Journal: Journal of Virology

Article Title: Resistance of endothelial cells to SARS-CoV-2 infection in vitro

doi: 10.1128/jvi.01205-25

Figure Lengend Snippet: mRNA expression of ACE2, TMPRSS2 , BSG , PPIA , and PPIB in human nasal epithelial cells (NECs) and endothelial cells (aortic, microvascular, and blood outgrowth). Expression levels for the genes ACE2 ( A ), TMPRSS2 ( B ), BSG ( C ), PPIA ( D ), and PPIB ( E ) were obtained from aortic (AoEC), microvascular (HMVEC), and blood outgrowth (BOEC) endothelial cells and NECs. Data for each donor were normalized using the average of the housekeepers (18S and Gapdh) and analyzed using a comparative Ct method (2ΔΔCt). Data are shown as the mean ± SEM fold change compared to nasal epithelium ( n = 3 wells using cells from two donors) for AoEC ( n = 3 wells using cells of three separate donors), HMVEC ( n = 3 wells using cells of three separate donors and BOECs ( n = 2 wells using cells of two separate donors).

Article Snippet: Nasal epithelial cells (Promocell) were maintained in Airway Epithelial Growth Media (Promocell) and differentiated nasal epithelial cells (MucilAir) (Epithelix, Switzerland) were grown in air-liquid interface culture using MucilAir Culture Medium (Epithelix).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Resistance of endothelial cells to SARS-CoV-2 infection in vitro

doi: 10.1128/jvi.01205-25

Figure Lengend Snippet: SARS-CoV-2 virus infection in human airway epithelial cells in air:liquid interface, Vero E6, and endothelial cells. Human airway epithelial cells grown in an air:liquid interface (MucilAir) were infected with SARS-CoV-2 live virus (MOI = 0.1). Infectious virus released to the apical side of the epithelium was determined over time (6, 24, 48, and 72 h post-infection) ( A ). In separate studies, the levels of SARS-CoV-2 nucleocapsid or spike protein in Vero E6 and endothelial cells (treated with media only [untreated] or IL-1β [10 ng/mL; 3 h]) at 24 ( B ) and 72 ( C ) h post-infection with SARS-CoV-2 (MOI = 0.1) were determined using fluorescent imaging. Mock controls (media only) experiments were run simultaneously using each endothelial cell line. Data are shown as n = 3 (pooled donors) for Mucilair cells ( A ) and n = 3 (separate donors) for human aortic (AoEC), lung microvascular (HMVEC), and blood outgrowth endothelial cells (BOECs). Data are shown as mean ± SEM for (A) and representative images shown for (B) and (C) (scale bar = 25 µm).

Article Snippet: Nasal epithelial cells (Promocell) were maintained in Airway Epithelial Growth Media (Promocell) and differentiated nasal epithelial cells (MucilAir) (Epithelix, Switzerland) were grown in air-liquid interface culture using MucilAir Culture Medium (Epithelix).

Techniques: Virus, Infection, Imaging

Journal: Advanced Science

Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses

doi: 10.1002/advs.202506764

Figure Lengend Snippet: Structure analysis and computational design strategy for thermostable and proteolysis‐resistant hIFN‐λ3. a) The 3D structure of the hIFN‐λ3/hIFN‐λR1/hIL‐10Rβ (PDB code: 5T5W) [ ⁵⁰ ] complex and its downstream signaling pathway. b) Induction of representative ISG expression ( Isg15, Mx1, and Oas1 ) in HNEpCs after 12‐h treatment with recombinant hIFN‐λs WT, analyzed by RT‐qPCR ( n = 3). mRNA levels were calculated relative to non‐treated controls and normalized to human 18s rRNA expression. c) First‐derivative plots (dF/dT) of thermal shift assays for calculating melting temperatures (Tm). 12.5 µg of each hIFN‐λ was mixed with 2.5 µL of diluted Protein Thermal Shift Dye. Tm values, corresponding to the peak dF/dT temperature, are indicated. d) Surface representation of hIFN‐λ3 (PDB code: 3HHC). [ ⁴⁹ ] The structure of the α3‐4 loop, which is missing in the crystal structure, is predicted by AlphaFold2 (AF2). The binding sites of hIFN‐λ3 for IL‐10Rβ and IFN‐λR1 are highlighted in cyan and lime, respectively. The exposed hydrophobic patch (magenta), thrombin cleavage site (red), and flexible α3–4 loop (dark green) are shown. e) Targeted backbone redesign using RFdiffusion. The designated hotspot residues (59 V, 118L, and 122L) on the exposed hydrophobic patch for RFdiffusion are highlighted in light gray. The resulting 100 designed backbones are categorized by scaffold length and the number of α‐helix turns at the redesigned backbone. f) Sequence design of 45 redesigned backbones (10 sequences/1 redesigned backbone) using ProteinMPNN, followed by structure prediction with AlphaFold2 (AF2). g) Scatter plot showing AlphaFold2‐predicted confidence (pLDDT) versus RMSD (AF2 prediction compared to the generated backbone) for 450 designed hIFN‐λ3 variants. Dashed lines indicate selection thresholds (pLDDT ≥ 93, RMSD ≤ 0.8), with 221 selected designs in the lower‐right quadrant. All data represent mean ± SD from independent experiments. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test (0.01<* P <0.1, 0.001<** P <0.01, 0.0001<*** P < 0.001, **** P <0.0001 versus control; ns, not significant).

Article Snippet: For ISG induction in HNEpCs (#C‐12620, PromoCell), starved cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs (wild type, designed and heat‐incubated proteins) for 12 h. For dose‐dependent ISG induction in Vero E6 cells, cells were starved with serum‐free MEM (#LM007‐08, Welgene) for 12 h and then incubated with G‐hIFN‐λ3‐DE1 (7.8–2000 ng mL −1 ; 4‐fold serial dilution) for 24 h. Following incubation for both protocols, total RNA was extracted using Trizol reagent (#15596026, Invitrogen) following the manufacturer's protocol.

Techniques: Expressing, Recombinant, Quantitative RT-PCR, Binding Assay, Sequencing, Generated, Selection, Control

Journal: Advanced Science

Article Title: Computational Design and Glycoengineering of Interferon‐Lambda for Nasal Prophylaxis Against Respiratory Viruses

doi: 10.1002/advs.202506764

Figure Lengend Snippet: Biological activity and thermal aggregation resistance of hIFN‐λ3‐DE1 under acute and long‐term heat stress. a) Relative mRNA expression of representative ISGs ( Isg15, Mx1, and Oas1 ) in HNEpCs following 12‐h treatment with hIFN‐λ3‐WT or hIFN‐λ3‐DE1 (100 ng mL −1 ), with or without short‐term heat stress (70 °C for 5 min). mRNA levels were analyzed by RT‐qPCR ( n = 3), normalized to 18s rRNA , and expressed relative to non‐treated controls. b) Short‐term thermal aggregation profiles of hIFN‐λ3‐WT and hIFN‐λ3‐DE1 after 5‐min incubation at the indicated temperatures (25, 50, 60, 70, 80, or 90 °C). Residual soluble protein concentrations were quantified (n = 3). c) Relative ISG expression ( Isg15, Mx1, and Oas1 ) in HNEpCs treated with hIFN‐λ3‐WT or hIFN‐λ3‐DE1 (100 ng mL −1 ) after long‐term incubation at 45 or 50 °C for 2 weeks. RT‐qPCR was performed as in (a) ( n = 3). d) Long‐term thermal aggregation of hIFN‐λ3‐WT and hIFN‐λ3‐DE1 during 2‐week incubation at 45 or 50 °C. Protein solubility was monitored over time ( n = 3). All data represent mean ± SD from independent experiments. Statistical analysis was performed by one‐way ANOVA followed by Sidak's multiple comparisons test (0.001<** P <0.01, 0.0001<*** P < 0.001, **** P <0.0001 vs control and ns is not significant). n.t., non‐treat; WT, hIFN‐λ3‐WT; DE1, hIFN‐λ3‐DE1.

Article Snippet: For ISG induction in HNEpCs (#C‐12620, PromoCell), starved cells were incubated with 100 ng mL −1 of recombinant hIFN‐λs (wild type, designed and heat‐incubated proteins) for 12 h. For dose‐dependent ISG induction in Vero E6 cells, cells were starved with serum‐free MEM (#LM007‐08, Welgene) for 12 h and then incubated with G‐hIFN‐λ3‐DE1 (7.8–2000 ng mL −1 ; 4‐fold serial dilution) for 24 h. Following incubation for both protocols, total RNA was extracted using Trizol reagent (#15596026, Invitrogen) following the manufacturer's protocol.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Incubation, Solubility, Control