Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: BMAd accumulation and sDPP4 release during skeletal aging activate BMM STING signaling. (a) Heatmap of dysregulated factors (≥1.5‐fold change) in the bone marrow supernatant of 3‐/18‐month‐old male rats identified through 4D label‐free technology ( n = 3 biological replicates). (b) Venn diagram of dysregulated factors in the bone marrow of rats. (c) ELISA of sDPP4 levels in conditioned medium collected from BMAds, Pre‐Ocs, BMMs, Ocs, BMSCs, Obs, and Neu ( n = 6 biological replicates; * p < 0.05 compared to BMAds‐CM). (d) Representative immunofluorescence staining showing that DPP4 was markedly detected in BMAds (Perilipin) in femora from 3‐/24‐month‐old male mice (scale bar: 100 µm). (e) ELISA of sDPP4 levels in conditioned medium collected in 3 M‐/18 M‐BMAds ( n = 6 biological replicates). (f,g) Representative micro‐CT images (f) and quantitative analysis of BV/TV, Tb. N, Tb.Th, and Tb.Sp (g) from PBS‐treated or rDPP4‐treated male mice ( n = 6 biological replicates). (h,i) Representative micro‐CT images (h) and quantitative analysis of BV/TV, Tb. N, Tb.Th, and Tb.Sp (i) from AAV9‐BMAd‐shNC‐treated or AAV9‐BMAd‐sh Dpp4 ‐treated male mice ( n = 6 biological replicates). (j) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs from PBS‐treated or rDPP4‐treated male mice ( n = 3 biological replicates). (k) Representative immunoblots of cGAS and STING protein/phosphorylation levels in BMMs treated with either rDPP4 alone or in combination with 18 M‐SN ( n = 3 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Micro-CT, Analysis, Western Blot, Phospho-proteomics

Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and rTLR4. (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Derivative Assay, Binding Assay, Activation Assay, Western Blot, Phospho-proteomics, Docking Assay, Analysis, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Knockdown

Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: STING signaling promotes NAD + decline in BMMs during skeletal aging by enhancing Cd38 transcription through IRF3 and P65. (a) WST‐8 analysis of NAD levels in 3, 10, 18, and 24 M‐BMMs ( n = 6 biological replicates). (b) Schematic of key enzymes in the NAD salvage biosynthesis pathway. (c,d) qPCR ( n = 6 biological replicates) and Western blotting ( n = 3 biological replicates) analyses of key enzymes in the NAD + salvage biosynthesis pathway in 3, 10, 18, and 24 M‐BMMs. (e) Fluorometric assay for CD38 NADase activity in 3 M‐/18 M‐BMMs ( n = 6 biological replicates). (f) WST‐8 analysis of NAD + levels in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (g,h) Fluorometric assay and WST‐8 analysis of CD38 NADase activity (g) or NAD + levels (h) in SN‐rDPP4‐treated BMMs with or without Cgas , Sting , Irf3, and Rela knockdown ( n = 6 biological replicates). (i) Representative immunoblots of CD38 protein levels in diABZI‐treated, SN‐rDPP4‐treated, or co‐cultured BMMs with or without Cgas , Sting , Irf3, and Rela knockdown ( n = 3 biological replicates). (j) Dual luciferase reporter assay in HEK 293T cells co‐transfected with CD38 luciferase reporter, IRF3 or P65 plasmid separately, and pRL‐CMV‐Renilla ( n = 6 biological replicates). (k) Chromatin immunoprecipitation assay showing that IRF3 or P65 bound to the CD38 promoter in BMMs. Binding levels were increased in BMMs treated with SN‐rDPP4 and were abolished by STING knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Analysis, Western Blot, Activity Assay, Knockdown, Cell Culture, Luciferase, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: A) IL-12 binding of CDR3-sequence clustered clones in ELISA with crude extracts shows four CDR3 clusters with IL-12 binding >2 times background (OD 480 nm of 0.05), selected clones are indicated in orange. Data shown is from a single experiment. B) ELISA dose-response curve of the wildtype QSY-2 clones binding to immobilised IL-12 Bound sdAbs were detected using rabbit-anti-VHH and HRP-conjugated donkey-anti-rabbit antibodies and OPD as substrate. Data represent mean values from a single experiment conducted in technical triplicates.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Binding Assay, Sequencing, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: A) Structure prediction of the IL-12 heterodimer in complex with the lead sdAb 2F3 (left panel) and coloured by plDDT score (right panel). Disulfides and N-linked glycans are shown as sticks. B) predicted Aligned Error (pAE): Expected Position Error versus Aligned Residue C) ELISA dose-response curve of the wildtype QSY-2F3 (2F3_wt) and single amino acid substitution mutants binding to immobilised IL-12. Bound sdAbs were detected using rabbit-anti-VHH and HRP-conjugated donkey-anti-rabbit antibodies and OPD as substrate. Data represent values from a singlet measurement from a single experiment.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Residue, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: A) ELISA dose-response curves of the wildtype 2F3_wt clone, single amino acid substitution and combination mutants binding to immobilised IL-12. Bound sdAbs were detected using rabbit-anti-VHH and HRP-conjugated donkey-anti-rabbit antibodies and OPD as substrate. Data represent mean values from a single experiment conducted in technical triplicates. B) The amino acid substitutions in the G31D (left), S54K (middle) and the A75R (right) mutant introduced extra salt bridges between the sdAb and IL-12, as determined through modeling of the sdAb/IL12 complexes.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Affinity maturation of a single-domain antibody via rational mutagenesis informed by artificial intelligence

doi: 10.64898/2026.10.13.758789

Figure Lengend Snippet: Binding affinities of the 2F3_wt and triple mutant for IL-12 by SPR. Association and dissociation rates from surface plasmon resonance, and binding affinity determined from these rates as well as from an equilibrium fit, in good agreement with each other.

Article Snippet: Phage display selections were performed on recombinant IL-12 (Sino Biological, CT011-H08H) in three panning rounds.

Techniques: Binding Assay, Mutagenesis, SPR Assay