Journal: Journal of Pharmaceutical Analysis

Article Title: New applications of clioquinol in the treatment of inflammation disease by directly targeting arginine 335 of NLRP3

doi: 10.1016/j.jpha.2024.101069

Figure Lengend Snippet: Clioquinol (CQ) affects the interaction between NOD-like receptor protein 3 (NLRP3) and flag-(never in mitosis gene a)-related kinase (NEK)/apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC). Lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BMDMs)/human monocyte leukemia cells (THP-1) cells were incubated with different doses of CQ for 1 h and then stimulated with nigericin for 1.5 h. (A, C) Immunofluorescence assay of ASC speck. BMDMs were stained with ASC antibody (green) and 4′,6-Diamidino-2-phenylindole (DAPI) (Blue). ASC speck formation was observed by immunofluorescence. BMDMs (A), and THP-1 cells (C). (B, D) The percentage of ASC specks was measured by ImageJ (relative to LPS + nigericin). BMDMs (B), and THP-1 cells (D). (E) Western blot analysis of ASC oligomerization level after cross-linking with disuccinimidyl suberate in NP-40-insoluble pellets from BMDMs. (F) NLRP3 self-oligomerization level was measured by native-polyacrylamide gel electrophoresis (PAGE). (G, H) Immunoprecipitation analysis of the interaction between NLRP3 and NEK7 in BMDMs. IP:NLRP3 (G), and IP:NEK7 (H). (I, J) Transfected HEK-293T with plasmid overnight and treated with CQ for 8 h, and then CO-immunoprecipitation (IP) with flag antibody and Western blot was used to evaluate the interaction between green fluorescent protein (GFP)-NLRP3 and FLAG-NEK7 (I) or FLAG-ASC (J). Data are representative of n = 5 (B, D), and are expressed as mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, ns: not significant, determined by Mann–Whitney test (relative to LPS + nigericin). Glyceraldehyde phosphate dehydrogenase (GAPDH) served as a loading control in (E–G).

Article Snippet: Then, 500 μL of disuccinimidyl suberate (S0657, Selleck) solution (2 mM) were added to the pellets and cross-linked for 60 min at room temperature on a shaker.

Techniques: Activation Assay, Derivative Assay, Incubation, Immunofluorescence, Staining, Western Blot, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection, Plasmid Preparation, Standard Deviation, MANN-WHITNEY, Control

Journal: Glycobiology

Article Title: Elucidating the unusual reaction kinetics of D-glucuronyl C5-epimerase

doi: 10.1093/glycob/cwaa035

Figure Lengend Snippet: C5-epimerase catalyzes the conversion of GlcA to IdoA. This reaction is reversible and is skewed towards the formation of the more thermodynamically favored GlcA. (A) Forward reaction using NSH and (B) pseudo-reverse reaction using CDNSH. The protons deuterium exchanged at the C5 positions of the GlcA and the IdoA residues are shown in blue. (C) NMR-based assay that is used to detect directly the presence of GlcA and IdoA within the reaction based on differences in chemical shifts of the anomeric proton of GlcA and IdoA. The peak area as a function of the internal standard (DSS) provides quantitative amounts (in mM) of GlcA and IdoA within the reaction. This figure is available in black and white in print and in color at Glycobiology online.

Article Snippet: For all NMR experiments, 10 μM DSS (Cambridge Isotope Laboratories, Andover, MA) was used as an internal standard for quantification of GlcA and IdoA concentrations.

Techniques:

Journal: iScience

Article Title: Ablation of Atp5if1 impairs metabolic reprogramming and proliferation of T lymphocytes and compromises mouse survival

doi: 10.1016/j.isci.2024.109863

Figure Lengend Snippet: Expression of IF1 in T cells and development of IF1-KO mice in CD4 + lymphocytes (A) Representative blot of IF1 and β-F1-ATPase expression in mouse tissue extracts of heart, kidney, liver, lymph nodes and spleen, and in spleen of mice treated with tamoxifen (Tam) or dextran sodium sulfate (DSS). α-tubulin is shown as loading control. (B) Representative blot of IF1 and β-F1-ATPase expression in naive and in vitro activated CD4 + mouse lymphocytes. CD4 is shown as loading control. (C) Quantification of IF1 mRNA levels in naive and activated CD4 + lymphocytes ( n = 4). (D) Extracellular acidification rates (ECAR) of naive and activated CD4 + lymphocytes ( n = 3). B–D, In vitro activation of CD4 + lymphocytes was performed by incubating the cells with anti-CD3 and anti-CD28 during 48 h. (E) Quantification of mRNA levels of IFNγ, IL17, TGFβ and IF1 in CD4 + lymphocytes polarized to the Th1, Th17 or Treg subsets (n = 3–4). (F) Representative blot of IF1 and β-F1-ATPase expression in CD4 + lymphocytes polarized to the Th1, Th17 or Treg subsets. CD4 is shown as loading control. (G) Spleen weight of mice 10 days after immunization with PBS (−) or BSA/CFA ( n = 4). (H) Representative plots of the percentage of IFNγ + immune cells (CD45 + ) in the spleen of mice 10 days after PBS (−) or BSA/CFA (+) injection. (I) Representative blot of IF1 and β-F1-ATPase expression in CD4 + lymphocytes 10 days after PBS (−) or BSA/CFA (+) injection. CD4 is shown as loading control. (J) Schematic of the generation of the CD4 + -IF1-KO mouse model. IF1-KO mice in CD4 + lymphocytes were obtained by breeding CD4-Cre with IF1-floxed mouse lines. (K) Histograms show the quantification of IF1 mRNA levels in CRL and IF1-KO CD4 + lymphocytes ( n = 4). (L) Representative blot of IF1 and β-F1-ATPase expression in extracts of CD4 + cells, immune CD4 − cells, brain and kidney of CRL and CD4 + -IF1-KO mice. The histograms show the mean and the error bars ±SEM. ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001 when compared to PBS (−) or CRL by Student’s t test. # p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001 when compared by one-way ANOVA test and the Tukey multiple comparison test. See also Figure S1 .

Article Snippet: Four-month-old CRL and CD4-IF-KO male mice were treated with 2.5% DSS (PanReac AppliChem) added in the drinking water to induce acute intestinal inflammation.

Techniques: Expressing, Control, In Vitro, Activation Assay, Injection, Comparison

Journal: iScience

Article Title: Ablation of Atp5if1 impairs metabolic reprogramming and proliferation of T lymphocytes and compromises mouse survival

doi: 10.1016/j.isci.2024.109863

Figure Lengend Snippet: Survival of CD4 + -IF1-KO mice is compromised in response to bacterial infection due to a restrained adaptive immune response (A) Progressive weight change of CRL ( n = 5) and IF1-KO ( n = 4) mice treated with 2.5% dextran sodium sulfate (DSS) in the drinking water for nine days. (B) Kaplan–Meier survival analysis of CRL ( n = 9) and IF1-KO mice ( n = 9) after treatment with 2.5% DSS in the drinking water. The p -value of the log rank test is shown. (C) Number of CD3 + (CD3 + ;CD45 + ;DAPI − ), CD4 + (CD4 + ;CD45 + ;DAPI − ) and CD8 + (CD8 + ;CD45 + ;DAPI − ) cells in the blood of CRL and IF1-KO mice after 6-day treatment with 2.5% DSS (n = 3–4). (D) Percentage of Th1 (INFγ + ;CD4 + ;CD45 + ), Th17 (IL17 + ;CD4 + ;CD45 + ) and Treg (FoxP3 + ;CD4 + ;CD45 + ) cells in the blood of CRL and IF1-KO mice after 6-day treatment with 2.5% DSS (n = 3–4). (E) Multiplexed quantitative analysis of cytokines and chemokines. Histograms show the mean and the error bars ±SEM of cytokine levels in the spleen and colon of CRL and IF1-KO mice after 6-day treatment with 2.5% DSS (n = 3–4). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01 when compared to CRL by Student’s t test. See also Table S1 .

Article Snippet: Four-month-old CRL and CD4-IF-KO male mice were treated with 2.5% DSS (PanReac AppliChem) added in the drinking water to induce acute intestinal inflammation.

Techniques: Infection