Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Preparation and characterization of engineered ADGRG1-targeting and hypoxia-treated EVs. (A)Induced fit docking analysis of ADGRG1-binding peptide (A1TP, 7 peptides) and extracellular domain of ADGRG1 protein (PDB database: 7SF8). (B) Analysis of the binding of the A1TP to purified ADGRG1 proteins using a microscale thermophoresis (MST) binding assay. (C) Induced fit docking analysis of A1TP-PEG and extracellular domain of ADGRG1 protein. (D) The binding free energy between A1TP or A1TP-PEG and ADGRG1 were calculated using molecular dynamics simulations. Lower values indicate more stable interactions, with values less than or equal to −20 considered as stable binding modes. (E) Schematic illustration of the conjugating reaction between DSPE-PEG-Alkyne and A1TP. Schematic illustration of the fabrication of A1TP-HX-EVs through external modification by A1TP anchoring. Specific steps for the synthesis of DSPE-PEG-A1TP (DPA) are shown in . (F) FT-IR analysis showed the characteristic peaks of the DSPE-PEG-A1TP. The new triazole ring itself showed a characteristic C=N stretching vibration, a peak at 1538 cm −1 revealed the successful conjugation of A1TP. (G) H Nuclear magnetic resonance (NMR) spectra of DSPE-PEG-A1TP in D2O. The hydrogen signatures of the phenyl and phenol groups at 7.5-8.0 ppm confirmed the successful conjugation of DSPE to A1TP. (H) Western blot analysis verified the presence of three EV marker proteins (ALIX, TSG101, and CD81) and one EV negative marker (GM130) in EVs, HX-EVs, and A1TP-HX-EVs. (I) Transmission electron microscopy (TEM) images of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 200 nm. (J) Zeta potentials of EVs, HX-EVs and A1TP-HX-EVs, n = 3. Two-tailed unpaired Student's t-test was used for statistical analysis. ns, not significant. A two-tailed unpaired Student's t-test was used for statistical analysis. (K) Representative images of the spherical morphology and dispersion states of EVs, HX-EVs and A1TP-HX-EVs. Scale bar, 500 nm. (L) Size distributions of EVs, HX-EVs and A1TP-HX-EVs.

Article Snippet: Finally, the presence of the characteristic EV markers Alix (92880, Cell Signaling Technology), CD81 (56039, Cell Signaling Technology) and TSG101 (sc-7964, Santa Cruz Biotechnology) was confirmed by Western blot analysis.

Techniques: Binding Assay, Purification, Microscale Thermophoresis, Modification, Conjugation Assay, Nuclear Magnetic Resonance, Western Blot, Marker, Transmission Assay, Electron Microscopy, Two Tailed Test, Dispersion

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Preparation and characterization of IWV and VLP (A) Schematic representation of the construction of plasmids encoding EV-D68 P1 and 3CD. (B) Workflow for the expression and purification of VLP. (C–F) SDS-PAGE analysis of purified IWV and VLP. (C and D) VLP expressed in (C) Expi293F cells and (D) ExpiCHO-S cells purified with sucrose. The Hsp and Hsc identified by mass spectrometry are labeled in red lines. (E) VLP was expressed in ExpiCHO-S cells and further purified using both sucrose and iodixanol (OptiPrep) gradients. (F) IWV was purified by sucrose gradient ultracentrifugation. (G) IWV and VLP particle size distribution was measured by dynamic light scattering. (H) Representative negative-stain TEM images of IWV and VLP. Scale bars, 100 nm. (I) The thermal stability of the non-inactivated virus, IWV, and VLP was assessed using differential scanning fluorimetry (DSF). Data represent the mean of four independent measurements ( n = 4) for each sample.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Expressing, Purification, SDS Page, Mass Spectrometry, Labeling, Staining, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Epitope specificities of IgG induced by IWV and VLP vaccines (A) Localization of neutralizing antigenic sites I–IV and corresponding epitope peptide sequences. Neutralizing antigenic sites I–IV were mapped onto a single icosahedral asymmetric unit of the EV-D68 MO strain capsid, based on the previously reported cryo-EM structure (PDB: 6CSG ). VP1, VP2, and VP3 are shown in gray, pink, and cyan, respectively. Neutralizing antigenic sites I, II, III, and IV are highlighted in yellow, green, blue, and magenta, respectively. The accompanying table lists the sequences of epitope peptides spanning the antigenic sites; residues constituting the neutralizing antigenic sites are indicated in red. Molecular graphics were generated using UCSF ChimeraX v1.9. (B) Plasma IgG levels specific to epitope peptides following boost immunization with either IWV or VLP. (C) Plasma IgG reactivity to mutant VLPs following boost immunization with IWV or wild-type VLP. Then, 1,000-fold diluted plasma samples were used. Details of the mutations in each mutant are summarized in the table on the right. (B–C) n = 5 per group. Data are presented as mean ± SD. (B) Statistical comparisons were performed using 50-fold diluted plasma samples. (B) “ns” indicates not significant. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, as determined by Tukey’s test. (C) ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, as determined by Dunnett’s multiple comparison test.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Vaccines, Cryo-EM Sample Prep, Generated, Clinical Proteomics, Mutagenesis, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Cryo-EM structure of EV-D68 MO strain VLP (A) Cryo-EM density maps showing the overall structures of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP of the EV-D68 MO strain. A schematic illustration of the viral particle is shown in the left-most. The 5-, 3-, and 2-fold symmetry axes are indicated by a pentagon, triangle, and circle, respectively. (B) Ribbon representations of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP structures around the 2-fold axis. The 3-fold and 2-fold axes are denoted by a triangle and circle, respectively. (C) Structural comparison of the icosahedral asymmetric units of the VLP with those of the mature virion (left, PDB: 6CSG ) and the empty particle (middle, PDB: 6CRU ). VP1, VP0, and VP3 of the VLP were superimposed onto the corresponding subunits of the mature virion and the empty particle. Representative structures are shown based on superposition via VP1. All structures are depicted as ribbon models. The table on the right summarizes the number of pruned atom pairs and root-mean-square deviation (RMSD) values for each superposition.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Cryo-EM Sample Prep, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Preparation and characterization of IWV and VLP (A) Schematic representation of the construction of plasmids encoding EV-D68 P1 and 3CD. (B) Workflow for the expression and purification of VLP. (C–F) SDS-PAGE analysis of purified IWV and VLP. (C and D) VLP expressed in (C) Expi293F cells and (D) ExpiCHO-S cells purified with sucrose. The Hsp and Hsc identified by mass spectrometry are labeled in red lines. (E) VLP was expressed in ExpiCHO-S cells and further purified using both sucrose and iodixanol (OptiPrep) gradients. (F) IWV was purified by sucrose gradient ultracentrifugation. (G) IWV and VLP particle size distribution was measured by dynamic light scattering. (H) Representative negative-stain TEM images of IWV and VLP. Scale bars, 100 nm. (I) The thermal stability of the non-inactivated virus, IWV, and VLP was assessed using differential scanning fluorimetry (DSF). Data represent the mean of four independent measurements ( n = 4) for each sample.

Article Snippet: The cryo-EM map of the EV-D68 MO strain VLP has been deposited in the Electron Microscopy DataBank with accession code EMD-65634.

Techniques: Expressing, Purification, SDS Page, Mass Spectrometry, Labeling, Staining, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Epitope specificities of IgG induced by IWV and VLP vaccines (A) Localization of neutralizing antigenic sites I–IV and corresponding epitope peptide sequences. Neutralizing antigenic sites I–IV were mapped onto a single icosahedral asymmetric unit of the EV-D68 MO strain capsid, based on the previously reported cryo-EM structure (PDB: 6CSG ). VP1, VP2, and VP3 are shown in gray, pink, and cyan, respectively. Neutralizing antigenic sites I, II, III, and IV are highlighted in yellow, green, blue, and magenta, respectively. The accompanying table lists the sequences of epitope peptides spanning the antigenic sites; residues constituting the neutralizing antigenic sites are indicated in red. Molecular graphics were generated using UCSF ChimeraX v1.9. (B) Plasma IgG levels specific to epitope peptides following boost immunization with either IWV or VLP. (C) Plasma IgG reactivity to mutant VLPs following boost immunization with IWV or wild-type VLP. Then, 1,000-fold diluted plasma samples were used. Details of the mutations in each mutant are summarized in the table on the right. (B–C) n = 5 per group. Data are presented as mean ± SD. (B) Statistical comparisons were performed using 50-fold diluted plasma samples. (B) “ns” indicates not significant. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, as determined by Tukey’s test. (C) ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, as determined by Dunnett’s multiple comparison test.

Article Snippet: The cryo-EM map of the EV-D68 MO strain VLP has been deposited in the Electron Microscopy DataBank with accession code EMD-65634.

Techniques: Vaccines, Cryo-EM Sample Prep, Generated, Clinical Proteomics, Mutagenesis, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Cryo-EM structure of EV-D68 MO strain VLP (A) Cryo-EM density maps showing the overall structures of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP of the EV-D68 MO strain. A schematic illustration of the viral particle is shown in the left-most. The 5-, 3-, and 2-fold symmetry axes are indicated by a pentagon, triangle, and circle, respectively. (B) Ribbon representations of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP structures around the 2-fold axis. The 3-fold and 2-fold axes are denoted by a triangle and circle, respectively. (C) Structural comparison of the icosahedral asymmetric units of the VLP with those of the mature virion (left, PDB: 6CSG ) and the empty particle (middle, PDB: 6CRU ). VP1, VP0, and VP3 of the VLP were superimposed onto the corresponding subunits of the mature virion and the empty particle. Representative structures are shown based on superposition via VP1. All structures are depicted as ribbon models. The table on the right summarizes the number of pruned atom pairs and root-mean-square deviation (RMSD) values for each superposition.

Article Snippet: The cryo-EM map of the EV-D68 MO strain VLP has been deposited in the Electron Microscopy DataBank with accession code EMD-65634.

Techniques: Cryo-EM Sample Prep, Comparison