Journal: EMBO Molecular Medicine

Article Title: Concurrent targeting of glycolysis in bacteria and host cell inflammation in septic arthritis

doi: 10.15252/emmm.202115284

Figure Lengend Snippet: A–C Primary osteoblast (OB), bone‐marrow‐derived osteoclast (BMOC), and bone‐marrow‐derived macrophage (BMDM) cells were infected with MRSA, the transcriptome expression profiles of which were analyzed with RNA‐sequencing. The expression of 85 genes increased in the setting of MRSA infection. Subsequently, the canonical pathways, upstream regulators, and corresponding category of carbohydrate metabolism were analyzed and are presented in a histogram. See Appendix Fig for detailed information. D Secretion of lactate, IL‐6, and TNF‐α were measured in BMDM cells infected with heat‐killed MRSA (HK‐MRSA; 4 × 10 6 CFU) for 24 h. E, F C57BL/6J mice were intraarticularly infected with different concentrations of MRSA ( n = 4 per group). (E) Physiological changes at 7 days were observed and representative images were captured. (F) mRNA expression levels of Slc2a1 and Slc16a1 were measured. G–K C57BL/6J mice were intraarticularly infected with MRSA (4 × 10 6 CFU; n = 3–4 per group) and left untreated for 24 h. (G) Representative images of physiological changes to the knee joint. (H) The amount of infiltrating immune cells in synovial fluid was quantified and detected using MRSA expressive of GFP within immune cells (Scale bar: 100 μm). See EV2 for individual images. MRSA bioburden in synovial fluid was quantified in colony‐forming units (CFU). (I) Lactate level in synovial fluid was measured and the relative luminescence intensity is shown. (J) Intraarticular inflammation including cytokines, chemokines, and growth factors was measured in synovial fluid; blue box indicates factors that changed significantly. See Appendix Fig for individual factor changes. (K) Paraffin‐embedded tissues were sectioned and used to measure inflammation score and synovial hyperplasia. GLUT1 and MCT4 expression was detected, and the percentage of positively staining cells were determined (Scale bar: 1,000 μm). L C57BL/6J mice were intraarticularly infected with MRSA (4 × 10 6 CFU) and left untreated for 14 days. Paraffin‐embedded tissues were sectioned and used to measure co‐expression and ‐localization levels of MCT4, GLUT1, IL‐1β, NLRP3, MMP3, p‐NF‐κB (Scale bar: 100 μm). See Appendix Fig for individual images. M Human cartilage and synovial tissue were infected with MRSA (4 × 10 6 CFU) for 24 h. The co‐expression and localization levels of GLUT1 and MCT4 in human cartilage tissue were measured and confirmed by line profile analysis (Scale bar: 100 μm). Expression of GLUT1, MCT4, IL‐1β, and MMP3 was measured in human synovial tissue (Scale bar: 1,000 μm). Data information: In vitro experiments were repeated at least three times with representative results. In vivo experiments were repeated in at least two independent experiments, with one or two experiments performed for each group. Error bars show means ± SD with individual data points. Two‐tailed unpaired t ‐test analysis was conducted to determine statistical significance (* P < 0.05 or ** P < 0.01; N.D. = not detected).

Article Snippet: We analyzed expression levels of proteins from synovial fluid and tissue in a septic arthritis model using a single‐cell RNA‐Sequencing data set of rheumatoid arthritis patients using the Web‐based Single Cell Portal hosted by Broad Institute of MIT and Harvard.

Techniques: Derivative Assay, Infection, Expressing, RNA Sequencing, Bioburden Testing, Staining, In Vitro, In Vivo, Two Tailed Test

Journal: Frontiers in Molecular Neuroscience

Article Title: MicroRNA-133b Negatively Regulates Zebrafish Single Mauthner-Cell Axon Regeneration through Targeting tppp3 in Vivo

doi: 10.3389/fnmol.2017.00375

Figure Lengend Snippet: miR-shRNA based gene silence of tppp3 diminishes regenerative length of M-cell (A) Diagram of miR-shRNA system. Sequence of ShRNA-5 was presented here, with the guide strand (bottom) highlighted in dark blue. (B) Quantitative RT-PCR analysis exhibited a deduction of tppp3 mRNA in shRNA-5 expressing embryos. (C) Confocal imaging of M-cell at 2 dpa. White asterisk: ablation point. Scale bar: 50 μm. (D) Regeneration length at 2 dpa. Student's two-tailed t -test, control vs. shRNA-1, P = 0.5807; control vs. shRNA-5, P = 0.0025. ** P < 0.01. Error bars represent S.E.M.

Article Snippet: Based on the Web-based shRNA design tool ( https://www.genscript.com ), five shRNAs (shRNA1-shRNA5) targeting the tppp3 gene were selected (Figure ). mCherry was used as a fluorescent reporter to mark the zebrafish embryos that expressed the miR-shRNA (Figure ).

Techniques: shRNA, Sequencing, Quantitative RT-PCR, Expressing, Imaging, Two Tailed Test