precision plus protein dual color standard protein marker  (Bio-Rad)

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: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Serum concentrations of the osteogenic marker osteocalcin (NOVUS, NBP2-68151) were also measured.

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Serum concentrations of the osteogenic marker osteocalcin (NOVUS, NBP2-68151) were also measured.

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

Journal: Virus Research

Article Title: Mycoviruses diversity in the black kōji mold, Aspergillus luchuensis (section Nigri ) isolated from liquor-production environments in Japan

doi: 10.1016/j.virusres.2026.199724

Figure Lengend Snippet: Identification of mycoviruses in A. luchuensis strains. (A) Origin of Aspergillus strains for dsRNA screening and the results of analysis. The number in parentheses indicates the number of dsRNA positive and tested Aspergillus spp. strains (Nanaji and Fujimori, unpublished data, see also the text). The map is provided by the Geospatial Information Authority of Japan ( https://www.gsi.go.jp ). The dsRNA-sequencing strains are shown on the right with an additional Okinawa strain (JCM22320, ). (B) Colony morphologies of selected A. luchuensis strains carrying virus-like dsRNA elements. (C) dsRNA profiles of the selected A. luchuensis strains. Each dsRNA fraction was analyzed by 1.0% agarose gel electrophoresis. Fungal strains subjected to dsRNA-seq analysis are indicated in red. Expected dsRNA bands for an alternavirus (AlAV1) and a partitivirus (AlPV1) genome, as well as AlPV1 satellite-like elements, were shown on the right (see ). The asterisks indicate the expected dsRNA bands for two other partitiviruses, AlPV2 and AlPV3. A 1 kb dsDNA ladder size marker (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific, Waltham, MA, USA) was used as a size standard.

Article Snippet: A 1 kb dsDNA ladder size marker (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific, Waltham, MA, USA) was used as a size standard.

Techniques: Sequencing, Virus, Agarose Gel Electrophoresis, Marker