Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: Computationally designed IL-21 mimic recapitulates receptor interactions with superior stability and human-mouse cross-reactivity. (A) The optimized IL-21 mimic, 21h10, is designed by recapitulating the helical bundle structure of the native hIL-21. Unstructured regions in the hIL-21 are removed, and short helices are extended to accommodate improved intramolecular packing. Some of the interface residues are conserved from the native hIL-21 for the design of initial hits. Other residues from the de novo scaffolds are further optimized, as shown. (B) Association and dissociation of 21h10 to human and murine IL-21R and 𝛾 c show concentration-dependent binding curves of 21h10. 21h10’s K D against hIL-21R is 482 pM, hIL-21R/h𝛾 c is 86.7 nM, mIL-21R is 7.87 nM, and mIL-21R/m𝛾 c is 72.8 nM. For measuring K D against hIL-21R and mIL-21R, IL-21R was immobilized on the biolayer interferometry (BLI) tip. For measuring K D against hIL-21R/h𝛾 c and mIL-21R/m𝛾 c , 𝛾 c was immobilized on the BLI tip and IL-21R was provided in solution with 1.5-fold molar excess to 21h10. Dotted lines show curve fits of raw data using a 1:1 Langmuir binding model. (C) The optimized IL-21 mimic, 21h10, exhibits monodispersed distribution when sized through size-exclusion chromatography using Superdex 75 10/300 GL column. (D) Wavelength scan from 200 nm to 250 nm confirmed 𝛼-helical secondary structure present in 21h10. Thermal melt from 25°C to 95°C with 222 nm scan revealed outstanding thermal stability of 21h10, with a T m around 75°C. (E) Crystal structure of 21h10 in complex with hIL-21R and h𝛾 c (PDB: XXXX). (F) 21h10 conserves key molecular interactions in the site I interface (left) and site IIa interface (right) observed in the structure of the human IL-21 receptor complex. (Human IL-21 receptor complex, PDB ID: 8ENT).

Article Snippet: To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 2-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Concentration Assay, Binding Assay, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 2-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

Journal: bioRxiv

Article Title: Characterization of BvlzGluc, a Novel Antifungal β-Glucanase from Bacillus velezensis , with Potential Agricultural and Industrial Applications

doi: 10.1101/2025.01.23.634490

Figure Lengend Snippet: Effect of BvlzGluc on Botrytis cinerea infection in plants. a . Immunocytochemical analysis using an anti-fungal monoclonal primary antibody and a GFP-conjugated anti-rabbit secondary antibody demonstrated the normal distribution of β-glucans in Botrytis cinerea hyphae. In the control samples, the β-glucans formed a regular, continuous, and uniform layer within the cell wall, which is crucial for maintaining cell wall integrity and structural support in the fungus. However, upon treatment with BvlzGluc, significant disruptions in the β-glucan layer were observed. These discontinuities suggest that BvlzGluc interferes with the synthesis or maintenance of β-glucans, likely weakening the fungal cell wall and increasing its vulnerability to external stress. b . Lesion size triggered by B. cinerea on melon leaves after 72 hpi was reduced by 80% upon treatment with BvlzGluc. The whisker plot shows all the measurements (orange dots), medians (black line), and minimum and maximum values (whisker ends). The datasets did not pass the Shapiro‒Wilk test for normality (P > 0.05) and were compared using a nonparametric two-tailed Mann‒Whitney test, with quadruple asterisks indicating significant differences at P < 0.0001.

Article Snippet: The blots were probed with a 1:1000 dilution of a rabbit monoclonal anti-His-tag antibody (Rockland).

Techniques: Infection, Control, Whisker Assay, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol for generation and regeneration of PEG-passivated slides for single-molecule measurements

doi: 10.1016/j.xpro.2022.101152

Figure Lengend Snippet:

Article Snippet: MBP Antibody Biotin Conjugated (Dilute to use ∼10 nM or 500–1000× times) , Rockland Antibodies & Assays , Cat#200406385S.

Techniques: Recombinant, Software, Adhesive, Microscopy

Journal: Biochimica et biophysica acta

Article Title: Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

doi: 10.1016/j.bbamcr.2014.03.020

Figure Lengend Snippet: Fig. 6. Exogenous MTP restores HGF stimulation of cell motility in MTP-knockdown NS/P cells. NS/P cells transfected with control siRNA (siN), with MTP-specific siRNA (siM), with MTP-specific siRNA plus expression plasmid containing full-length wild-type MTP (pM) or plus expression plasmid containing protease inactive MTP (pMN) were subjected to cell motility assay in the absence (–) or presence (+) of HGF stimulation. “rM” indicates the motility of cells that was assayed in the presence of the recombinant protein of MTP pro- tease domain. Data were normalized to that of NS/P cells without HGF treatment that is set at 1. Data are mean ± SD from three to five independent experiments. *, P ≦0.05; **, P ≦0.01; ***, P ≦0.001.

Article Snippet: The recombinant protein of mouse MTP protease domain was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Knockdown, Transfection, Control, Expressing, Plasmid Preparation, Motility Assay, Recombinant

Journal: Nature Communications

Article Title: Circadian fluctuation of soluble CD26 dictates the impact of the timing of cord blood transplantation on acute graft-versus-host disease

doi: 10.1038/s41467-026-68958-4

Figure Lengend Snippet: a Experimental design. Blood samples were collected at 7:00 am ± 15 min ( n = 10) or 11:30 am ± 15 min ( n = 10) from recipients with malignant hematological diseases in complete remission prior to cord blood infusion. b , c Serum concentrations of cytokines before cord blood infusion. Normalized z score ( b ) and absolute concentrations ( c ) of cytokines between 7:00 am and 11:30 am. The P values are two-sided and reported as exact values. d Absolute concentrations of soluble CD26/DPPIV in serum post-MAC. Blood samples were collected from recipients at 7:00 am ± 15 min ( n = 19), 11:30 am ± 15 min ( n = 19), or 4:00 pm ± 15 min ( n = 29). e – i Associations between serum soluble CD26/DPPIV (sCD26/DPPIV) levels and inflammatory cytokines before cord blood infusion on day 0. Pearson’s correlation coefficient ( r ) was calculated for the relationships between serum DPPIV levels and IL-1Ra ( e ) ( r = 0.476, P = 0.034), IL-1β ( f ) ( r = 0.450, P = 0.046), LIF ( g ) ( r = 0.517, P = 0.020), IL-18 ( h ) ( r = −0.811, P < 0.001) and IL-1α ( i ) ( r = 0.006, P = 0.741) concentrations. The P values are two-sided and reported as exact values.The data are presented as the means ± SEMs and were analyzed by unpaired t test followed by Bonferroni-Dunn correction ( c ), one-way ANOVA with Bonferroni multiple comparisons ( d ) and Pearson’s correlation ( e – i ). Source data are provided as a Source Data file.

Article Snippet: In the experimental groups, recombinant human CD26/DPPIV protein (1000 ng/mL, 11244-SE, R&D Systems) or Sitagliptin phosphate monohydrate (DPPIV inhibitor, 200 μg/mL, S4002, Selleck) was added to the respective groups.

Techniques:

Journal: Nature Communications

Article Title: Circadian fluctuation of soluble CD26 dictates the impact of the timing of cord blood transplantation on acute graft-versus-host disease

doi: 10.1038/s41467-026-68958-4

Figure Lengend Snippet: a , b Representative immunofluorescence images ( a ) and quantification ( b ) of CD26/DPPIV levels in intestinal epithelial cells from a healthy population at different times ( n = 3 patients per group) ( P < 0.001). Intestinal samples were collected at 10 am (CT10), 2 pm (CT14), and 6 pm (CT18). Scale bars, 50 μm. c Bmal1 and DPP4 expression in primary mouse epidermal cells postsynchronization in vitro ( n = 3 biological replicates, each from an independent primary culture prepared and synchronized on separate days). The Gapdh gene was used as an internal control. The relative expression levels were computed via the 2 −ΔΔCT method. CT6 is double plotted. d The concentration of CD26/DPPIV in the supernatant of primary mouse epidermal cells postsynchronization in vitro ( n = 3 biological replicates, each from an independent primary culture prepared and synchronized on separate days) ( P < 0.001). CT6 is double plotted. e Diurnal mRNA expression of DPP4 in mouse epidermal cells. Bmal1 ΔEC , epithelium cell-specific Bmal1 knockout mice. Statistics were obtained from a publicly available dataset ( GSE190035 ), with values representing the log2-transformed mean RPKM from 4 biological replicates per time point. ZT0 is double plotted.The data are presented as the means ± SEMs and were analyzed by one-way ANOVA with Bonferroni multiple comparisons ( b , d ) and JTK_Cycle ( c , e ). Source data are provided as a Source Data file.

Article Snippet: In the experimental groups, recombinant human CD26/DPPIV protein (1000 ng/mL, 11244-SE, R&D Systems) or Sitagliptin phosphate monohydrate (DPPIV inhibitor, 200 μg/mL, S4002, Selleck) was added to the respective groups.

Techniques: Immunofluorescence, Expressing, In Vitro, Control, Concentration Assay, Knock-Out, Transformation Assay

Journal: Nature Communications

Article Title: Circadian fluctuation of soluble CD26 dictates the impact of the timing of cord blood transplantation on acute graft-versus-host disease

doi: 10.1038/s41467-026-68958-4

Figure Lengend Snippet: PBMCs from healthy donors were cultured with PHA-L (control group), PHA-L plus CD26/DPPIV (sCD26 group), or PHA-L plus Sitagliptin (DPPIV inhibitor, DPPIVi group) for 72 h. a Experimental design. PBMCs from healthy donors were cultured with PHA-L (control group), PHA-L plus CD26/DPPIV (sCD26 group), or PHA-L plus Sitagliptin (DPPIV inhibitor, DPPIVi group) for 72 h. b , c Representative plots (left) and quantified percentages (right) of CD86 + ( b ) and CD80 + ( c ) CD14 + cells after 72 h of PBMCs coculture in different groups (pooled data from 8 healthy donors across four independent experiments). d – i Representative plots (left) and quantified percentages (right) of Ki-67 + , IFNγ + , and CD38 + cells among CD4 + T cells ( d , f , h ) and CD8 + T cells ( e , g , i ) after 72 h of PBMCs coculture in different groups (pooled data from 6 healthy donors across three independent experiments).The data are presented as the means ± SEMs and were analyzed by one-way ANOVA with the Bonferroni correction for multiple comparisons ( b – i ). All P values are two-sided and reported as exact values unless <0.001. Source data are provided as a Source Data file.

Article Snippet: In the experimental groups, recombinant human CD26/DPPIV protein (1000 ng/mL, 11244-SE, R&D Systems) or Sitagliptin phosphate monohydrate (DPPIV inhibitor, 200 μg/mL, S4002, Selleck) was added to the respective groups.

Techniques: Cell Culture, Control