV; sample cone, 150 V; extraction cone, 150 V; cone gas, 40 l/h; source temperature, 80 °C; trap collision voltage, 4–160 V; transfer collision voltage, 4 V; trap DC bias, 60 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 ml/min; IMS gas flow, 80 ml/min.
T173 2752-2849 Sentence denotes Data were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters).
T174 2851-2885 Sentence denotes Mass spectrometry of glycopeptides
T175 2886-2993 Sentence denotes Aliquots of 30–50 μg of coronavirus spikes were denatured, reduced and alkylated as described previously36.
T176 2994-3138 Sentence denotes Proteins were proteolytically digested with trypsin (Promega), chymotrypsin (Promega), alpha-lytic protease (Sigma-Aldrich) and Glu-C (Promega).
T177 3139-3282 Sentence denotes Reaction mixtures were dried and peptides/glycopeptides were extracted using C18 Zip-tip (MerckMilipore) following the manufacturer’s protocol.
T178 3283-3494 Sentence denotes Samples were resuspended in 0.1% formic acid prior to analysis by liquid chromatography-mass spectrometry using an Easy-nLC 1200 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific).
T179 3495-3723 Sentence denotes Glycopeptides were separated using an EasySpray PepMap RSLC C18 column (75 μm × 75 cm) with a 240-min linear solvent gradient of 0–32% acetonitrile in 0.1% formic acid, followed by 35 min of 80% acetonitrile in 0.1% formic acid.
T180 3724-3871 Sentence denotes Other settings include an LC flow rate of 200 nL/min, spray voltage of 2.8 kV, capillary temperature of 275 °C, and an HCD collision energy of 50%.
T181 3872-4003 Sentence denotes Precursor and fragmentation detection were performed using an Orbitrap at the following resolution: MS1 = 100,000 and MS2 = 30,000.
T182 4004-4122 Sentence denotes The automatic gain control (AGC) targets were MS1 = 4e5 and MS2 = 5e4, and injection times were MS1 = 50 and MS2 = 54.
T183 4123-4265 Sentence denotes The following cleavage sites were used for the respective proteases; trypsin=R/K, chymotrypsin=F/Y/W, alpha lytic protease=T/A/S/V, Glu C=E/D.
T184 4266-4307 Sentence denotes Number of missed cleavages were set at 3.
T185 4308-4355 Sentence denotes The following modifications were also included:
T186 4356-4653 Sentence denotes Carbamidomethyl (+57.021464, target=C, fine control=fixed), Oxidation (+15.994915, target=M, fine control=variable rare 1), Glu to pyro-Glu (−18.010565, target=peptide N-term E, fine control=variable rare 1), and Gln to pyro-Glu (−17.026549, target peptide N-term Q, fine control=variable rare 1).
T187 4654-4801 Sentence denotes Glycopeptide fragmentation data were extracted form raw files using ByonicTM (Version 3.5.0) and ByologicTM (Version 3.5-15; Protein Metrics Inc.).
T188 4802-5013 Sentence denotes Glycopeptide fragmentation data were manually evaluated with true-positive assignments given when correct b- and y-fragments and oxonium ions corresponding to the peptide and glycan, respectively, were observed.
T189 5014-5108 Sentence denotes The precursor mass tolerance was set at 4 ppm for precursor ions and 10 ppm for fragment ions.
T190 5109-5202 Sentence denotes MS data were searched using a glycan library (SI Fig. 9) with the identical peptide sequence.
T191 5203-5247 Sentence denotes A 1% false discovery rate (FDR) was applied.
T192 5248-5465 Sentence denotes The extracted ion chromatographic areas for each true-positive glycopeptide, with the same amino-acid sequence, were compared to determine the relative quantitation of glycoforms at each specific N-linked glycan site.
T193 5467-5485 Sentence denotes Model construction
T194 5486-5759 Sentence denotes Structural models of N-linked glycan presentation on SARS, MERS and HKU1 S were created using electron microscopy structures (PDB ID 5X58, 5X59, and 5I08, respectively)9,11, along with complex-, hybrid-, and oligomannose-type N-linked glycans (PDB ID 4BYH, 4B7I, and 2WAH).
T195 5760-5883 Sentence denotes The most dominant glycoform presented at each site was modelled on to the N-linked carbohydrate attachment sites in Coot68.
T196 5885-5913 Sentence denotes Molecular evolution analysis
T197 5914-6052 Sentence denotes Publicly available sequences encoding full-length GPC spike gene for SARS-CoV (3765 bp) were downloaded from GenBank and manually aligned.
T198 6053-6134 Sentence denotes For MERS-CoV, we leveraged the whole genome alignment collated by Dudas et al.69.
T199 6135-6261 Sentence denotes Specifically, the alignment corresponding to the spike gene was extracted (4059 bp), excluding sequences isolated from humans.
T200 6262-6353 Sentence denotes Final alignments for SARS- and MERS-CoV corresponded to 70 and 100 sequences, respectively.
T201 6354-6493 Sentence denotes For the dN/dS analysis, we first estimated Bayesian molecular clock phylogenies for SARS- and MERS-CoV independently using BEAST v 1.8.470.
T202 6494-6661 Sentence denotes For both viruses, we assumed an uncorrelated log-normal distributed molecular clock71, Bayesian Skyline coalescent prior72 and a codon-structured substitution model73.
T203 6662-6792 Sentence denotes Multiple independent MCMC runs of 10–20 million steps were executed to ensure that stationarity and convergence had been achieved.
T204 6793-7086 Sentence denotes Empirical distributions of time-scaled phylogenies were obtained by combining (after the removal of burnin) the posterior tree distributions from the separate runs, which were subsequently used to estimate dN/dS ratios using the renaissance counting approach74,75 implemented in BEAST v 1.8.4.
T205 7087-7300 Sentence denotes We also estimated per-site amino-acid diversity, which was calculated as the average number of amino-acid difference between two sequences at an amino-acid position in all possible pairs in the sequence alignment.
T206 7302-7341 Sentence denotes Cryo-EM data analysis and visualization
T207 7342-7473 Sentence denotes Single-particle cryo-EM data analysis of BG505 SOSIP.664 in complex with RM20A3 Fab was reproduced directly from Berndsen et al.51.
T208 7474-7700 Sentence denotes Data for the SARS-CoV S 2P ectodomain was previously published52 and the final particle stack and alignment parameters from the published reconstruction were used for 3D variability analysis in the SPARX software package76,77.
T209 7701-7835 Sentence denotes All metadata for these reconstructions along with raw data images and FSC resolution curves can be found in the original publications.
T210 7836-7993 Sentence denotes In summary, both datasets were acquired on a FEI Titan Krios (Thermo Fisher) operating at 300 KeV equipped with a K2 Summit Direct Electron Detector (Gatan).
T211 7994-8106 Sentence denotes Movie micrographs were aligned and dose weighted with MotionCor278 and CTF estimation was performed with Gctf79.
T212 8107-8192 Sentence denotes Single-particle data processing was performed using CryoSparc v.280 and Relion v.381.
T213 8193-8278 Sentence denotes Maps were low-pass filtered using a Gaussian kernel and visualized in UCSF chimera82.
T214 8279-8445 Sentence denotes A detailed description of the auto-thresholding method used to set the isosurface value for visualisation of low-pass filtered maps can be found in Berndsen et al.51.
T215 8447-8490 Sentence denotes Clustering analysis of viral glycan shields
T216 8491-8704 Sentence denotes Solvent-accessible residues and interactions between N-linked glycans and amino-acid residues were calculated using Proteins, Interfaces, Structures and Assemblies (PISA) European Bioinformatics Institute (EBI)83.
T217 8705-8874 Sentence denotes Glycan shield density was calculated by the number of amino-acid residues interacting with glycans divided by the total number of solvent-accessible amino-acid residues.
T218 8876-8893 Sentence denotes Reporting summary
T219 8894-9010 Sentence denotes Further information on research design is available in the Nature Research Reporting Summary linked to this article.