Journal: Food Chemistry: Molecular Sciences

Article Title: Usage of nanobody-beta-galactosidase fusion in immunoassays and its application in detecting a peanut allergen

doi: 10.1016/j.fochms.2026.100357

Figure Lengend Snippet: Tagging a nanobody to the N-terminus of β-gal does not affect its activity. (A) Colonies of dH5α bacteria transformed with a plasmid containing the coding sequence for β-gal with Nb16 fused to its N-terminus via a 4AA (GSHV) linker and plated on an LB/Kan plate with IPTG and X-gal. The plate was photographed 24 h after incubation at 37 °C for 16 h. (B), (C), and (D) are the same as (A), except that the bacteria were induced to express β-gal with Nb16 fused to its N-terminus via a longer flexible peptide (GSGASGSHV), a Strep-tag-containing peptide (GSWSHPQFEKHV), or β-gal with purification tags, respectively.

Article Snippet: Milli-Q water was purified in-house using a Milli-Q Advantage A10 system (Millipore, Bedford, MA, USA) and used throughout. o -Nitrophenyl-β-galactoside (ONPG), Isopropyl β-D-1-thiogalactopyranoside (IPTG), Kanamycin (Kan), and X-Gal were purchased from GoldBio (St Louis, MO, USA).

Techniques: Activity Assay, Bacteria, Transformation Assay, Plasmid Preparation, Sequencing, Incubation, Strep-tag, Purification

Journal: iScience

Article Title: GAL and F2R as immune diagnostic biomarkers for fetal growth restriction

doi: 10.1016/j.isci.2026.115228

Figure Lengend Snippet: Targeted additional analysis of the three key IR-DEGs (A) Chromosomal positions of the key IR-DEGs are presented. (B) A PCA plot illustrates the distribution of samples based on the expression profiles of the 3 key IR-DEGs. The x axis and y axis correspond to the first two principal components (PC1 and PC2), respectively, and the percentage of total variance explained by each component is indicated in parentheses adjacent to the axis labels. (C) Comparative expression levels of three crucial IR-DEGs in FGR, which integrates datasets GSE24129 , GSE100415 , and GSE147776 . (D) ROC curves were used to validate the efficacy of three crucial IR-DEGs in predicting FGR, quantifying the diagnostic performance of each gene for FGR identification. (E) A nomogram for predicting the risk of FGR is constructed based on three IR-DEGs, specifically F2R, GAL, and CXCL10. For each of these genes, a corresponding point value is assigned according to its expression level; the total point score is calculated by summing the individual gene points, and this total score is further converted to the predicted risk of developing FGR. (F) A calibration curve for the nomogram is shown, comparing the nomogram-predicted risk of FGR ( x axis) with the actually observed risk ( y axis). The diagonal line represents an ideal prediction scenario where predicted and observed risks are identical. The dashed line (“Apparent”) denotes the model’s performance before bias correction, while the solid line (“Bias-corrected”) represents performance after bias correction.

Article Snippet: IHC staining was performed according to previously established protocols using primary antibodies against GAL (Bioss, Beijing, China, catalog number: bs-0017M, RRID: AB_10855141) and F2R (Bioss, Beijing, China, catalog number: bs-0828R, RRID: AB_10857704).

Techniques: Expressing, Diagnostic Assay, Construct

Journal: iScience

Article Title: GAL and F2R as immune diagnostic biomarkers for fetal growth restriction

doi: 10.1016/j.isci.2026.115228

Figure Lengend Snippet: An evaluation focusing on immune infiltration related to two IR-DEGs (A) Stacked bar plot showing the relative abundance of 22 immune cell subtype proportions between FGR and AGA samples. (B) A boxplot is employed to visualize the differentiation in ratios of 22 immune cell types, with a specific focus on comparisons between FGR and AGA. (C) A Spearman correlation network is constructed to illustrate the correlative relationships between two IR-DEGs (namely GAL and F2R) and infiltrating immune cells in FGR. This network visualization explicitly displays how each of the four target IR-DEGs correlates with the 22 types of infiltrating immune cells, facilitating intuitive recognition of positive or negative correlation patterns between the genes and immune cell subsets. (D) Expression levels of F2R are significantly higher in FGR tissues ( n = 11) compared to AGA samples ( n = 25). Data are presented as mean ± SEM. (E) Expression levels of GAL are significantly elevated in FGR tissues ( n = 11) relative to AGA specimens ( n = 25). Data are presented as mean ± SEM. (F) qRT-PCR analysis demonstrates that F2R mRNA expression is significantly upregulated in FGR placental tissues compared to AGA controls ( p = 0.0019). Data are presented as mean ± SEM. (G) qRT-PCR analysis reveals a significant downregulation of GAL mRNA in FGR placental tissues compared to AGA controls ( p = 0.0015). Data are presented as mean ± SEM. Additionally, representative images of IHC staining for F2R and GAL in FGR and AGA patients are presented, illustrating both high and low expression levels of the two genes. All staining images are shown at magnifications of ×40 and ×200, with scale bars clearly indicated for reference. Statistical p values were calculated via the chi-square test, where ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: IHC staining was performed according to previously established protocols using primary antibodies against GAL (Bioss, Beijing, China, catalog number: bs-0017M, RRID: AB_10855141) and F2R (Bioss, Beijing, China, catalog number: bs-0828R, RRID: AB_10857704).

Techniques: Construct, Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining

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 attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

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 suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 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 an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

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 modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. 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 an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

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: Comparative analysis of SCS and D + Q drugs on glucocorticoid-induced bone marrow senescence inhibition. ( A ) Schematic diagram showing the treatment of SCS and D + Q after glucocorticoid-induced senescence. ( B and C ) Representative flow cytometry images of bone marrow SA-β-Gal for senescence detection on day 42 (B), with corresponding quantification analysis (C). n = 6 biological replicates. ( D and E ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( F ) Schematic diagram of SCS and D + Q treatment in the early stage of glucocorticoid-induced senescence. ( G and H ) Representative flow cytometry images of p16-positive senescent cells in bone marrow on day 42 (G), with corresponding quantification analysis (H). n = 6 biological replicates. ( I and J ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( K-M ) Representative images of HE staining of the distal femur with macro and high-magnification images (K), and quantification of trabecular and cortical bone empty lacunae (L and M). n = 6 biological replicates. (Scale bars, 550 μm and 25 μm) ( N and O ) Representative ALP staining images of in vitro osteogenic differentiation of bone marrow LepR + MSCs after 14 days (N), with corresponding quantification analysis (O). n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) 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 , H , I , J , L , M and O ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Inhibition, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Poultry Science

Article Title: miRNA profiling reveals that gga-let-7i/COL1A2 axis induces cell cycle arrest and triggers cellular senescence to accelerate ovarian aging in laying hens by suppressing the PI3K/AKT/MDM2 pathway

doi: 10.1016/j.psj.2026.106542

Figure Lengend Snippet: gga-let-7i promotes the senescence of chicken granulosa cells. (A and B) Functional impact of gga-let-7i overexpression and interference on the relative expression of senescence-related genes, n = 9. (C–E) Overexpression of gga-let-7i upregulates protein levels of the core senescence regulators p53 and p21, while downregulating the senescence inhibitor MDM2. Conversely, gga-let-7i knockdown exhibits the opposite effects, n = 3. (F and G) Effect of gga-let-7i overexpression or knockdown on the proportion of SA-β-gal-positive chicken granulosa cells, n = 3, scale bar = 100 μm. All data were derived from at least three independent replicates and are presented as the mean ± SEM. *, P < 0.05; ⁎⁎ , P < 0.01.

Article Snippet: Senescence of GCs was assessed using the SA-β-gal staining kit (Beyotime) following manufactures’ instructions.

Techniques: Functional Assay, Over Expression, Expressing, Knockdown, Derivative Assay

Journal: Poultry Science

Article Title: miRNA profiling reveals that gga-let-7i/COL1A2 axis induces cell cycle arrest and triggers cellular senescence to accelerate ovarian aging in laying hens by suppressing the PI3K/AKT/MDM2 pathway

doi: 10.1016/j.psj.2026.106542

Figure Lengend Snippet: gga-let-7i bound with COL1A2 to promote GC senescence via suppressing PI3K/AKT/MDM2 signaling pathway. (A) Validation of COL1A2 knockdown efficiency, n = 9. (B) qPCR detected relative proliferation-related gene levels regulated by si-COL1A2, n = 9. (C) Interference of COL1A2 blocked cell cycle of GCs, n = 3. (D and E) COL1A2 inhibition downregulated EdU positive cell ration in GCs, n = 3, scale bar = 200 μm. (F) qPCR detected relative senescence-related gene levels regulated by si-COL1A2, n = 9. (G–I) si-COL1A2 effectively suppresses COL1A2 protein, concomitantly upregulating p53 and downregulating MDM2, and ultimately increasing the proportion of SA-β-gal-positive cells, n = 3, scale bar = 100 μm. (J–M) Western blot assay revealed protein levels of COL1A2, p53, CDK2, p-AKT/AKT and p-MDM2/MDM2 following co-treatment with si-COL1A2, gga-let-7i inhibitor or empty vector, n = 3. All data were derived from at least three independent replicates and are presented as the mean ± SEM. *, P < 0.05; ⁎⁎ , P < 0.01.

Article Snippet: Senescence of GCs was assessed using the SA-β-gal staining kit (Beyotime) following manufactures’ instructions.

Techniques: Biomarker Discovery, Knockdown, Inhibition, Western Blot, Plasmid Preparation, Derivative Assay