Journal: Molecular and Clinical Oncology

Article Title: Epstein-Barr virus infection mediated TP53 and Bcl-2 expression in nasopharyngeal carcinoma pathogenesis

doi: 10.3892/mco.2021.2422

Figure Lengend Snippet: Representative immunohistochemical staining profile images of Bcl-2, EBV and TP53. Representative images of (A) a Bcl-2 positive nasopharyngeal carcinoma sample; (B) EBV positive nasopharyngeal carcinoma sample; and (C) TP53 positive laryngeal carcinoma sample. Magnification, x40. Bcl-2, B cell lymphoma-2; EBV, Epstein Barr virus.

Article Snippet: An EBV positive control slide was commercially procured (cat. no. CSE0125P; StatLab).

Techniques: Immunohistochemical staining, Staining, Virus

Journal: Molecular and Clinical Oncology

Article Title: Epstein-Barr virus infection mediated TP53 and Bcl-2 expression in nasopharyngeal carcinoma pathogenesis

doi: 10.3892/mco.2021.2422

Figure Lengend Snippet: Representative positive and negative immunohistochemical staining profile images of Bcl-2, EBV and TP53. (A) Bcl-2 negative control; (B) TP53 negative control; (C) EBV negative control; (D) Bcl-2 positive control; (E) TP53 positive control; and (F) EBV positive control. Bcl-2, B cell lymphoma-2; EBV, Epstein Barr virus.

Article Snippet: An EBV positive control slide was commercially procured (cat. no. CSE0125P; StatLab).

Techniques: Immunohistochemical staining, Staining, Negative Control, Positive Control, Virus

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Choice of cell line for sACE2 2 .v2.4-IgG1 production impacts pharmacokinetics and glycosylation (A and B) sACE2 2 .v2.4-IgG1 was expressed and purified from human Expi293F (gray) or nonhuman ExpiCHO-S (black) cultures that were both transiently transfected. A 10 mg/kg amount of sACE2 2 .v2.4-IgG1 was injected into the tail vein of human FcRn transgenic mice. (A) ACE2 catalytic activity and (B) protein concentrations based on ELISA were measured in plasma. Data are mean ± SEM, n = 3 mice per time point. (C) N-glycan types from glycomics analysis of sACE2 2 .v2.4-IgG1 produced in ExpiCHO-S vs. Expi293F cells. (D) Abundance of sialylated (Neu5Ac) and fucosylated N-glycan structures. (E and F) O-Glycan analysis of sACE2 2 .v2.4-IgG1 produced in (E) ExpiCHO-S and (F) Expi293F cells. After PNGaseF treatment of protein, O-glycans were released, purified, permethylated, and analyzed by MALDI-TOF-MS. O-glycan structures were assigned using Glycoworkbench software based on precursor masses and the common mammalian biosynthetic pathway. Glycan structures are indicated on the x axis by their m/z ratio. GlcNAc, blue squares; GalNAc, yellow squares; Gal, yellow circles; Neu5Ac, purple diamonds.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Drug discovery, Glycoproteomics, Purification, Transfection, Injection, Transgenic Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Produced, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: An engineered derivative of sACE2 2 .v2.4-IgG1 and its glycosylation (A) Left, the structure (PDB: 6M17 ) of dimeric ACE2 (chains “A” and “B” in dark and light green) bound to RBD (gray ribbons). Glycans are shown as orange sticks. PD, protease domain; CLD, collectrin-like dimerization domain. Center and right, residues mutated to fill cavities (blue spheres), introduce disulfides (yellow spheres), or add N-glycosylation motifs (purple spheres) are shown on a single ACE2 subunit. Lead candidate sACE2 2 .S19-IgG1 has mutations V491I, M662T, N720S. (B) N-glycan types on sACE2 2 .S19-IgG1 produced in Expi293F cells. (C) Abundance of sialylated and fucosylated N-glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells. (D) O-Glycan structures on sACE2 2 .S19-IgG1 produced in Expi293F cells following O-glycan release and MALDI-TOF-MS analysis. (E) Occupancy of the N-glycosylation sites based on glycopeptidomics analysis of sACE2 2 .S19-IgG1 from Expi293F (green), sACE2 2 .v2.4-IgG1 from Expi293F (pale gray), and sACE2 2 .v2.4-IgG1 from ExpiCHO-S (dark gray). sACE2 2 .S19-IgG1 has added glycosylation sites at positions 660 and 718. (F) Percent of the glycoforms at each N-glycosylation site that have at least one sialic acid.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Glycoproteomics, Introduce, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: BLI kinetics for monovalent binding of decoy receptors to Spike RBD

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Binding Assay, Mutagenesis

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: High sialylation and YTE mutations in the Fc region enhance the pharmacokinetics of sACE2 2 .S19-IgG1 (A) Intravenous administration of proteins at 10 mg/kg into human FcRn mice. Blood was collected into heparin via retroorbital route at the plotted time points. Plasma levels of the indicated proteins were measured by ELISA. (B and C) Proteins were injected s.c. in the flank of human FcRn mice at a dose of 10 mg/kg (solid lines) or 100 mg/kg (broken line). (B) ACE2 catalytic activity in plasma and (C) protein concentrations based on ELISA. (D) sACE2 2 .S19-IgG1(YTE) was treated with PNGase F or neuraminidase. Proteins (20 μg) were analyzed without further purification by SDS-PAGE under non-reducing conditions and stained with Coomassie. The calculated molecular weight (MW) of the mature polypeptide (excluding glycans) is 218 kD for the dimer. PNGase F has an MW of 36 kD. A. ureafaciens neuraminidase is a mixture of isoenzymes. (E) Proteins (10 μg) were analyzed by IEF gel electrophoresis. Glycosidase-treated proteins were analyzed without (−) and with (+) purification by gel filtration following treatment. (F) Purified proteins were injected i.v. at 10 mg/kg into human FcRN mice and concentrations in plasma were measured by ELISA for 5 days. For PK studies in this figure, data are mean ± SEM for n = 3 per group and proteins were purified from transiently transfected Expi293F.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Drug discovery, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection, Activity Assay, Purification, SDS Page, Staining, Molecular Weight, Nucleic Acid Electrophoresis, Filtration, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Pharmacokinetic properties of optimized decoy receptors in Tg32 mice

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Produced, Transfection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: A single dose of sACE2 2 .S19-IgG1(YTE) sourced from Expi293F culture protects K18-hACE2 mice from lethal SARS-CoV-2 challenge (A and B) K18-hACE2 mice were inoculated intranasally with 1 × 10 4 PFU 2019n-CoV/USA_WA1/2020 virus. Mice were administered a single i.v. dose (10 mg/kg) of sACE2 2 .S19-IgG1(YTE) (red; purified from transiently transfected Expi293F culture) or IgG1 control (gray) 24 h post-inoculation. Survival (A) and weights (B) for N = 10 mice per treatment group. p value determined by Gehan-Breslow-Wilcoxon test. (C and D) Lungs of inoculated mice were harvested at day 7 and relative viral loads were determined by qPCR for mRNA expression levels of SARS-CoV-2 Spike (C) and Nsp (D). Mean ± SEM, N = 4 per treatment group. p values determined by unpaired t test.

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Virus, Purification, Transfection, Control, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: Sialylation of decoy receptors purified from different sources

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Purification

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation

doi: 10.1016/j.omtm.2024.101301

Figure Lengend Snippet: The v2.4 and S19 mutations in ACE2 are predicted to have low immunogenicity (A) Computational analysis of calculated affinity of peptides to HLA-II allotypes. The wild-type sACE2 2 -IgG1 sequence (top row) is scanned for peptides predicted to be displayed on a common set of 14 HLA-II allotypes. In this analysis, the minimum threshold for a peptide to be considered an antigen is predicted affinity for four HLA-II allotypes (Nhits ≥4) from a set of 14 common alleles. For sACE2 2 -IgG1 derivatives, the sequence is grayed out except for the regions where mutations are introduced to highlight whether a mutation is within a predicted epitope and/or changes the probability of presentation. Peptides that were analyzed experimentally by yeast display are indicated below with dark red bars. (B) Peptides were displayed on yeast and binding to HLA-II following an HLA-DM-dependent peptide loading reaction was measured by flow cytometry. The correlation plot shows the agreement between two independent replicates measuring mean fluorescence units (MFU) for bound HLA-II. Polyserine negative control reactions are blue, positive control peptide/HLA-II pairs are orange, and ACE2 peptides are gray. (C) Yeast display measurements of HLA-II binding to ACE2 peptides is plotted from no signal (dark blue) to high binding signal (orange).

Article Snippet: The IgG1 is an isotype-matched control antibody (InVivoPlus human IgG1 isotype control, Cat. No. BP0297, Bio X Cell).

Techniques: Immunopeptidomics, Sequencing, Mutagenesis, Binding Assay, Flow Cytometry, Fluorescence, Negative Control, Positive Control

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: Involvement of LRP1 in Tat-mediated HIV-1 LTR transactivation. a The LRP1 antagonist, receptor-associated protein (RAP), concentration dependently reduced Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05). b Protein expression levels of LRP1 were significantly ( n = 3, p < 0.05) reduced in U87MG cells transfected with LRP1 siRNA (100 nM) compared to cells transfected with control (scrambled) siRNA. c siRNA knockdown of LRP1 reduced significantly ( n = 3; * p < 0.05) Tat-mediated HIV-1 LTR transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Concentration Assay, Expressing, Transfection

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE-HDL isoform dependently affected Tat-mediated HIV-1 LTR transactivation. U87MG cells stably transfected with a luciferase gene under the control of HIV-1 Tat responsive LTR promoter were incubated for 48 h with ApoE2-HDL ( a ), ApoE3-HDL ( b ), or ApoE4-HDL ( c ) in the presence of HIV-1 Tat protein (2 μg/ml) and 100 μM chloroquine (CQ). ApoE2-HDL, ApoE3-HDL, and ApoE4-HDL significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001) ( d ). In comparison to ApoE2-HDL and ApoE3-HDL, ApoE4-HDL was less potent and effective at restricting Tat-mediated HIV-1 transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Stable Transfection, Transfection, Luciferase, Incubation, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: HDL affects the ability of ApoE to restrict Tat-mediated HIV-1 LTR transactivation. a ApoE2 significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3; * p < 0.05; ** p < 0.01). b ApoE3 significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3; ** p < 0.01). c ApoE4 concentration dependently enhanced Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3, ** p < 0.01). d In the presence of HDL, ApoE2 was more potent and effective at restricting Tat-mediated HIV-1 LTR transactivation. e In the presence of HDL, ApoE3 was more potent and effective at restricting Tat-mediated HIV-1 LTR transactivation. f In presence of HDL, ApoE4 concentration dependently restricted Tat-mediated HIV-1 LTR transactivation; however, in the absence of HDL, ApoE4 concentration dependently enhanced Tat-mediated HIV-1 LTR transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE2 and ApoE3, but not ApoE4, inhibited HIV-1 Tat internalization. Representative images ( a ) and quantified internalized fluorescence intensities ( b ) show that ApoE2 and ApoE3 at 5 and 10 μg/ml inhibited HIV-1 Tat internalization as evidenced by lower levels of punctate staining of internalized FITC-labeled Tat ( n = 30; *** p < 0.001, **** p < 0.0001), whereas ApoE4 did not inhibit HIV-1 Tat internalization

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Fluorescence, Staining, Labeling

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE mimetic peptide decreased Tat-mediated HIV-1 LTR transactivation. a U87MG cells were treated with an ApoE mimetic peptide in the presence of HIV-1 Tat and chloroquine (CQ) for 48 h. ApoE mimetic peptide significantly reduced Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05). b Incubation with an ApoE4 structure corrector, that makes the structure and function of ApoE4 more like ApoE3, enhanced the ability of ApoE4 to restrict Tat-mediated LTR transactivation ( n = 3; * p < 0.05)

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Incubation