LitCovid-sample-sentences  

PMC:7443692 / 48512-70536 JSONTXT 13 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T330 0-14 Sentence denotes Method Details
T331 16-102 Sentence denotes Expression, Purification, and Characterization of SARS-CoV-2 S and Human ACE2 Proteins
T332 103-530 Sentence denotes To express a stabilized ectodomain of Spike protein, a synthetic gene encoding residues 1−1208 of SARS-CoV-2 Spike with the furin cleavage site (residues 682–685) replaced by a “GGSG” sequence, proline substitutions at residues 986 and 987, and a foldon trimerization motif followed by a C-terminal 6xHisTag was created and cloned into the mammalian expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg, MD).
T333 531-660 Sentence denotes The expression construct was transiently transfected in HEK293F cells using polyethylenimine (Polysciences, Inc, Warrington, PA).
T334 661-905 Sentence denotes Protein was purified from cell supernatants using Ni-NTA resin (QIAGEN, Germany), the eluted fractions containing S protein were pooled, concentrated, and further purified by gel filtration chromatography on a Superose 6 column (GE Healthcare).
T335 906-1003 Sentence denotes Negative stain electron microscopy (EM) analysis was performed as described (Shaik et al., 2019).
T336 1004-1297 Sentence denotes Briefly, analysis was performed at room temperature with a magnification of 52,000x and a defocus value of 1.5 μm following low-dose procedures, using a Philips Tecnai F20 electron microscope (Thermo Fisher Scientific) equipped with a Gatan US4000 CCD camera and operated at voltage of 200 kV.
T337 1298-1443 Sentence denotes The DNA fragment encoding human ACE2 (1-615) with a 6xHis tag at C terminus was synthesized by Genscript and cloned to the vector pCMV-IRES-puro.
T338 1444-1525 Sentence denotes The expression construct was transfected in HEK293F cells using polyethylenimine.
T339 1526-1602 Sentence denotes The medium was discarded and replaced with FreeStyle 293 medium after 6-8 h.
T340 1603-1728 Sentence denotes After incubation in 37°C with 5.5% CO2 for 5 days, the supernatant was collected and loaded to Ni-NTA resin for purification.
T341 1729-1804 Sentence denotes The elution was concentrated and further purified by a Superdex 200 column.
T342 1806-1861 Sentence denotes In-Gel Analysis of SARS-CoV-2 S and Human ACE2 Proteins
T343 1862-2122 Sentence denotes A 3.5-μg aliquot of SARS-CoV-2 S protein as well as a 2-μg aliquot of human ACE2 were combined with Laemmli sample buffer, analyzed on a 4%–12% Invitrogen NuPage Bis-Tris gel using the MES pH 6.5 running buffer, and stained with Coomassie Brilliant Blue G-250.
T344 2124-2216 Sentence denotes Analysis of N-linked and O-linked Glycans Released from SARS-Cov-2 S and Human ACE2 Proteins
T345 2217-2371 Sentence denotes Aliquots of approximately 25-50 μg of S or ACE2 protein were processed for glycan analysis as previously described (Aoki et al., 2007; Aoki et al., 2008).
T346 2372-2442 Sentence denotes For N-linked glycan analysis, the proteins were digested with trypsin.
T347 2443-2575 Sentence denotes Following trypsinization, glycopeptides were enriched by C18 Sep-Pak and subjected to PNGaseF digestion to release N-linked glycans.
T348 2576-2713 Sentence denotes Following PNGaseF digestion, released glycans were separated from residual glycosylated peptides bearing O-linked glycans by C18 Sep-Pak.
T349 2714-2833 Sentence denotes O-glycosylated peptides were eluted from the Sep-Pak and subjected to reductive β-elimination to release the O-glycans.
T350 2834-2951 Sentence denotes Another 25-50 μg aliquot of each protein was denatured with SDS and digested with PNGaseF to remove N-linked glycans.
T351 2952-3092 Sentence denotes The de-N-glycosylated, intact protein was precipitated with cold ethanol and then subjected to reductive β-elimination to release O-glycans.
T352 3093-3193 Sentence denotes The profiles of O-glycans released from peptides or from intact protein were found to be comparable.
T353 3194-3377 Sentence denotes N- and O-linked glycans released from glycoproteins were permethylated with methyliodide according to the method of Anumula and Taylor prior to MS analysis (Anumula and Taylor, 1992).
T354 3378-3499 Sentence denotes Glycan structural analysis was performed using an LTQ-Orbitrap instrument (Orbitrap Discovery, Thermo Fisher Scientific).
T355 3500-3810 Sentence denotes Detection and relative quantification of the prevalence of individual glycans was accomplished using the total ion mapping (TIM) and neutral loss scan (NL scan) functionality of the Xcalibur software package version 2.0 (Thermo Fisher Scientific) as previously described (Aoki et al., 2007; Aoki et al., 2008).
T356 3811-3924 Sentence denotes Mass accuracy and detector response was tuned with a permethylated oligosaccharide standard in positive ion mode.
T357 3925-4046 Sentence denotes For fragmentation by collision-induced dissociation (CID in MS2 and MSn), normalized collision energy of 45% was applied.
T358 4047-4153 Sentence denotes Most permethylated glycans were identified as singly or doubly charged, sodiated species in positive mode.
T359 4154-4258 Sentence denotes Sulfated N-glycans were detected as singly or doubly charged, deprotonated species in negative ion mode.
T360 4259-4355 Sentence denotes Peaks for all charge states were deconvoluted by the charge state and summed for quantification.
T361 4356-4408 Sentence denotes All spectra were manually interpreted and annotated.
T362 4409-4533 Sentence denotes The explicit identities of individual monosaccharide residues have been assigned based on known human biosynthetic pathways.
T363 4534-4730 Sentence denotes Graphical representations of monosaccharide residues are consistent with the Symbol Nomenclature for Glycans (SNFG), which has been broadly adopted by the glycomics community (Varki et al., 2015).
T364 4731-4919 Sentence denotes The MS-based glycomics data generated in these analyses and the associated annotations are presented in accordance with the MIRAGE standards and the Athens Guidelines (Wells et al., 2013).
T365 4920-5135 Sentence denotes Data annotation and assignment of glycan accession identifiers were facilitated by GRITS Toolbox, GlyTouCan, GNOme, and GlyGen (Kahsay et al., 2020; Tiemeyer et al., 2017; Weatherly et al., 2019; York et al., 2020).
T366 5137-5198 Sentence denotes Analysis of Disulfide Bonds for SARS-Cov-2 S Protein by LC-MS
T367 5199-5384 Sentence denotes Two 10-μg aliquots of SARS-CoV-2 S protein were denatured by incubating with 20% acetonitrile at room temperature and alkylated by 13.75 mM of iodoacetamide at room temperature in dark.
T368 5385-5519 Sentence denotes The two aliquots of proteins were then digested respectively using alpha lytic protease, or a combination of trypsin, Lys-C and Glu-C.
T369 5520-5595 Sentence denotes Following digestion, the proteins were deglycosylated by PNGaseF treatment.
T370 5596-5819 Sentence denotes The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min.
T371 5820-5965 Sentence denotes The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid.
T372 5966-6063 Sentence denotes The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C.
T373 6064-6244 Sentence denotes Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following electron transfer dissociation (ETD) were collected in the Orbitrap at 15k resolution.
T374 6245-6385 Sentence denotes The raw spectra were analyzed by Byonic (v3.8.13, Protein Metrics Inc.) with mass tolerance set as 20 ppm for both precursors and fragments.
T375 6386-6466 Sentence denotes The search output was filtered at 1% false discovery rate and 10 ppm mass error.
T376 6467-6549 Sentence denotes The spectra assigned as cross-linked peptides were manually evaluated for Cys0015.
T377 6551-6649 Sentence denotes Analysis of Site-Specific N-linked Glycopeptides for SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
T378 6650-6829 Sentence denotes Four 3.5-μg aliquots of SARS-CoV-2 S protein were reduced by incubating with 10 mM of dithiothreitol at 56°C and alkylated by 27.5 mM of iodoacetamide at room temperature in dark.
T379 6830-7005 Sentence denotes The four aliquots of proteins were then digested respectively using alpha lytic protease, chymotrypsin, a combination of trypsin and Glu-C, or a combination of Glu-C and AspN.
T380 7006-7177 Sentence denotes Three 10-μg aliquots of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark.
T381 7178-7321 Sentence denotes The three aliquots of proteins were then digested respectively using alpha lytic protease, chymotrypsin, or a combination of trypsin and Lys-C.
T382 7322-7545 Sentence denotes The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min.
T383 7546-7691 Sentence denotes The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid.
T384 7692-7789 Sentence denotes The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C.
T385 7790-8157 Sentence denotes Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following higher-energy collisional dissociation (HCD) with stepped collision energy (15%, 25%, 35%) were collected in the Orbitrap at 15k resolution. pGlyco v2.2.2 (Liu et al., 2017) was used for database searches with mass tolerance set as 20 ppm for both precursors and fragments.
T386 8158-8266 Sentence denotes The database search output was filtered to reach a 1% false discovery rate for glycans and 10% for peptides.
T387 8267-8366 Sentence denotes Quantitation was performed by calculating spectral counts for each glycan composition at each site.
T388 8367-8462 Sentence denotes Any N-linked glycan compositions identified by only one spectra were removed from quantitation.
T389 8463-8548 Sentence denotes N-linked glycan compositions were categorized into 22 classes (including Unoccupied):
T390 8549-8891 Sentence denotes HexNAc(2)Hex(9∼5)Fuc(0∼1) was classified as M9 to M5 respectively; HexNAc(2)Hex(4∼1)Fuc(0∼1) was classified as M1-M4; HexNAc(3∼6)Hex(5∼9)Fuc(0)NeuAc(0∼1) was classified as Hybrid with HexNAc(3∼6)Hex(5∼9)Fuc(1∼2)NeuAc(0∼1) classified as F-Hybrid; Complex-type glycans are classified based on the number of antenna, fucosylation, and sulfation:
T391 8892-9630 Sentence denotes HexNAc(3)Hex(3∼4)Fuc(0)NeuAc(0∼1) is assigned as A1 with HexNAc(3)Hex(3∼4)Fuc(1∼2)NeuAc(0∼1) assigned as F-A1; HexNAc(4)Hex(3∼5)Fuc(0)NeuAc(0∼2) is assigned as A2/A1B with HexNAc(4)Hex(3∼5)Fuc(1∼5)NeuAc(0∼2) assigned as F-A2/A1B; HexNAc(5)Hex(3∼6)Fuc(0)NeuAc(0∼3) is assigned as A3/A2B with HexNAc(5)Hex(3∼6)Fuc(1∼3)NeuAc(0∼3) assigned as F-A3/A2B; HexNAc(6)Hex(3∼7)Fuc(0)NeuAc(0∼4) is assigned as A4/A3B with HexNAc(6)Hex(3∼7)Fuc(1∼3)NeuAc(0∼4) assigned as F-A4/A3B; HexNAc(7)Hex(3∼8)Fuc(0)NeuAc(0∼1) is assigned as A5/A4B with HexNAc(7)Hex(3∼8)Fuc(1∼3)NeuAc(0∼1) as F-A5/A4B; HexNAc(8)Hex(3∼9)Fuc(0) is assigned as A6/A5B with HexNAc(8)Hex(3∼9)Fuc(1) assigned as F-A6/A5B; any glycans identified with a sulfate are assigned as Sulfated.
T392 9632-9704 Sentence denotes Analysis of Deglycosylated SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
T393 9705-9885 Sentence denotes Three 3.5-μg aliquots of SARS-CoV-2 S protein were reduced by incubating with 10 mM of dithiothreitol at 56°C and alkylated by 27.5 mM of iodoacetamide at room temperature in dark.
T394 9886-10002 Sentence denotes The three aliquots were then digested respectively using chymotrypsin, Asp-N, or a combination of trypsin and Glu-C.
T395 10003-10172 Sentence denotes Two 10-μg aliquots of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark.
T396 10173-10280 Sentence denotes The two aliquots were then digested respectively using chymotrypsin, or a combination of trypsin and Lys-C.
T397 10281-10415 Sentence denotes Following digestion, the proteins were deglycosylated by Endoglycosidase H followed by PNGaseF treatment in the presence of 18O water.
T398 10416-10639 Sentence denotes The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min.
T399 10640-10785 Sentence denotes The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid.
T400 10786-10883 Sentence denotes The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C.
T401 10884-11070 Sentence denotes Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following collision-induced dissociation (CID) at 38% collision energy were collected in the ion trap.
T402 11071-11211 Sentence denotes The spectra were analyzed using SEQUEST (Proteome Discoverer 1.4) with mass tolerance set as 20 ppm for precursors and 0.5 Da for fragments.
T403 11212-11342 Sentence denotes The search output was filtered using ProteoIQ (v2.7) to reach a 1% false discovery rate at protein level and 10% at peptide level.
T404 11343-11561 Sentence denotes Occupancy of each N-linked glycosylation site was calculated using spectral counts assigned to the 18O-Asp-containing (PNGaseF-cleaved) and/or HexNAc-modified (EndoH-cleaved) peptides and their unmodified counterparts.
T405 11563-11661 Sentence denotes Analysis of Site-Specific O-linked Glycopeptides for SARS-Cov-2 S and Human ACE2 Proteins by LC-MS
T406 11662-11879 Sentence denotes Three 10-μg aliquots of SARS-CoV-2 S protein and one 10-μg aliquot of ACE2 protein were reduced by incubating with 5 mM of dithiothreitol at 56°C and alkylated by 13.75 mM of iodoacetamide at room temperature in dark.
T407 11880-11997 Sentence denotes The four aliquots were then digested respectively using trypsin, Lys-C, Arg-C, or a combination of trypsin and Lys-C.
T408 11998-12117 Sentence denotes Following digestion, the proteins were deglycosylated by PNGaseF treatment and then digested with O-protease OpeRATOR®.
T409 12118-12341 Sentence denotes The resulting peptides were separated on an Acclaim PepMap RSLC C18 column (75 μm x 15 cm) and eluted into the nano-electrospray ion source of an Orbitrap Fusion Lumos Tribrid mass spectrometer at a flow rate of 200 nL/min.
T410 12342-12487 Sentence denotes The elution gradient consists of 1%–40% acetonitrile in 0.1% formic acid over 370 min followed by 10 min of 80% acetonitrile in 0.1% formic acid.
T411 12488-12585 Sentence denotes The spray voltage was set to 2.2 kV and the temperature of the heated capillary was set to 280°C.
T412 12586-12860 Sentence denotes Full MS scans were acquired from m/z 200 to 2000 at 60k resolution, and MS/MS scans following higher-energy collisional dissociation (HCD) with stepped collision energy (15%, 25%, 35%) or electron transfer dissociation (ETD) were collected in the Orbitrap at 15k resolution.
T413 12861-12979 Sentence denotes The raw spectra were analyzed by Byonic (v3.8.13) with mass tolerance set as 20 ppm for both precursors and fragments.
T414 12980-13081 Sentence denotes MS/MS filtering was applied to only allow for spectra where the oxonium ions of HexNAc were observed.
T415 13082-13162 Sentence denotes The search output was filtered at 1% false discovery rate and 10 ppm mass error.
T416 13163-13234 Sentence denotes The spectra assigned as O-linked glycopeptides were manually evaluated.
T417 13235-13334 Sentence denotes Quantitation was performed by calculating spectral counts for each glycan composition at each site.
T418 13335-13430 Sentence denotes Any O-linked glycan compositions identified by only one spectra were removed from quantitation.
T419 13431-13624 Sentence denotes Occupancy of each O-linked glycosylation site was calculated using spectral counts assigned to any glycosylated peptides and their unmodified counterparts from searches without MS/MS filtering.
T420 13626-13683 Sentence denotes Sequence Analysis of SARS-CoV-2 S and Human ACE2 Proteins
T421 13684-13829 Sentence denotes The genomes of SARS-CoV as well as bat and pangolin coronavirus sequences reported to be closely related to SARS-CoV-2 were downloaded from NCBI.
T422 13830-14001 Sentence denotes The S protein sequences from all of those genomes were aligned using EMBOSS needle v6.6.0 (Rice et al., 2000) via the EMBL-EBI provided web service (Madeira et al., 2019).
T423 14002-14102 Sentence denotes Manual analysis was performed in the regions containing canonical N-glycosylation sequons (N-X-S/T).
T424 14103-14422 Sentence denotes For further sequence analysis of SARS-CoV-2 S variants, the genomes of SARS-CoV-2 were downloaded from NCBI and GISAID and further processed using Biopython 1.76 to extract all sequences annotated as “surface glycoprotein” and to remove any incomplete sequence as well as any sequence containing unassigned amino acids.
T425 14423-14646 Sentence denotes For sequence analysis of human ACE2 variants, the single nucleotide polymorphisms (SNPs) of ACE2 were extracted from the NCBI dbSNP database and filtered for missense mutation entries with a reported minor allele frequency.
T426 14647-14808 Sentence denotes Manual analysis was performed on both SARS-CoV-2 S and human ACE2 variants to further examine the regions containing canonical N-glycosylation sequons (N-X-S/T).
T427 14809-14918 Sentence denotes LibreOffice Writer and its macro capabilities was used to shade regions on the linear sequence of S and ACE2.
T428 14920-15029 Sentence denotes 3D Structural Modeling and Molecular Dynamics Simulation of Glycosylated SARS-CoV-2 S and Human ACE2 Proteins
T429 15030-15332 Sentence denotes SARS-CoV-2 Spike (S) protein structure and ACE2 co-complex – A 3D structure of the prefusion form of the S protein (RefSeq: YP_009724390.1, UniProt: P0DTC2 SPIKE_SARS2), based on a Cryo-EM structure (PDB code 6VSB) (Wrapp et al., 2020), was obtained from the SWISS-MODEL server (swissmodel.expasy.org).
T430 15333-15399 Sentence denotes The model has 95% coverage (residues 27 to 1146) of the S protein.
T431 15400-15561 Sentence denotes The receptor binding domain (RBD) in the “open” conformation was replaced with the RBD from an ACE2 co-complex (PDB code 6M0J) by grafting residues C336 to V524.
T432 15562-15830 Sentence denotes Glycoform generation – Glycans (detected by glycomics) were selected for installation on glycosylated S and ACE2 sequons (detected by glycoproteomics) based on three sets of criteria designed to reasonably capture different aspects of glycosylation microheterogeneity.
T433 15831-16170 Sentence denotes We denote the first of these glycoform models as “Abundance.” The glycans selected for installation to generate the Abundance model were chosen because they were identified as the most abundant glycan structure (detected by glycomics) that matched the most abundant glycan composition (detected by glycoproteomics) at each individual site.
T434 16171-16556 Sentence denotes We denote the second glycoform model as “Oxford Class.” The glycans selected for installation to generate the Oxford Class model were chosen because they were the most abundant glycan structure, (detected by glycomics) that was contained within the most highly represented Oxford classification group (detected by glycoproteomics) at each individual site (Figure S7; Tables S1 and S8).
T435 16557-16991 Sentence denotes Finally, we denote the third glycoform model as “Processed.” The glycans selected for installation to generate the Processed model were chosen because they were the most highly trimmed, elaborated, or terminally decorated structure (detected by glycomics) that corresponded to a composition (detected by glycoproteomics) which was present at ≥ 1/3rd of the abundance of the most highly represented composition at each site (Table S1).
T436 16992-17168 Sentence denotes 3D structures of the three glycoforms (Abundance, Oxford Class, Processed) were generated for the SARS-CoV-2 S protein alone, and in complex with the glycosylated ACE2 protein.
T437 17169-17532 Sentence denotes The glycoprotein builder available at GLYCAM-Web (www.glycam.org) was employed together with an in-house program that adjusts the asparagine side chain torsion angles and glycosidic linkages within known low-energy ranges (Nivedha et al., 2014) to relieve any atomic overlaps with the core protein, as described previously (Grant et al., 2016; Peng et al., 2017).
T438 17533-17732 Sentence denotes Energy minimization and Molecular dynamics (MD) simulations – Each glycosylated structure was placed in a periodic box of TIP3P water molecules with a 10 Å buffer between the solute and the box edge.
T439 17733-17928 Sentence denotes Energy minimization of all atoms was performed for 20,000 steps (10,000 steepest decent, followed by 10,000 conjugant gradient) under constant pressure (1 atm) and temperature (300 K) conditions.
T440 17929-18171 Sentence denotes All MD simulations were performed under nPT conditions with the CUDA implementation of the PMEMD (Götz et al., 2012; Salomon-Ferrer et al., 2013) simulation code, as present in the Amber14 software suite (University of California, San Diego).
T441 18172-18340 Sentence denotes The GLYCAM06j force field (Kirschner et al., 2008) and Amber14SB force field (Maier et al., 2015) were employed for the carbohydrate and protein moieties, respectively.
T442 18341-18534 Sentence denotes A Berendsen barostat with a time constant of 1 ps was employed for pressure regulation, while a Langevin thermostat with a collision frequency of 2 ps-1 was employed for temperature regulation.
T443 18535-18587 Sentence denotes A nonbonded interaction cut-off of 8 Å was employed.
T444 18588-18697 Sentence denotes Long-range electrostatics were treated with the particle-mesh Ewald (PME) method (Darden and Pedersen, 1993).
T445 18698-18832 Sentence denotes Covalent bonds involving hydrogen were constrained with the SHAKE algorithm, allowing an integration time step of 2 fs to be employed.
T446 18833-18946 Sentence denotes The energy minimized coordinates were equilibrated at 300K over 400 ps with restraints on the solute heavy atoms.
T447 18947-19195 Sentence denotes Each system was then equilibrated with restraints on the Ca atoms of the protein for 1ns, prior to initiating 4 independent 250 ns production MD simulations with random starting seeds for a total time of 1 μs per system, with no restraints applied.
T448 19196-19223 Sentence denotes Antigenic surface analysis.
T449 19224-19578 Sentence denotes A series of 3D structure snapshots of the simulation were taken at 1 ns intervals and analyzed in terms of their ability to interact with a spherical probe based on the average size of hypervariable loops present in an antibody complementarity determining region (CDR), as described recently (https://www.biorxiv.org/content/10.1101/2020.04.07.030445v2).
T450 19579-19732 Sentence denotes The percentage of simulation time each residue was exposed to the AbASA probe was calculated and plotted onto both the 3D structure and primary sequence.
T451 19734-19799 Sentence denotes Analysis of SARS-CoV-2 Spike VSV Pseudoparticles (ppVSV-SARS-2-S)
T452 19800-19907 Sentence denotes 293T cells were transfected with an expression plasmid encoding SARS-CoV-2 Spike (pcDNAintron-SARS-2-SΔ19).
T453 19908-20020 Sentence denotes To increase cell surface expression, the last 19 amino acids containing the Golgi retention signal were removed.
T454 20021-20102 Sentence denotes Two SΔ19 constructs were compared, one started with Met1 and the other with Met2.
T455 20103-20236 Sentence denotes Twenty-four h following transfection, cells were transduced with ppVSVΔG-VSV-G (particles that were pseudotyped with VSV-G in trans).
T456 20237-20319 Sentence denotes One h following transduction cells were extensively washed and media was replaced.
T457 20320-20434 Sentence denotes Supernatant containing particles were collected 12-24 h following transduction and cleared through centrifugation.
T458 20435-20490 Sentence denotes Cleared supernatant was frozen at −80°C for future use.
T459 20491-20588 Sentence denotes Target cells Vero E6 were seeded in 24-well plates (5x105 cells/mL) at a density of 80% coverage.
T460 20589-20778 Sentence denotes The following day, ppVSV-SARS-2-S/GFP particles were transduced into target cells for 60 min, particles pseudotyped with VSV-G, Lassa virus GP, or no glycoprotein were included as controls.
T461 20779-20999 Sentence denotes 24 h following transduction, transduced cells were released from the plate with trypsin, fixed with 4% formaldehyde, and GFP-positive virus-transduced cells were quantified using flow cytometry (Bectin Dickson BD-LSRII).
T462 21000-21236 Sentence denotes To quantify the ability of various SARS-CoV-2 S mutants to mediate fusion, effector cells (HEK293T) were transiently transfected with the indicated pcDNAintron-SARS-2-S expression vector or measles virus H and F (Brindley et al., 2014).
T463 21237-21357 Sentence denotes Effector cells were infected with MVA-T7 four h following transduction to produce the T7 polymerase (Paal et al., 2009).
T464 21358-21590 Sentence denotes Target cells naturally expressing the receptor ACE2 (Vero) or ACE2 negative cells (HEK293T) were transfected with pTM1-luciferase, which encodes for firefly luciferase under the control of a T7 promoter (Brindley and Plemper, 2010).
T465 21591-21696 Sentence denotes 24 h following transfection, the target cells were lifted and added to the effector cells at a 1:1 ratio.
T466 21697-21827 Sentence denotes 4 h following co-cultivation, cells were washed, lysed and luciferase levels were quantified using Promega’s Steady-Glo substrate.
T467 21828-21943 Sentence denotes To visualize cell-to-cell fusion, Vero cells were co-transfected with pGFP and the pcDNAintron-SARS-2-S constructs.
T468 21944-22024 Sentence denotes 24 h following transfection, syncytia was visualized by fluorescence microscopy.