Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Schematic representation of ( A ) SARS-CoV-2 S pseudotyped lentivirus infection aided by S protein ACE2 binding and TMPRSS2. Knocking down of ACE2 and TMPRSS2 using LNP-siRNA LNP-trim inhibiting lentivirus entry ( B ). The presence of LNPs with surface ACE2 peptide (LNP-trap1), rhACE2 (LNP-trap2), and mAb (LNP-trap3) bind to the lentivirus, thereby inhibiting cell entry and infection ( C ).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Infection, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Cytotoxicity of LNP-COOH analyzed using ( A ) cell impedance and ( B ) MTT assays in Calu-3 cells. Cytotoxicity MTT assays of LNP-PEP ( C ), LNP-rhACE2 ( D ), LNP-mAb ( E ), and LNP-si ACE2 ( F ) in Calu-3 cells. Values are expressed as the mean ± standard error of the mean (n = 3).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques:

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Quantitative determination of cellular accumulation of ( A ) LNP-PEP and ( B ) LNP-rhACE2 in Calu-3 cells over time. Representative confocal microscopy images from Calu-3 cells treated (3 hours) with cell-impermeable LNP-PEP ( C ‒ F ) and cell-permeable LNP- si ACE2 ( G ‒ J ). Plasma membrane from Calu-3 cells was stained with WGA Alexa Fluor TM 594 ( C and G ); LNP localization in Calu-3 cells was visualized with green fluorescent DiO dye ( D and H ); and cell nuclei were stained using Hoestch ( E and I ). Composite overlay of all three images showing cell membrane, nuclei, and LNPs ( F and J ). Orthogonal projections of Calu-3 cells treated with LNP-si ACE2 ( K ) for 3 hours. Two-dimensional images in z-position where red lines indicate cross-section through y-position and green lines indicate cross-section through x-position. The white dashed box in image K is enlarged. Values are expressed as the mean ± standard error of the mean (n = 3).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Confocal Microscopy, Membrane, Staining

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Relative ACE2 ( A ) and TMPRSS2( B ) mRNA levels in HEK-293-hACE2 cells at different time points after transfection with LNP-si ACE2 (40 nM si ACE2 ) and LNP-si TMPRSS2 (40 nM si TMPRSS2 ), respectively. The control cells in ( A ) and ( B ) received LNP-siSCR. Relative ACE2 mRNA level in Calu-3 cells 72 hours after receiving LNP-si ACE2 (40 nM si ACE2 ) against control cells that received LNP-siSCR ( C ). Relative ACE2 protein level in Calu-3 cells 72 hours after receiving LNP-si ACE2 (40 nM si ACE2 ) against control cells that received LNP-siSCR ( D ). The uncropped blots are shown in . Values are expressed as the mean ± standard error of the mean (n = 3). Statistical significance between the control and treatment groups were analyzed using t -test (**p < 0.01, ***p<0.001, ****p<0.0001; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Transfection

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Transduction efficiency (luciferase expression) of SARS-CoV-2 S pseudotyped luciferase lentivirus at the given MOIs in HEK-293 and HEK-293-hACE2 cell lines ( A ). Percentage inhibition of infection in ACE2 KD and TMPRSS2 KD HEK-293-hACE2 cells ( B ). Percentage inhibition of infection in ACE2 KD ( C ) and TMPRSS2 KD ( D ) in airway epithelial Calu-3 cell line at different siRNA concentrations (LNP-siRNA: 1, 2, 10, 20 and 40 nM; presented in the graph as log 10 values). The nonlinear regression curve fit analysis of siRNA concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n =3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, **p<0.01; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Transduction, Luciferase, Expressing, Inhibition, Infection, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Percentage inhibition of SARS-CoV-2 S pseudotyped luciferase lentivirus (MOI = 2) infection in Calu-3 cells in the presence of different concentrations (Log ( C ) of ACE2 peptide and LNP-PEP ( A ), rhACE2 and LNP-rhACE2 ( B ), mAb and LNP-mAb ( C ). The nonlinear regression curve fit analysis of concentration vs inhibition is presented in the graphs. Values are expressed as the mean ± standard error of the mean (n = 3).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Inhibition, Luciferase, Infection, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Percentage inhibition of SARS-Cov-2 S pseudotyped luciferase lentivirus infection (MOI = 2) in airway epithelial Calu-3 cell line following a combination of conditions including LNP-PEP treatment and ACE2 and TMPRSS2 knockdown. The extent of infection was measured following a luciferase assay. Values are expressed as the mean ± standard error of the mean (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Inhibition, Luciferase, Infection

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Percentage inhibition of SARS-Cov-2 S pseudotyped luciferase LV infection (MOI = 2) in airway epithelial Calu-3 cell line following treatment with different concentrations of lactoferrin ( A ), camostat mesylate ( B ), and carrageenan ( C ). Percentage inhibition of infection in Calu-3 cells with various combinatory treatments of LNP-PEP (0.1 µg/mL), lactoferrin (10 µM), camostat mesylate (1 µM), and carrageenan (1 µM) ( D ). The extent of infection was measured following a luciferase assay. Values are expressed as the mean ± standard error of the mean (n = 3). Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test (*p < 0.05, ****p<0.0001; p < 0.05 was considered statistically significant).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Inhibition, Luciferase, Infection

Journal: International Journal of Nanomedicine

Article Title: Lipid Nanoparticle-Based Inhibitors for SARS-CoV-2 Host Cell Infection

doi: 10.2147/IJN.S448005

Figure Lengend Snippet: Distribution of LNP-PEP ( A ) and LNP-si ACE2 ( B ) at different time points up to 24 hours after intranasal administration in nude mice. The LNPs were tagged with IR dye (DiR) to aid visualization. The NIR fluorescence images (Ex/Em 745/800) were acquired and analyzed using IVIS imager. The quantitative analysis of fluorescent LNP-PEP in the head ( C ) and body ( D ) regions at different time points. The quantitative analysis of fluorescent LNP-si ACE2 in the head ( E ) and body ( F ) regions at different time points. Values are expressed as the mean ± standard error of the mean (n = 4).

Article Snippet: The ability of LNP-Trap to bind to SARS-CoV-2 spike protein was evaluated using SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit (Cell Signalling Technology) following the manufacturer’s protocol.

Techniques: Fluorescence

Journal: Foods

Article Title: Unripe Black Raspberry ( Rubus coreanus Miquel) Extract and Its Constitute, Ellagic Acid Induces T Cell Activation and Antitumor Immunity by Blocking PD-1/PD-L1 Interaction

doi: 10.3390/foods9111590

Figure Lengend Snippet: Effect of Rubus coreanus extract (RCE) on programmed cell death protein 1 (PD-1)/PD-1 ligand-1 (PD-L1) protein interaction using competitive enzyme-linked immunosorbent assay (ELISA) or cell-based assay. ( A , B ) The effect of RCE on the binding of PD-1 and PD-L1 in vitro using a competitive ELISA assay. ( C , D ) The effect of RCE on cell viability of PD-1/NFAT Jurkat cells ( C ) and PD-L1/aAPC CHO-K1 cells ( E ) using the Cell Counting Kit-8 (CCK) assay. Each of the cells were treated according to the indicated concentrations of RCE for 24 h. ( E ) The effect of RCE on cellular PD-1/PD-L1 blockade activity. PD-1/NFAT Jurkat effector cells and PD-L1/aAPC CHO-K1 target cells were co-cultured with RCE or αPD-L1 for 24 h at the indicated concentrations. The relative PD-1/PD-L1 blockade activity were measured by PD-1/NFAT luciferase reporter assay. ( F ) Effect of RCE on IL-2 cytokine release in co-cultured cell model. Cell culture media was analyzed to measure IL-2 level by cytokine ELISA. Data are presented as mean ± S.E. of three representative independent experiments. Asterisks indicate significant inhibition of PD-1/PD-L1 binding activity by each test inhibitor as compared with the vehicle-treated group; *** p < 0.001, ** p < 0.01, * p < 0.05, compared with the vehicle group.

Article Snippet: The human PD-1/PD-L1 competitive enzyme-linked immunosorbent assay (ELISA) kit and antagonist antibody to human PD-L1 (αPD-L1) were purchased from BPS Bioscience Inc. (San Diego, CA, USA).

Techniques: Competitive ELISA, Enzyme-linked Immunosorbent Assay, Cell Based Assay, Binding Assay, In Vitro, Cell Counting, Activity Assay, Cell Culture, Luciferase, Reporter Assay, Inhibition

Journal:

Article Title: Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Monocytes Involves the Raf-1/MEK1-MEK2/ERK1-ERK2 Pathway

doi:

Figure Lengend Snippet: Effect of PD-098059 on LPS-induced TNF-α mRNA expression in human monocytes. Human monocytes were pretreated with PD-098059 (50 μM, 30 min) and subsequently incubated with buffer (white bars) or 10 ng of LPS/ml (black bars) for 1 h. Thereafter, reverse transcriptase PCR was performed on RNA isolates, and PCR fragments were quantified with a digoxigenin-labeled probe as described in Materials and Methods. Results are expressed as fold induction [(TNF-αx/GAPDHx)/(TNF-αcontrol/GAPDHcontrol)]. Results are expressed as the means from four independent experiments ± standard errors. ∗, significant difference (P < 0.05) by Student’s t test for paired samples.

Article Snippet: Random hexamer primers, DNase I (RNase free), and the digoxigenin detection fluorescence ELISA kit were obtained from Boehringer (Mannheim, Germany).

Techniques: Expressing, Incubation, Labeling