Journal: Microorganisms

Article Title: Polyethylene Terephthalate Hydrolases in Human Gut Microbiota and Their Implications for Human Health

doi: 10.3390/microorganisms12010138

Figure Lengend Snippet: Effects of PET MPs and hydrolysates on cell viability and macrophage polarization. ( A , B ) Relative expression levels of IL-1β, IL-6, TNF-α, and Arg-1 (IL-4 stimulation) in mouse monocyte macrophages RAW 264.7 cells treated with TPA (0.01 mM) ( A ) and TPA (1.00 mM) ( B ) for 24 h followed by LPS/IL-4 stimulation for 6 h. ( C ) Relative expression levels of IL-1β, IL-6, TNF-α, and Arg-1 (IL-4 stimulation) in mouse bone-marrow-derived macrophages (BMDMs) treated with TPA (1.00 mM) for 24 h followed by LPS/IL-4 stimulation for 6 h. ( D , E ) Relative expression levels of IL-1β ( D ) and Arg-1 ( E ) in RAW 264.7 cells treated with PET MPs (<400 μm) at a gradient dose at 0.01, 0.10, 0.50, and 1.00 mg/mL for 24 h followed by LPS/IL-4 stimulation for 6 h. ( F , G ) Relative expression levels of IL-1β ( F ) and Arg-1 ( G ) in RAW 264.7 cells treated with PET MPs (<400 μm) at a gradient dose at 0.01, 0.10, 0.50, and 1.00 mg/mL for 24 h without any stimulation. *** p -value < 0.001; ** p -value < 0.01; * p -value < 0.05.

Article Snippet: Macrophage colony-stimulating factor (M-CSF) (product code: HY-P7085), lipopolysaccharides (LPS) (product code: HY-D1056), and murine interleukin-4 (IL-4) (product code: HY-P70644) were obtained from MedChemExpress, Princeton, NJ, USA.

Techniques: Expressing, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: GDF9 His209GlnfsTer6/S428T and GDF9 Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

doi: 10.1186/s12964-024-01616-8

Figure Lengend Snippet: Effects of GDF9 variants on gene expression and GDF9-BMP15 interactions. A Western blots of mature GDF9 proteins expressed in follicular fluid collected from the first and second retrieval cycle of individual P1 and age-matched controls ( n =3). B ELISA of GDF9 in follicular fluid collected from the first retrieval cycle of individual P1 and age-matched controls ( n =6). C Western blots of GDF9 expression in HEK293T cells transfected with human GDF9 WT , GDF9 Q321X , GDF9 S428T , and GDF9 His209GlnfsTer6 respectively and their relative mature-GDF9 expression in culture medium (CM) (3 independent experiments). The amount of concentrated culture medium used for the detection was 35μl per sample per assay. lnfsTer6, His209GlnfsTer6. * P < 0.05. D Western blots of GDF9 precursors and mature GDF9 over time during in vitro cleavage assay (3 independent experiments). Total GDF9=GDF9 precursor+mature GDF9. E Western blots of BMP15 expression in HEK293T cells co-transfected with human BMP15 and human GDF9 WT , GDF9 Q321X , GDF9 S428T , and GDF9 His209GlnfsTer6 respectively and their relative BMP15 precursor expression in CM (3 independent experiments). lnfsTer6, His209GlnfsTer6. * P < 0.05. F Co-IP assay of the binding of GDF9 proteins to BMP15 protein. lnfsTer6, His209GlnfsTer6. * P < 0.05. G Western blots of p-smad2 and smad2 expression in human primary luteinized granulosa cells treated with purified recombinant pro-GDF9 WT -BMP15 and pro-GDF9 S428T -BMP15 protein respectively (3 independent experiments)

Article Snippet: GDF9 was measured in follicular fluid collected at the first oocyte retrieval from individual P1 and in follicular fluid from other six control women using an ELISA kit (CSB-E12925h, CUSABIO, China).

Techniques: Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, In Vitro, Cleavage Assay, Co-Immunoprecipitation Assay, Binding Assay, Purification, Recombinant

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Killing efficiency of different physicochemical treatments on recombinant L. lactis strains displaying different binders of proinflammatory cytokines on their surface. The killing efficiency was determined by culturing and counting colonies on agar plates. Complete killing of bacteria i.e., 0 CFU/mL (no bacterial colonies on agar plates) was considered as 100% efficiency. pSD-ZIL6, L. lactis displaying anti-IL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; pNZ8148, L. lactis harboring empty plasmid. The results presented are means ± standard deviation (SD) of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Recombinant, Bacteria, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Cytokine binding ability of recombinant L. lactis bacteria displaying different binders of proinflammatory cytokines on their surface upon exposure to bacteria-killing treatments. ELISA-determined concentrations of recombinant IL6 (A) , TNF (B) , IL17 (C) , and IL8 (D) that remained in the solution following incubation with the corresponding strain of bacteria before and after treatment with heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. The results are presented as means ± SD of three technical replicates of a representative experiment. ns, p = 0.09; *, p ≤ 0.05; **, p = 0.002; ***, p < 0.001 (unpaired two-tailed t -test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Sonication, Irradiation, Control, Plasmid Preparation, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Determination of binding affinity of ZIL6-displaying L. lactis for human IL6. A constant number of 8 × 10 6 CFU bacteria was incubated with increasing concentrations of human IL6 for 2 h. MFI values were measured by flow cytometry and binding curves were fitted to the data using the Hill equation in the GraphPad Prism v.10.3.1. The dissociation constants (Kd) values were calculated from the binding curves for non-treated, live ZIL6-displaying L. lactis bacteria and bacteria treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. Data are means ± SD of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Bacteria, Incubation, Flow Cytometry, Sonication, Irradiation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Quantification of the maximum binding capacity of ZIL6-displaying L. lactis . Increasing concentrations of recombinant human IL6 (0.45, 4.5, 45 and 450 ng) were incubated with a constant number of bacteria (4.5 × 10 7 CFU equivalent to 0.1 mg dry cell weight) in 450 μl. Residual IL6 in the supernatants was quantified by ELISA for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. The percentage of bound IL6 was calculated from the difference measured in the presence of ZIL6-displaying L. lactis and control bacteria (carrying empty plasmid pNZ8148). The data are means ± SD of two biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Incubation, Bacteria, Enzyme-linked Immunosorbent Assay, Sonication, Irradiation, Control, Plasmid Preparation, Concentration Assay, Comparison

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: (A) Inhibition of STAT3 signaling in HEKblue-IL6R cells by non-viable ZIL6-displaying L. lactis bacteria in comparison to live non-treated strain. ZIL6-displaying L. lactis cells (1 × 10 8 CFU/mL or 1 × 10 9 CFU/mL) were preincubated with IL6 (1 ng/mL) for 2 h. After removal of bacterial cells, the cell-free supernatant containing remainder of IL6 was added to HEKblue-IL6R cells to induce the reporter system. The percentage of STAT3 inhibition was calculated relative to the STAT3 signaling induced by IL6 in the absence of bacteria. The inhibition of STAT3 signaling was determined for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. An anti-IL6 monoclonal antibody (Ab) was used as a positive control. The data are means ± SD of four biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test). (B) The effect of lactic acid production by L. lactis on viability of HEKblue-IL6R cells. HEKblue-IL6R cells (100 000 cells/well) were incubated with ZIL6-displaying L. lactis (2 × 10 7 bacteria/well) for 6, 12, and 24 h, and the viability of HEKblue-IL6R cells was determined with trypan blue. The data are means ± SD of three technical replicates of a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Inhibition, Bacteria, Comparison, Sonication, Irradiation, Control, Plasmid Preparation, Positive Control, Concentration Assay, Incubation

Journal: Journal of Tissue Engineering

Article Title: Therapeutic potential and mechanisms of umbilical cord mesenchymal stem cells differentiating into tendon cells and promotion of rotator cuff tendon-bone healing

doi: 10.1177/20417314251315185

Figure Lengend Snippet: Analysis of the induction effect of BMP-12, GDF-7, and CTGF on the differentiation of UCMSCs: (a) morphological changes of UCMSCs after induction treatment, (b) mRNA expression of MKX, SCX, and TNC detected by qPCR, (c) bands and protein expression of MKX, SCX, and TNC detected by WB, and (d) COL-I and COL-III expression detected by immunofluorescence (400×). ** p < 0.01 and *** p < 0.001 compared with the control group.

Article Snippet: Rat umbilical cord mesenchymal stem cells (UCMSCs, CP-R302, Pricella, China), 293T cells (Pricella, China), DMEM medium (L110KJ, Yuanpei, China), Fetal bovine serum(AC03L055, life-ilab, China), GDF-6 (HY-P79333, MedChemexpress, USA), GDF-7 (92004ES10, Yisheng, China), CTGF (HY-P72154, MedChemexpress Biotechnology, USA), GoldenstarTM RT6 cDNA Synthesis Kit Ver.2 (TSK302M, Tsingke, China), qPCR Mix (SYBR Green I) kit and PCR primer (TSE002, Tsingke, China), RIPA lysis buffer (Beyotime, China), TNC (A1927, abclonal, China), MKX (ab236400, abcam, UK), SCX (ab58655, abcam, UK), GAPDH (A19056, abcam, UK), Hes1 (ab71559, abcam, UK), Hras (ab32417, abcam, UK), GAPDH Rabbit mAb (A19056, abclonal, China), Lentiviral Packaging Kit (41102ES10, Yisheng, China), Fetal bovine serum (26050070, Thermo, USA), trypsin (LP0042, Thermo, USA), PBS (10010001, Thermo, USA).

Techniques: Expressing, Immunofluorescence, Control