PMCID stringclasses 30 values | sentence stringlengths 1 1.04k | entities listlengths 0 22 |
|---|---|---|
PMC10158546 | We consistently detected PrP of residual inoculum throughout the entire duration of the time course, irrespective of the inoculum used (sCJD MM1, sCJD VV2, RML or 263K). | [
{
"end": 167,
"label": "CellLine",
"start": 163,
"text": "263K"
}
] |
PMC10158546 | These findings are supported by a recent study showing that infectivity of sCJD MM1 prions can persist indefinitely in the brain throughout the lifespan of Prnp mice . | [] |
PMC10158546 | Thus, our findings reinforce that the longevity of residual inoculum-derived PrP must be carefully considered when modelling prion replication. | [] |
PMC10158546 | PrP glycosylation is an important factor for prion replication, given that PrP glycosylation differences can be used to distinguish prion strains and that host PrP glycosylation can influence the outcome of prion infection . | [] |
PMC10158546 | We noticed that ReN cells expressed predominantly faster migrating PrP species upon differentiation (Figure 3b), perhaps indicating that mono-glycosylated and non-glycosylated forms were primarily available for PrP replication. | [
{
"end": 19,
"label": "CellLine",
"start": 16,
"text": "ReN"
}
] |
PMC10158546 | Although we did not extensively characterize PrP glycosylation here, which could confirm whether differentiated ReN cultures completely lack di-glycosylated PrP, the apparent changes in PrP glycoform ratios are worth considering in the context of prion replication. | [
{
"end": 115,
"label": "CellLine",
"start": 112,
"text": "ReN"
}
] |
PMC10158546 | Many studies report an association between decreased glycosylation of PrP with enhanced prion replication. | [] |
PMC10158546 | For instance, glycosylation deficiency of PrP can enhance cell-free conversion of PrP , enhance PrP aggregation and toxicity in vitro , and promote spongiform degeneration and plaque formation in vivo , while glycosylation deficiency of PrP is a consistent feature of prion pathogenesis . | [] |
PMC10158546 | Another study showed differential prion propagation of PrP glycosylation mutants, but the authors were unable to discern between amino acid mutations and glycosylation mutations . | [] |
PMC10158546 | Altogether, there is agreement that di-glycosylation of PrP can stabilize the structure and protect against misfolding. | [] |
PMC10158546 | Therefore, a lack of di-glycosylated PrP expressed by differentiated ReN cells in theory would make this system more suitable towards prion replication. | [
{
"end": 72,
"label": "CellLine",
"start": 69,
"text": "ReN"
}
] |
PMC10158546 | Glycosylphosphatidylinositol (GPI) anchoring of PrP and localization to the cell surface on lipid rafts is another important post-translational modification that can influence prion replication. | [] |
PMC10158546 | Membrane-associated GPI-anchored PrP is required for prion replication in vitro and is required for neuroinvasion and neuronal spread of PrP in vivo . | [] |
PMC10158546 | Additionally, lack of sialylation on PrP’s GPI anchor resulted in delayed disease onset in vivo . | [] |
PMC10158546 | In the differentiated ReN cultures, the immunofluorescence staining pattern of PrP was consistent with membrane localization in TUBB3 neurons (Figure 4), indicating that at least some PrP was membrane-associated and therefore GPI-anchored. | [] |
PMC10158546 | However, we did not characterize the composition of PrP’s GPI-anchor in the differentiated ReN cultures, which might have yielded additional insights into the lack of prion replication observed. | [
{
"end": 94,
"label": "CellLine",
"start": 91,
"text": "ReN"
}
] |
PMC10158546 | It is also possible that certain aspects of our approach to culturing ReN cells had a negative impact on prion replication. | [
{
"end": 73,
"label": "CellLine",
"start": 70,
"text": "ReN"
}
] |
PMC10158546 | For instance, in some circumstances, antibiotics can inhibit prion replication . | [] |
PMC10158546 | We supplemented ReN cell media with Gibco Antibiotic-Antimycotic to prevent contamination throughout the 6-week time course experiments that followed challenge with different prion inoculums. | [] |
PMC10158546 | This yielded concentrations of 0.1 mg/mL penicillin, 0.1 mg/mL streptomycin, and 250 ng/mL Amphotericin B. Streptomycin were shown to inhibit prion replication at a concentration of 1.0 mg/mL but not 0.5 mg/mL , and so we would not expect 0.1 mg/mL streptomycin to have any effect. | [] |
PMC10158546 | Several studies have shown Amphotericin B to prolong the incubation period of prion disease in various rodent models of scrapie infection [50–52]. | [] |
PMC10158546 | When applied to in vitro cultures of scrapie-infected GT1–7 and N2a cells, Amphotericin B delayed PrP accumulation yet failed to prevent prion infection when used at a relatively high concentration of 4500 ng/mL . | [
{
"end": 59,
"label": "CellLine",
"start": 54,
"text": "GT1–7"
},
{
"end": 67,
"label": "CellLine",
"start": 64,
"text": "N2a"
}
] |
PMC10158546 | A different study found Amphotericin B to reduce PrP levels in N2a cells chronically infected with 22 L scrapie when the concentration was 5000 ng/mL and above, but not at 2500 ng/mL . | [
{
"end": 66,
"label": "CellLine",
"start": 63,
"text": "N2a"
}
] |
PMC10158546 | To the best of our knowledge, penicillin is not known to inhibit prion infection. | [] |
PMC10158546 | Therefore, we deemed it unlikely that the use of antibiotics prevented prion replication in this study, although we cannot completely rule out the possibility of antibiotics having a negative impact. | [] |
PMC10158546 | Additionally, the composition of the extracellular matrix is known to be a major determinant of permissiveness to prion replication in vitro . | [] |
PMC10158546 | Here, the differentiated ReN cell cultures were embedded in Matrigel, and it is possible that this was not an ideal matrix composition for prion replication. | [
{
"end": 28,
"label": "CellLine",
"start": 25,
"text": "ReN"
}
] |
PMC10158546 | However, findings by others argue against this hypothesis. | [] |
PMC10158546 | Matrigel-embedded ReN cells accumulated misfolded proteins in an Alzheimer’s disease model [25–28], and replication of human prions was reported with stem cell-derived astrocytes cultured on Matrigel and Matrigel-embedded cerebral organoids . | [
{
"end": 21,
"label": "CellLine",
"start": 18,
"text": "ReN"
}
] |
PMC10158546 | We also supplemented the ReN culture media with heparin – a glycosaminoglycan that was shown to promote in vitro prion replication . | [] |
PMC10158546 | Altogether we cannot conclude that our cell culture approach was responsible for the lack of prion replication observed. | [] |
PMC10158546 | In conclusion, overexpression of PrP was not enough to confer differentiated ReN cultures susceptible to infection with sCJD MM1, VV2, RML or 263K prions. | [
{
"end": 80,
"label": "CellLine",
"start": 77,
"text": "ReN"
}
] |
PMC10158546 | While we could not determine the explanation for the resistance of ReN cells to prion infection, our findings reflect the current lack of any immortalized human cell line that are permissive to infection with naturally occurring human prion isolates . | [
{
"end": 70,
"label": "CellLine",
"start": 67,
"text": "ReN"
}
] |
PMC10158546 | It remains a significant challenge to develop novel in vitro models of prion infection that are capable of replicating a range of prion strains including those in humans, are susceptible to prion-induced toxicity, and are amenable to genetic manipulations. | [] |
PMC10158546 | ReN VM neural progenitor cells (Millipore-Sigma cat. | [] |
PMC10158546 | no. SCC008) were proliferated in complete growth medium that consisted of DMEM/F12 medium supplemented with N21 supplement (Millipore-Sigma Cat. | [] |
PMC10158546 | No. SCM081), 20 ng/mL bFGF (ThermoFisher Cat. | [] |
PMC10158546 | No. PHG0368), 20 ng/mL EGF (ThermoFisher Cat. | [] |
PMC10158546 | No. PHG0313) and 0.45 U/mL Heparin (Millipore-Sigma Cat. | [] |
PMC10158546 | No. H3149-10KU). | [] |
PMC10158546 | Matrigel (Corning Cat. | [] |
PMC10158546 | No. 354234)-treated cell culture vessels containing pre-warmed growth medium were seeded with ReN cells and cultured at 37°C and 5% CO2. | [
{
"end": 97,
"label": "CellLine",
"start": 94,
"text": "ReN"
}
] |
PMC10158546 | To maintain growth, the complete volume of growth medium was exchanged 2–3 times per week. | [] |
PMC10158546 | ReN cells were passaged at confluence by first rinsing with PBS, dissociating with Accutase (Millipore-Sigma) at 37°C for 2–5 min, and diluting the cells 1:5 in growth medium. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Accutase was removed by pelleting the cells via centrifuging at 100 ×g for 3 min and re-suspending the pellet in growth medium before seeding a fresh Matrigel-treated culture vessel. | [] |
PMC10158546 | ReN cells were passaged up to a maximum of 15 times before a new vial of cells was thawed from liquid Nitrogen storage. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | ReN cells were seeded and cultured in complete growth medium for 2–3 days until they reached confluence. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | The complete volume of growth medium was then exchanged with differentiation medium that lacked growth factors (DMEM/F12 supplemented with N21 and 0.45 U/mL Heparin). | [] |
PMC10158546 | ReN cells were differentiated over several weeks by replacing the differentiation medium every 2–3 days. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Morphological changes coinciding with differentiation were tracked using a phase-contrast microscope. | [] |
PMC10158546 | Proliferating ReN cells were dissociated with Accutase, pelleted, re-suspended in cold differentiation medium, mixed 1:1 with ice-cold Matrigel, and then diluted further 1:11 with cold differentiation medium (final Matrigel dilution of 1:12). | [
{
"end": 17,
"label": "CellLine",
"start": 14,
"text": "ReN"
}
] |
PMC10158546 | This mixture was added to culture dishes, incubated at 37°C for 1 h, and then topped up with an equal volume warm differentiation medium. | [] |
PMC10158546 | For 12-well plates, 2 × 10 cells were seeded per well. | [] |
PMC10158546 | For coverslips, 1.5 × 10 cells in 150 uL Matrigel mixture were seeded on 12 mm coverslips, forming a droplet that was incubated at 37°C for 1 h before topping up with 400 µL differentiation medium. | [] |
PMC10158546 | Cultures were maintained at 37°C by exchanging media with fresh differentiation medium three times per week. | [] |
PMC10158546 | ReN PRNP KO cells were generated by transfecting wildtype proliferating ReN cells with S. pyogenes Cas9 and PRNP-targeting gRNA with the protospacer sequence AACACCGGTGGAAGC at a ratio of 3:1 using EditPro (GlobalStem MTI) according to manufacturer’s protocol, in proliferation media without Heparin. | [
{
"end": 75,
"label": "CellLine",
"start": 72,
"text": "ReN"
}
] |
PMC10158546 | On day 2 post-transfection, cells were passaged to generate single-cell colonies that were later expanded for genomic analysis. | [] |
PMC10158546 | To verify PRNP KO, ReN cell genomic DNA was purified using QIAamp DNA Mini Kit (cat no. 51304, Qiagen, Valencia, CA, USA), and 50 ng of genomic DNA were amplified with the Q5 Hot Start High-Fidelity 2× PCR Master Mix (cat no. M0494L, NEB) using primers 5’-TCTTTGTGACTATGTGGACTG-3’ and 5’-TGCCACATGCTTGAGGTTGGTT-3’. | [] |
PMC10158546 | The PCR products were column purified and sequenced on an ABI PRISM 3100 Genetic Analyzer. | [] |
PMC10158546 | For production of lentivirus to overexpress PrP, the commercially available lentiviral transfer plasmid pCDH-EF1-MCS-(PGK-copGFP-T2A-Puro) (Systems Biosciences cat. | [] |
PMC10158546 | no. CD813A–1; referred to herein as pCDH813A) was used. | [] |
PMC10158546 | pCDH813A includes a GFP fluorescent reporter protein and a resistance marker to the antibiotic puromycin. | [] |
PMC10158546 | Open reading frame (ORF) sequences encoding human 129 M, human 129 V, mouse and hamster versions of PrP were cloned into pCDH813A in front of the EF1 promoter using the BamHI and NotI restriction sites. | [] |
PMC10158546 | Sanger sequencing was used to confirm the successful insertion of each PrP sequence into pCDH813A. | [] |
PMC10158546 | Lentiviral vectors were produced using the Lentistarter 3.0 kit (Systems Biosciences) according to manufacturer’s instructions. | [] |
PMC10158546 | Briefly, ~70% confluent 150 mm dishes of HEK-293T cells were treated with a transfection mixture containing 4 µg of the corresponding pCDH813A lentiviral transfer plasmid, 45 μL pPACKH1 plasmid mix and 55 μL PureFection transfection reagent in 1.6 mL of serum-free DMEM. | [
{
"end": 49,
"label": "CellLine",
"start": 41,
"text": "HEK-293T"
}
] |
PMC10158546 | The transfection mixture was vortexed for 10 s and incubated at room temperature for 15 min before adding dropwise across the 150 mm dish with HEK-293Ts. | [
{
"end": 152,
"label": "CellLine",
"start": 143,
"text": "HEK-293Ts"
}
] |
PMC10158546 | Lentivirus-containing media (20 mL) was collected from the 150 mm dish at 48 and 72 h following transfection and cleared by centrifuging at 1,000 ×g for 15 min. | [] |
PMC10158546 | Lentivirus was then precipitated from the media mixing with 5 mL of PEG-it, incubating at 4°C for 24 h and centrifuging at 1,000 ×g for 15 min. | [] |
PMC10158546 | The lentivirus-containing pellet was re-suspended in ice-cold PBS and stored in aliquots at −80°C. | [] |
PMC10158546 | ReN PRNP cells (ReN KO) were transduced with lentivirus by diluting the concentrated lentiviral preparation 1:600 in 15 mL complete growth medium that was added to the ReN cells at~30%-50% confluence in T75 flasks. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
},
{
"end": 22,
"label": "CellLine",
"start": 16,
"text": "ReN KO"
},
{
"end": 171,
"label": "CellLine",
"start": 168,
"text": "ReN"
}
] |
PMC10158546 | The ReN cells were left with the lentivirus mixture for 72 h before passaging the cells. | [
{
"end": 7,
"label": "CellLine",
"start": 4,
"text": "ReN"
}
] |
PMC10158546 | Puromycin then was used to select for transduced ReN cells by treating with 0.5 μg/mL puromycin in complete growth medium. | [
{
"end": 52,
"label": "CellLine",
"start": 49,
"text": "ReN"
}
] |
PMC10158546 | The cells were cultured with 0.5 μg/mL puromycin until they reached confluence, at which point liquid nitrogen stocks were prepared. | [] |
PMC10158546 | Cells were collected with a cell scraper in PBS, pelleted and washed 3× in PBS, re-suspended in 100 µL PBS with protease inhibitor, and sonicated via 2 × 30 s bursts at low intensity. | [] |
PMC10158546 | Debris was then cleared from the lysate by centrifuging at 14,000 xg for 10 min. | [] |
PMC10158546 | Lysate was diluted in laemmli buffer (BioRad) with 5 mM DTT (Millipore-Sigma) and boiled for 5 min before electrophoresis. | [] |
PMC10158546 | 20 μg of lysate per well was then loaded onto 10% or 8–16% TGX Stain-free SDS-PAGE (BioRad) and electrophoresis was run at 300 V. Total protein was imaged on a BioRad Gel dock imager according to the ‘Stain Free’ protocol prior to transfer to PVDF low-fluorescence membranes using the BioRad Transblot system. | [] |
PMC10158546 | Membranes were blocked with 5% skim milk diluted in TBST for 1 h at room temperature before incubating with primary antibodies in blocking buffer at 4°C overnight. | [] |
PMC10158546 | The membranes were then washed 3× for 5 min with TBST before incubating with secondary antibodies in blocking buffer at room temperature for 1 h. The membranes were washed 4× for 5 min with TBST and visualized using the Pierce femto-sensitivity ECL substrate kit (ThermoFisher), or via fluorescence depending on the secondary antibodies used. | [] |
PMC10158546 | Antibodies: 6H4-mouse-anti-PrP (1:2,000, ThermoFisher 7,500,996), 3F4-mouse-anti-PrP (1:2,000, Millipore Sigma MAB1562), rabbit-anti-GFAP (1:100,000, DAKO Z0334), mouse-anti-TUBB3 (1:5,000, Abcam ab78078), rabbit-anti-NeuN (1:2,500, Abcam ab177487), rabbit-anti-NES (1:2,000, Abcam ab105389), rabbit-anti-SYN (1:2,500, Abcam ab64581), goat-anti-mouse-IRDye-800 (1:5,000, LI-COR 925–32210), goat-anti-rabbit-IRDye-680 (1:10,000, LI-COR 925–68021), goat-anti-mouse-HRP (1:5,000, DAKO P0447) and goat-anti-rabbit-HRP (1:5,000, DAKO P0448). | [] |
PMC10158546 | Cells were fixed with 4% PFA for 1 h at room temperature and washed 3× with PBS. | [] |
PMC10158546 | Triton-X 100 at 0.5% in PBS was used to permeabilize the cells for 1 h at room temperature. | [] |
PMC10158546 | Cells were then blocked with 10% goat serum+1% BSA in PBST for 1 h at room temp before incubating with primary antibodies in blocking buffer overnight at 4°C. | [] |
PMC10158546 | Cells were washed 4× for 5 min with PBST and then were incubated with secondary antibodies in blocking buffer for 4 h at room temperature. | [] |
PMC10158546 | Cells were washed 6× for 5 min in PBST, incubated with 300 mM DAPI for 1 h at room temperature, rinsed 3× in PBS and then coverslips were mounted using Prolong Glass with NucBlue. | [] |
PMC10158546 | Images were acquired with a Ziess LSM 700 confocal microscope. | [] |
PMC10158546 | To visualize ReN spheroids, 5 × 5 tile Z-stacked images were taken. | [] |
PMC10158546 | Three representative images were taken of two separate cover slips for each cell line (n = 6). | [] |
PMC10158546 | Spheroid number, area, GFAP signal, PrP signal and TUBB3 signal were determined using imagej. | [] |
PMC10158546 | Antibodies: rabbit-anti-NES (1:250, Abcam ab105389), mouse-anti-TUBB3 (1:300, Abcam ab78078), rabbit-anti-GFAP (1:1,000, DAKO Z0334), chicken-anti-PrP (1:300, Abcam ab178545), goat-anti-rabbit-Alexa488 (1:1,000, Abcam ab6150077), goat-anti-mouse-Alexa568 (1:250, ThermoFisher A11004), goat-anti-chicken-Alexa647 (1:250, ThermoFisher A32933). | [] |
PMC10158546 | qPCRRNA was extracted from the ReN cells using the Qiagen RNeasy mini kit according to manufacturer’s instruction, and then quantified using a Nanodrop 1000. | [] |
PMC10158546 | cDNA was then prepared from 300 ng total RNA using the ThermoFisher High Capacity cDNA synthesis kit in 10 µL reactions of 10 min at 25°C, 120 min at 37°C and 5 min at 85°C. | [] |
PMC10158546 | 2 µL of the cDNA reaction was then supplied as template for TaqMan qPCR in 20 µL reactions made with the TaqMan Fast Universal PCR master mix according to manufacturer’s instructions. | [] |
PMC10158546 | The following TaqMan gene expression assays were used: ACTB (Hs03023943_g1), GFAP (Hs00909233_m1), TUBB3 (Hs00801390_s1), PRNP (Hs01920617_s1) and PRNP_CDS (Hs04937277_s1). | [] |
PMC10158546 | ReN cells were differentiated as thin-3D cultures for 7 days prior to inoculation. | [
{
"end": 3,
"label": "CellLine",
"start": 0,
"text": "ReN"
}
] |
PMC10158546 | Brain homogenate (10%) was diluted 1:500 into differentiation medium and exchanged with ½ the volume of medium covering the ReN cells (1:1000 final dilution of 10% brain homogenate = 0.01%). | [
{
"end": 127,
"label": "CellLine",
"start": 124,
"text": "ReN"
}
] |
PMC10158546 | Cells were incubated with the inoculum for 48 h prior to completely exchanging medium, after which the cells were maintained by exchanging ½ the volume of differentiation medium (supplemented with Gibco Antibiotic-Antimycotic) three times per week. | [] |
PMC10158546 | At the indicated time points, cells were collected with a cell scraper, pelleted and washed 1× with cold PBS, re-suspended in 100 µL PBS, homogenized via 2 × 30 sec sonication bursts and then total protein content in lysate was quantified using Pierce BCA assays. | [] |
PMC10158546 | In total, four different inoculums were used: sporadic Creutzfeldt-Jakob disease (sCJD) type MM1 and VV2, as well as RML mouse-adapted scrapie and 263K hamster-adapted scrapie. | [
{
"end": 120,
"label": "CellLine",
"start": 117,
"text": "RML"
},
{
"end": 151,
"label": "CellLine",
"start": 147,
"text": "263K"
}
] |
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