Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: SDS-PAGE and Western blot analysis of hScFv-IL-17A proteins expressed in various E. coli strains. (a) SDS-PAGE of insoluble fractions; (b) SDS-PAGE of soluble fractions; (c) Western blot of insoluble fractions; (d) Western blot of soluble fractions. Lane 1: M (molecular weight marker); Lanes 2–4: hScFv-IL-17A-WT expressed in E. coli Origami B (DE3), SHuffle, and BL21 (DE3) with DisCoTune, respectively; Lanes 5–7: hScFv-IL-17A-C97S expressed in the same strains; Lane 8: purified hScFv M6-1B9 (positive control). Western blot detection was performed using an anti-His tag antibody. The arrow indicates the expected size of hScFv-IL-17A (∼28 kDa). Estimated amounts of hScFv-IL-17A are labeled. < LOD indicates signals below the limit of detection.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: SDS Page, Western Blot, Molecular Weight, Marker, Purification, Positive Control, Labeling

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: The expression efficiency of soluble hScFv-IL-17A-WT (orange) and hScFv-IL-17A-C97S (green) in different expression systems: (WT/Ori and C97S/Ori) from Origami B (DE3); (WT/SHuffle and C97S/SHuffle) from SHuffle; and (WT/BL21Dc and C97S/BL21Dc) from BL21 (DE3) with DisCoTune. Data represent mean ± SD from three independent biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Expressing

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Indirect ELISA of hScFv-IL-17A binding to hIL-17A using an avidin–biotin system. Purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S were tested at 1 and 5 µg/mL, expressed from SHuffle (WT/SHuffle-1, WT/SHuffle-5; C97S/SHuffle-1, C97S/SHuffle-5; light/dark green) and BL21 (DE3) with DisCoTune (WT/BL21Dc-1, WT/BL21Dc-5; C97S/BL21Dc-1, C97S/BL21Dc-5; light/dark orange). An IL-17A mAb (dark grey) was used as a positive control. Data represent mean ± SD from three independent biological replicates. Statistical analysis was determined by unpaired two-tailed Student’s t -test. Significance levels are denoted as: ** p < 0.01, *** p < 0.001, **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Indirect ELISA, Binding Assay, Avidin-Biotin Assay, Purification, Positive Control, Two Tailed Test

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Binding assessment of hScFv-IL-17A-WT (a) and hScFv-IL-17A-C97S (b) from BL21 (DE3) with DisCoTune by competitive ELISA. Various concentrations (0.1, 1, and 2 µg/mL) of IL-17A mAb (neutralizing antibody) were tested in competition with hScFv-IL-17A. WT was used at 1 µg/mL, whereas C97S was used at 5 µg/mL to compensate for its reduced binding activity observed in indirect ELISA. An anti-IFN-γ mAb (clone B27) was used as an irrelevant antibody control. Data represent mean ± SD from three independent biological replicates.

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Binding Assay, Competitive ELISA, Activity Assay, Indirect ELISA, Control

Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Comparative evaluation of E. coli expression systems for soluble hScFv-IL-17A with an unpaired CDR-L3 cysteine

doi: 10.1016/j.jgeb.2025.100613

Figure Lengend Snippet: Structural alignment of hScFv-IL-17A-WT and hScFvIL-17A-C97S. The predicted structures were generated using ABodyBuilder2. The conformational structures of hScFv-IL-17A-WT and hScFv-IL-17A-C97S were compared using UCSF ChimeraX software. The red ribbon indicates hScFv-IL-17A-WT structure, and the gold ribbon indicates hScFv-IL-17A-C97S structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: After washing, purified hScFv-IL-17A-WT and hScFv-IL-17A-C97S (1 and 5 μg/mL) were added and incubated for 1 h. In this experiment, mouse anti-IL-17A mAb (neutralizing antibody mAb clone M237, Sino Biological, Beijing, China) (Cat.12047-M237) (1 μg/mL, 50 μL), which recognizes hIL-17A, was used as a positive control.

Techniques: Generated, Software

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: Epigenetic metabolite lipid nanoparticles alleviate venous thrombosis via bone marrow reprogramming

doi: 10.1016/j.bvth.2025.100086

Figure Lengend Snippet: Immunomodulatory effects of ITA-LNP in vitro and in vivo. (A) Immunoblot probing for IL-1β in BMDMs treated with 100 ng/mL LPS in the presence of various doses of ITA-LNPs. (B) Schematic of the experiments in C57BL/6 mice subjected to LPS-induced endotoxemia. (C) Kaplan-Meier survival analysis after LPS-induced (15 mg/kg) endotoxemia in C57BL/6 mice pretreated with Ctrl/ITA-LNPs or anti–IL-1β mAb. (D) Rectal temperature measured in the mice from panel C before and 6 hours after LPS injections. (E-F) The levels of IL-1β and IL-6 in the serum from mice of a separate cohort injected as in panel B but with a sublethal dose of 8 mg/kg LPS; n = 10 to 15 mice per group. (G) NETs in primary human neutrophils incubated for 5 hours in vitro with 50 μM LNPs as indicated and in the presence of 100 nM PMA followed by immunofluorescence analysis; n = 6 for Ctrl-LNP and ITA-LNP without PMA; n = 8 for Ctrl-LNP with PMA; n = 9 for ITA-LNP with PMA; and n = 4 for PBS groups with or without PMA. (H) Cytokine analysis in supernatants from experiments in panel G. Statistical analysis is through pairwise t test with Holm post hoc test . N.d., not detected; PBS, phosphate-buffered saline; ROI, region of interest; T, temperature.

Article Snippet: C57BL/6 male mice (10 weeks old, n = 15 per group) were IV injected with 50 mg/kg ITA- or control (Ctrl)-LNPs or 50 mg/kg anti-mouse IL-1β monoclonal antibody (clone 7E3, InvivoGen mil1b-mab9-10).

Techniques: In Vitro, In Vivo, Western Blot, Injection, Incubation, Immunofluorescence, Saline

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: Epigenetic metabolite lipid nanoparticles alleviate venous thrombosis via bone marrow reprogramming

doi: 10.1016/j.bvth.2025.100086

Figure Lengend Snippet: ITA-LNPs alleviate venous thrombosis in IVC ligation model. (A) Schematic of the experiments. WT mice IV pretreated (50 mg/kg, bolus) with ITA- or Ctrl-LNPs were subjected to IVC ligation surgery followed by the isolation of the blood clots; n = 6 for Ctrl-LNP and n = 8 for ITA-LNP. (B) Gross pathology with clot weight quantification. (C) Hematoxylin and eosin staining of blood clot sections from these experiments. (D) Immunofluorescence staining of the same sections using Ly6G/C mAb (Red) and nuclei (blue). (E) MPO (top, alkaline phosphatase immunohistochemistry) and IL-1β (bottom, immunofluorescence) staining in the same clots. (F) Quantification of the staining in panel E. For each biological replicate (n = 6 for Ctrl-LNP and n = 8 for ITA = LNP), 3 to 4 histopathological locations at different depths (100 μm apart) were analyzed. (G) NET staining in the same sections using triple-positive identification through colocalization of the signals from MPO (green), H4Cit3 (red), and DNA (DAPI; blue). (H) Quantification of the staining in panel G. Quantification was accomplished using 2 to 3 different histopathological locations and the same aforementioned biological replicates. Statistical analysis is through a 2-tailed t test. DAPI, 4′,6-diamidino-2-phenylindole; H4Cit3, citrullinated histone H4; MPO, myeloperoxidase; WT, wild type.

Article Snippet: C57BL/6 male mice (10 weeks old, n = 15 per group) were IV injected with 50 mg/kg ITA- or control (Ctrl)-LNPs or 50 mg/kg anti-mouse IL-1β monoclonal antibody (clone 7E3, InvivoGen mil1b-mab9-10).

Techniques: Ligation, Isolation, Staining, Immunofluorescence, Immunohistochemistry