Journal: International Journal of Dentistry

Article Title: The ROS/CaMK II/β-Catenin Signaling Axis Affects the Osteogenic Potential of BMSCs and Disrupts Implant Osseointegration: An In Vitro Study

doi: 10.1155/ijod/5566776

Figure Lengend Snippet: Determination of optimum concentration of KN-93 and Mito TEMPOL. (a) The Cell Counting Kit-8 (CCK-8) assay revealed that a concentration of 30 and 40 μM Mito TEMPOL impaired the proliferation capacity of BMSCs. ⁣ ∗ p < 0.05; ⁣ ∗∗ p < 0.01; ⁣ ∗∗∗ p < 0.001. (b) Live/dead cell staining demonstrated that Mito TEMPOL at concentrations ≤20 μM did not exhibit significant cytotoxicity toward BMSCs. (c) The ROS assay determined that the optimal concentration of Mito TEMPOL for inhibiting ROS was 20 μM. (d) Quantitative analysis of fluorescence intensity determined that the optimal concentration of Mito TEMPOL for ROS inhibition was 20 μM. ⁣ ∗ p < 0.05; ⁣ ∗∗ p < 0.01; ⁣ ∗∗∗ p < 0.001. (e) The CCK-8 assay revealed that 20 and 40 μM concentrations of KN-93 impaired the proliferation capacity of BMSCs at 48 and 72 h. ⁣ ∗ p < 0.05; ⁣ ∗∗ p < 0.01; ⁣ ∗∗∗ p < 0.001.

Article Snippet: The first one consisted of the normal glucose (NG) group, the HG group, the ROS-inhibited group, and the ROS-activated group: (a) NG: Osteogenic induction medium (OIM; Meilun Bio, Dalian, China) with NG concentration (5.5 mM D-glucose); (b) HG: OIM with HG concentration (35 mM D-glucose); (c) ROS inhibition: OIM (35 mM D-glucose) + Mito TEMPOL (20 μM; MCE, USA); (d) ROS activation: OIM (5.5 mM D-glucose) + H 2 O 2 (25 μM; Lierkang, Shandong, China).

Techniques: Concentration Assay, Cell Counting, CCK-8 Assay, Staining, ROS Assay, Fluorescence, Inhibition

Journal: PLoS ONE

Article Title: Permeability Transition Pore-Mediated Mitochondrial Superoxide Flashes Regulate Cortical Neural Progenitor Differentiation

doi: 10.1371/journal.pone.0076721

Figure Lengend Snippet: (A) Mitochondrial membrane potential in differentiated cells at day 2. Cells were treated with 100 nM CsA, 1µM ATR or vehicle at the time of growth factor removal. Values are expressed as a percentage of the mean of the control condition in NPCs (day 0). *p<0.05 compared to the control value in cells (day 2). (B) Mitochondrial superoxide in differentiated cells at day 2. Cells were treated with 1µM Mito-Tempol (MitoTEMO), 1µM PQ or vehicle at the time of growth factor removal. Values are expressed as a percentage of the mean of the control condition in NPCs (day 0), *p<0.05 compared to the control value in cells (day 2). (C) Superoxide flash incidence in differentiated cells at day2. Cells were treated with CsA, ATR, Mito-Tempol and PQ as indicated in A and B. Values are expressed as a percentage of the mean of the control condition in NPCs (day 0); Perinuclear or whole cell flash: *p<0.05 compared to the control value in cells (day 2); Flash at process: #p<0.05 compared to the control value in cells (day 2). (D, E and F) Percentage of NPCs, neurons and astrocytes at different days of differentiation. NPCs, neurons and astrocytes were identified by antibodies against Sox2, Tuj1 and GFAP at different days after differentiation. Total number of cells was counted by counterstaining nuclei with DAPI. n = 4 separate experiments performed on NPCs cultured from 4 pregnant mice. *p<0.05 compared to the control value of each individual day.

Article Snippet: Treatments included: 100 nM cyclosporine A (CsA; Sigma, St. Louis, MO); 1 µM atractyloside (ATR; Sigma, St. Louis, MO), 1 µM Mito-Tempol (Sigma, St. Louis, MO); and 1µM paraquat (PQ; Sigma, St. Louis, MO).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Inflammation-induced mitochondrial and metabolic disturbances in sensory neurons control the switch from acute to chronic pain

doi: 10.1101/2022.08.29.505682

Figure Lengend Snippet: A) Supervised partial least square discriminant analysis of DI-HRMS of lumbar DRG isolated from untreated mice (day 0) and 1 or 7 days after intraplantar carrageenan (each dot represents a metabolic signature of lumbar DRG isolated from one mouse). B/C) Intensity of metabolites measured with DI-HRMS B) involved in generation of NAD + C) and used to measure 3-hydroxybutyrate/acetoacetate ratio as an indirect measure of mitochondrial NAD + /NADH ratio (n=4). D) Extracellular acidification rate (ECAR) in cultured DRG neurons from untreated mice (non-primed, n=22) or mice that had resolved from carrageenan-induced inflammatory hyperalgesia (primed, day 7, n=20). E) Course of PGE 2 -induced mechanical hyperalgesia after intraperitoneal injection with nicotinamide riboside (NR, 500 mg/kg) at day 7 in carrageenan-primed or non-primed mice and 15 min prior to intraplantar PGE 2 . F/G) Detection of mtROS formation in DRG neurons of untreated mice (day 0) or mice that had resolved from carrageenan-induced inflammatory hyperalgesia (day 7). mtROS was visualised by an intrathecal injection with MitoTracker Red CM-H2Xros (100 uM) that accumulates in mitochondria and generates fluorescence upon oxidation by mtROS. F) Mean MitoTracker Red CM-H2Xros fluorescence intensity in small and medium/large-diameter neurons at indicated days. Data visualized with a violin plot to show distribution of all data, median (black horizontal line) and quartiles (dotted line; small-diameter n= 700-1000 cells, medium/large-sized = 250-550 cells). G) Representative pictures of F. Neurons are visualised with neurotrace (blue; scale bar = 100 µM). H) Course of PGE 2 -induced mechanical hyperalgesia after intrathecal administration of mito-tempol (25 μg) at day 7 in carrageenan-primed or non-primed mice and 15 min prior to intraplantar PGE 2 . Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed by Student’s T-test (D), one-way ANOVA (B, C, F) followed by Dunnett’s multiple comparison test or two-way repeated measures ANOVA followed by a post-hoc Sidak’s multiple comparison test (E/H, stars indicate significance comparing primed conditions).

Article Snippet: In the CFA experiment, we measured hyperalgesia 4 hours after injection of NR. Intrathecal injections (5 μl) with NR (50 μg), myxothiazol (50 uM, Sigma-Aldrich), mito-tempol[ ] (25 μg, Sanbio) and oligodeoxynucleotide (3 μg/μl day 4, 5 and 6 after carrageenan, Sigma-Aldrich), were performed under light isoflurane anesthesia as described.

Techniques: Isolation, Cell Culture, Injection, Fluorescence

Journal: bioRxiv

Article Title: Inflammation-induced mitochondrial and metabolic disturbances in sensory neurons control the switch from acute to chronic pain

doi: 10.1101/2022.08.29.505682

Figure Lengend Snippet: A) Unsupervised principle component analysis (PCA) of whole metabolomics DI-HMRS dataset of HAP1 cells expressing ATPSc-KMT (WT), cells deficient for ATPSc-KMT (KO) and KO cells reconstituted with WT (KO + WT) or with enzyme dead mutant ATPSc-KMT (KO + E117A) B) Heat map of metabolite levels that were significantly changed between WT and ATPSc-KMT -/- cells. Blue = reduced intensity, red = increased intensity. Intensity of lactic acid and GA3P in C) HAP1 cells and D) N2A cells with and without ATPSc-KMT. E) Ratio of 3-hydroxybutyrate and acetoacetate which was measured with DI-HMRS and which is an indirect measure of mitochondrial NAD + /NADH balance. F/G) NAD + /NADH ratio calculated from direct measurement of cellular NAD + and NADH in F) HAP1 WT and ATPSc-KMT deficient cells and G) N2A cells after overexpression of ATPSc-KMT or control empty vector (EV). H) Course of PGE 2 -induced mechanical hyperalgesia after an intraperitoneal injection of nicotinamide riboside (NR), 15 min prior to intraplantar PGE 2 injection, in mice overexpressing ATPSc-KMT in DRG neurons. Intraplantar HSV injections were administrated at day −3 and −1 (35.000 pfu/paw) I) Same as H, but after intrathecal injection of mito-tempol, 15 min prior to intraplantar PGE 2 injection. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed by Student’s t-test (D, F and G), one-way ANOVA (C and E) or two-way repeated measures ANOVA followed by a post-hoc Sidak’s multiple comparison test (H and I).

Article Snippet: In the CFA experiment, we measured hyperalgesia 4 hours after injection of NR. Intrathecal injections (5 μl) with NR (50 μg), myxothiazol (50 uM, Sigma-Aldrich), mito-tempol[ ] (25 μg, Sanbio) and oligodeoxynucleotide (3 μg/μl day 4, 5 and 6 after carrageenan, Sigma-Aldrich), were performed under light isoflurane anesthesia as described.

Techniques: Expressing, Mutagenesis, Over Expression, Plasmid Preparation, Injection