Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rational Design of Natural Xanthones Against Gram-negative Bacteria.

doi: 10.1002/advs.202411923

Figure Lengend Snippet: Figure 1. Screening of broad-spectrum antibacterial hits. A) Scheme of the screening of antibacterial scaffolds. S. aureus ATCC 29213, LPS deficient A. baumannii 176, and LPS deficient A. baumannii 7-2 were used as model strains to screen broad-spectrum antibacterial natural products. B) Property- guided design of antibacterial substances against Gram-negative bacteria. Functional groups were highlighted in red. S. aureus ATCC 29213 and E. coli ATCC 2922 were used as model strains. (C,D) Time-killing curves of A20 against C) S. aureus ATCC 29213 and D) E. coli ATCC 25922. Vancomycin (VAN) and colistin (COL) were used as controls. Experiments in B, C, and D were performed as three biologically independent experiments (n = 3), and the mean ± SD is shown.

Article Snippet: To quantify the intracellular accumulation of A20, we explored the penetration dynamics by assessing the whole-cell accumulation of A20 in E. coli ATCC 25922.

Techniques: Bacteria, Functional Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rational Design of Natural Xanthones Against Gram-negative Bacteria.

doi: 10.1002/advs.202411923

Figure Lengend Snippet: Figure 2. Self-promoted uptake of A20 for transmembrane transportation. A) Intracellular accumulation kinetics of A20 in E. coli. B) Scheme of an- tibacterial agents crosses the OM. 1) OMP-depedent fluoquinolones and 2) LPS-dependent colistin. C) MICs of antibacterial compounds against ΔLPS- mutants. WT, wild-type. D) Competitive binding assay between A20 and the fluorescent probe BODIPY-TR-cadaverine in the presence of lipid A. E) Syn- ergy of A20 and colistin with hydrophobic rifampicin. F) Simulation of A20 interacting with OM in E. coli. Experiments in A, C, D, and E were performed as three biologically independent experiments (n = 3), and mean ± SD is shown.

Article Snippet: To quantify the intracellular accumulation of A20, we explored the penetration dynamics by assessing the whole-cell accumulation of A20 in E. coli ATCC 25922.

Techniques: Competitive Binding Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rational Design of Natural Xanthones Against Gram-negative Bacteria.

doi: 10.1002/advs.202411923

Figure Lengend Snippet: Figure 3. A20 binds to phospholipids to cross the inner membrane. A) Schematic diagram of chemicals crossing the inner membrane. B) Intracellular accumulation of A20 under the treatment of PMF disruptors including 3-chlorophenylhydrazone (CCCP) and triclosan (TCL). C) Fold changes in MICs of A20 in the presence of bacterial phospholipids including phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). D) Leakage of PG liposomes under the treatment of A20 for 10 min. E) The affinity between A20 and PG based on the isothermal titration calorimetry test. F) The permeability of the inner membrane probed with propidium iodide (PI) for E. coli under the treatment of A20. Experiments in A, C, D, and E were performed as three biologically independent experiments (n = 3), and mean ± SD is shown. P values were determined using the One-way ANOVA test, and P < 0.05 was considered statistically significant.

Article Snippet: To quantify the intracellular accumulation of A20, we explored the penetration dynamics by assessing the whole-cell accumulation of A20 in E. coli ATCC 25922.

Techniques: Membrane, Liposomes, Isothermal Titration Calorimetry, Permeability

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rational Design of Natural Xanthones Against Gram-negative Bacteria.

doi: 10.1002/advs.202411923

Figure Lengend Snippet: Figure 4. A20 disrupts bacterial respiration. A) Metabolism-dependent bactericidal activities of A20 against E. coli. B) Antibacterial activities of A20 under aerobic and anaerobic conditions. C) Scheme of the respiratory chain in E. coli. D) Activity of respiratory complex IV in E. coli treated with A20. E) Enzyme activity of horseradish catalase in the presence of A20. F) MICs of A20 with exogenous hemin. G) Scheme of the experimental design and growth curves of E. coli under the treatment of A20 and hemin. H) The representative color and the UV−Vis spectra of hemin (20 μM) with A20 in HEPES (200 mM, pH 7.0). Experiments were performed as three biologically independent experiments (n = 3). Data are presented as mean ± SD.

Article Snippet: To quantify the intracellular accumulation of A20, we explored the penetration dynamics by assessing the whole-cell accumulation of A20 in E. coli ATCC 25922.

Techniques: Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rational Design of Natural Xanthones Against Gram-negative Bacteria.

doi: 10.1002/advs.202411923

Figure Lengend Snippet: Figure 5. Therapeutic efficacy of A20 in vivo. A) Scheme of the experimental protocol for the G alleria mellonella infection model and mouse skin wound infection model. B) Time-survival curves of G. mellonella larva during six days after infection (n = 8). C) Bacterial loads in G. mellonella at 6 days post- infection (n = 8). Data are representative of eight independent experiments and values are expressed in mean ± SEM. D) Representative pictures of wound healing records. E) Bacterial loads in wounds on day 12. Data are presented as mean ± SD. P values were determined using an unpaired, two tailed Stuent’s t-test.

Article Snippet: To quantify the intracellular accumulation of A20, we explored the penetration dynamics by assessing the whole-cell accumulation of A20 in E. coli ATCC 25922.

Techniques: In Vivo, Infection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: The Efficiency of Neurospheres Derived from Human Wharton’s Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats

doi: 10.3390/ijms24043846

Figure Lengend Snippet: Cell viability of MSCs after P7C3 or Isx9 treatment. Cell viability was assessed by MTT assay at 0, 1, 2.5, 5, 10, and 20 µM for ( A ) P7C3 treatment for 48 h, and at 0, 0.25, 0.50, 1, 2.5, and 5 µM for ( B ) Isx9 treatment for 48 h. Data were shown as mean ± S.D. with different lower-case letters, and are significantly different at p < 0.05.

Article Snippet: The cytotoxicity of neurogenesis-enhancing small molecules, Isx9 and P7C3-A20 (MedChemExpress, NJ, USA), was evaluated by addition into the culture medium at 0, 1, 2.5, 5, 10, and 20 µM for Isx9, and at 0, 0.25, 0.50, 1, 2.5, and 5 µM for P7C3-A20.

Techniques: MTT Assay

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 8. Decreased proliferation and increased chemokine production of epidermal keratinocytes from miR-132–KO mice. (A) H&E staining of the skin from miR-132–KO mice (n = 9) and their WT littermate controls (n = 5) and epidermis thickness were analyzed. Scale bars: 100 μm. (B) Immunostaining of Ki-67 and HB-EGF in skin from WT (n = 9) and KO mice (n = 5). Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67–positive cells was counted. Scale bars: 50 μm. (C) Epidermal keratinocytes were isolated from the skin of WT (n = 3) and KO (n = 3) mice. Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, Il1b, and Hbegf expression levels were analyzed using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Staining, Immunostaining, Isolation, Expressing, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 9. Skin wound healing is impaired in miR-132–KO mice. (A) Representative images of wounds on the backs of miR-132–KO mice (n = 9) and their WT littermate controls (n = 6) on days 0, 2, 4, 6, and 8 after wounding. Wound closures were quantified and are presented as the percentage of the initial wound area size. (B) qRT-PCR was performed to detect Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, and Il1b expression levels in the intact and wound-edge skin of both WT (n = 5) and KO (n = 9) mice 2 days after injury. *P < 0.05 and **P < 0.01 by Student’s t test. (C) H&E staining (left panels) and immunostaining of Gr-1 (mid- dle and right panels) in the wound area in WT and KO mice 2 days after injury. White arrows demarcate wound edges. Scale bars: 50 μm. (D) Chemotaxis of neutrophils isolated from the peripheral blood of WT (n = 5) and KO mice (n = 7) toward the medium containing 8 μM N-formylmethionyl-leucyl-phenyla- lanine (fMLP). Migrating cells were quantified by flow cytometry. Plots show the FSC/SSC of the migrated cells. (E) Immunostaining of Ki-67 and HB-EGF in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67– positive cells was counted. (F) Immunostaining of p-STAT3 and p-ERK in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Scale bars: 50 μm (E and F). *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Staining, Immunostaining, Chemotaxis Assay, Isolation, Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 11. Inhibition of miR-132 delays reepithelialization of human ex vivo skin wounds. Human ex vivo skin wounds were treated topically with anti–miR-132 (n = 4) or anti–miR-Ctrl (n = 4) after injury, and miR-132 blocking efficiency was confirmed by qRT-PCR (A). (B) H&E staining of the ex vivo skin wounds 3 days after injury. Black arrows demarcate the initial wound edges, while the red arrows indicate a newly formed epidermis. Scale bars: 200 μm. (C) Immunostaining of Ki-67 in ex vivo skin wounds 3 days after injury. Sections were counterstained with DAPI. The number of Ki-67– positive cells was counted. White arrows demarcate the wound edges. Scale bars: 50 μm. CXCL1, CXCL5, CCL20 (D), and HB-EGF (E) expression levels were detected by qRT-PCR in ex vivo wounds treated with anti–miR-Ctrl (n = 4) or anti–miR-132 (n = 4) 3 days after injury. (F) Schematic summary of the regulation and function of miR-132 during skin wound healing. *P < 0.05 and **P < 0.01 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Inhibition, Ex Vivo, Blocking Assay, Quantitative RT-PCR, Staining, Immunostaining, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: Inflammatory cytokines induce A20 expression in MSC s. MSC s ( A ) and C3H/10T1/2 ( B and C ) were treated with 0, 2, 5 and 10 ng/ml IFN ‐γ and TNF ‐α for 24 hrs. A20 mRNA and protein levels were examined by qRT ‐ PCR and Western blot analysis. MSC s ( D ) and C3H/10T1/2 ( E and F ) were treated with 5 ng/ml IFN ‐γ and TNF ‐α for 0, 6, 12 and 24 hrs, and mRNA and protein expression levels were determined by qRT ‐ PCR and Western blot analysis, respectively.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: Morphological and phenotypic characterization of C3 MSC s after A20 knockdown. ( A ) qRT ‐ PCR analysis of A20 mRNA levels with or without A20 knockdown. ( B ) Morphology and ( C ) size of cultured sh CTRL C3 MSC s and shA20 C3 MSC s were analysed by microscopy and flow cytometry. ( D ) Cell surface markers were analysed by flow cytometry, ** P < 0.01. All experiments were repeated three times.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, Quantitative RT-PCR, Cell Culture, Microscopy, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 knockdown attenuates immunosuppressive capacity of C3 MSC s in vitro . CFSE ‐labelled CD 3 + T cell was cultured alone ( A ) or co‐cultured with different numbers of C3 MSC s ( B ) in RPMI 1640 complete medium supplemented with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 48 hrs. Cells were subjected to flow cytometry for T cell proliferation as detected by the CFSE signal.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, In Vitro, Cell Culture, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 knockdown inhibits tumorigenesis in vivo . ( A ) Representative tumours photographed 13 days after C57 BL /6 mice were injected with PBS , sh CTRL C3 MSC s or shA20 C3 MSC s. ( B ) On day 0, 5 × 10 5 B16‐F0 cells in 100‐μl PBS were injected subcutaneously into the right posterior flanks, with or without co‐injection of sh CTRL C3 MSC s or shA20 C3 MSC s (1 × 10 6 cells). Tumour growth was measured at 2‐day intervals, and tumour volume was calculated. ( C ) Thirteen days later, all mice were killed and tumours were sectioned and weighted. Each treatment group included four mice, and data are representative of three independent experiments, ** P < 0.01.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, In Vivo, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 knockdown showed no significant effect on ICAM ‐1, VCAM ‐1, PD ‐L1, PGE 2 or nitric oxide expression. Sh CTRL C3 MSC s and shA20 C3 MSC s were cultured with or without stimulation with 5 ng/ml IFN ‐γ and TNF ‐α. 12 hrs later, the expression of ( A ) ICAM ‐1, ( B ) VCAM ‐1 and ( C ) PD ‐L1 were analysed by flow cytometry. ( D ) Nitric oxide production was measured by Griess assay. ( E ) PGE 2 production was measured by ELISA .

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, Expressing, Cell Culture, Flow Cytometry, Griess Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 inhibits TNF ‐α and promotes IL ‐10 production in C3 MSC s, and the p38/ MAPK pathway is involved in A20‐induced immunomodulation. TNF ‐α ( A ) and IL ‐10 ( B , left) expression was examined with or without stimulation with 5 ng/ml IFN ‐γ and TNF ‐α by qRT ‐ PCR . IL ‐10 protein level was determined by ELISA ( B , right). ( C ) The time courses of p38 phosphorylation in response to 5 ng/ml IFN ‐γ and TNF ‐α was determined by immunoblotting, ** P < 0.01, * P < 0.05.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot