Superoxide anion concentration, as detected with coelenterazine, serves as an <i>in vivo</i> reporter of beta-cell mass.

<p>(A) Images of a representative nonprogressive mouse at 10, 12, 14 and 16 weeks of age. (B) Images of a representative progressive mouse at 10, 12, 14 and 16 weeks of age. The progressive mouse was normoglycemic at the time of the images. (C) The signal intensities for the two groups of mice, nonprogressive (white) (n = 8) and progressive (black) (n = 7), for a given age normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. (D) The signal intensities for the nonprogressive mice during the last week of the study normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. The signal intensities for progressive mice for the last day of the study during which their fasting blood glucose levels were not exceeding 5.6, 6.9 or 8.3 mmol/L normalized to the first day of imaging for the corresponding mouse and expressed as the fold change in signal intensity. (E) The mean of the fasting blood glucose levels that were less than 8.3 mmol/L for nonprogressive and progressive mice. (Error bars represent the mean ± SEM, and * denotes a significant difference between the groups of P < 0.039.)</p

Acute hyperglycemia results in dynamic changes in the superoxide anion concentration of HeLa cells.

<p>(A) 5x10⁵ HeLa cells were exposed to glucose concentrations ranging from 4 mmol/L (control) to 20 mmol/L and sequentially imaged. Representative image is 15 minutes post treatment. (B) Quantification of coelenterazine chemiluminescence at each time point for each sample (n = 3). (C) 5x10⁵ RIN-5F cells were exposed to glucose concentrations ranging from 4 mmol/L (control) to 20 mmol/L and sequentially imaged. Representative image is 15 minutes post treatment. (D) Quantification of coelenterazine chemiluminescence at each time point for each sample (n = 3). (E) 5x10⁵ INS-1 cells were exposed to glucose concentrations ranging from 4 mmol/L (control) to 20 mmol/L and sequentially imaged. Representative image is 15 minutes post treatment. (F) Quantification of coelenterazine chemiluminescence at each time point for each sample (n = 3). (G) 5x10⁵ HeLa cells were exposed to glucose concentrations ranging from 13.2 mmol/L (control) to 27.8 mmol/L and sequentially imaged. Representative image is 120 minutes post treatment. (H) Quantification of coelenterazine chemiluminescence for HeLa, RIN-5F and INS-1 cells treated as described in G at 120 minutes post-treatment (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 13.2 mmol/L HeLa cells of P < 0.015, + denotes a significant difference from the 13.2 mmol/L RIN-5F cells of P < 0.015, and # denotes a significant difference from the 13.2 mmol/L INS-1 cells of P < 0.015.) (I) Dihydroethidium assay of 5x10⁵ HeLa cells exposed to glucose concentrations of 4 mmol/L (control) or 20 mmol/L with the quantification of 2-hydroxyethidium fluorescence as each time point minus the pre-treatment (-5 minutes) fluorescence intensity for each sample (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 4 mmol/L control of P < 0.04.) (J) Coelenterazine assay of 5x10⁵ HeLa cells exposed to glucose concentrations of 4 mmol/L (control) or 20 mmol/L with the quantification as the cumulative area under the curve at each time point minus the pre-treatment (-5 minutes) chemiluminescence for each sample (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 4 mmol/L control of P < 0.004.)</p

HeLa cells produce coelenterazine chemiluminescence <i>in vitro</i>.

<p>(A) 5x10⁵ HeLa cells were imaged sequentially, at 5 min intervals for 1 hour. (B) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence at each time point (n = 3). (C) 5x10⁵ HeLa cells were imaged at 15 minutes following the addition of native coelenterazine ranging in concentration from 0 μmol/L to 100 μmol/L. (D) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence (n = 3). (E) A range of 500 to 1x10⁶ HeLa cells were imaged 15 minutes following the addition of 10 μmol/L of native coelenterazine. (F) A range of 500 to 1x10⁶ HeLa cells were imaged 1 minute following the addition of 1.07 mmol/L luciferin. (G) Correlation graph of coelenterazine chemiluminescent signal intensity (y-axis) versus the corresponding luciferase signal intensity (x-axis) for a given cell concentration. (Error bars represent the mean ± SEM.)</p

Treatment with mitochondrial shuttle inhibitors, GKA50, Ranolazine and glucose results in significant decreases in superoxide concentration as detected by coelenterazine chemiluminescence.

<p>(A) Bold, red font indicates methods of perturbation. The physiological response of cells was assessed by addition of glucose, GKA50, an activator of glycolysis, AOAC, an inhibitor of the malate-aspartate shuttle, 4-OHCA, an inhibitor of the pyruvate and lactate shuttle, ranolazine, an inhibitor of beta-oxidation and an activator of pyruvate dehydrogenase. (B) HeLa cells were treated with vehicle control, 250 μmol/L of 4-OHCA, 100 μmol/L AOAC, a combination of 4-OHCA and AOAC, 1 μmol/L GKA50 or 1 μmol/L Ranolazine and the final glucose concentrations adjusted from 4 mmol/L to 4 mmol/L, 11.7 mmol/L or 30 mmol/L and sequentially imaged. Representative image is 95 minutes post treatment. (C) Quantification of coelenterazine chemiluminescence 95 minutes post-treatment (n = 3). (Error bars represent the mean ± SEM, * denotes a significant difference from the 4 mmol/L control of P < 0.05, # denotes a significant difference from the 11.7 mmol/L control of P < 0.015, and + denotes a significant difference from the 30 mmol/L control of P < 0.015.)</p

Coelenterazine chemiluminescence is dependent upon superoxide anion concentrations <i>in vitro</i>.

<p>(A) HeLa cells were treated with range of 0 units/mL to 300 units/mL of PEG-SOD and imaged sequentially. The image was taken at 95 minutes post PEG-SOD addition. (B) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence for each concentration 95 minutes post PEG-SOD addition (n = 3). (C) HeLa cells were treated with range of 0 units/mL to 600 units/mL of PEG-CAT and imaged sequentially. Image is 95 minutes post PEG-CAT addition. (D) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence for each concentration 95 minutes post PEG-CAT addition (n = 3). (E) HeLa cells were treated with range of 0 μmol/L to 500 μmol/L of uric acid and imaged sequentially. Image is 95 minutes post uric acid addition. (F) Quantification of HeLa (black) or buffer (white) coelenterazine chemiluminescence for each concentration 95 minutes post uric acid addition (n = 3). (Error bars represent the mean ± SEM, and * denotes significant differences of P < 0.02.)</p

Coelenterazine administration <i>in vivo</i> results in chemiluminescent detection of superoxide anion in a dose-dependent manner that is of pancreatic origin.

<p>(A) Healthy mice were administered 0, 1, 2.5, 5, 7.5 and 10 mg/kg coelenterazine and imaged for chemiluminescence. (B) Quantification of the chemiluminescent signal for each coelenterazine dose (n = 1). (C) The fold change in the abdominal chemiluminescent signal from before to 20 hours following treatment with 600 units of PEG-SOD (SOD) or vehicle control (CON) (n = 3). (D) Tissues collected from a mouse euthanized 3 minutes post-administration of 5 mg/kg native coelenterazine. (E) Tissues from the mouse in D fluorescently imaged with an excitation of 430 nm and emission of 520 nm. (F) Isolated tissues treated with 20 μg of native coelenteraine <i>ex vivo</i>. (G) Quantification of the chemiluminescent signal from each well (n = 3). (Error bars represent the mean ± SEM, and * denotes a significant difference of P < 0.02.) (H) Whole mouse pancreas was excised and incubated in 20 μmol/L coelenterazine, fixed and stained with anti-insulin antibody followed by an Alexa Fluor 750 secondary. (Scale bar represents 400 μm.)</p

Magnetothermal Control of Temperature-Sensitive Repressors in Superparamagnetic Iron Nanoparticle-Coated <i>Bacillus subtilis</i>

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI), and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression resulted in a 14.6-fold (12 hours; h) and 1.8-fold (1 h) increase in reporter transcripts with a 10.0-fold (12 h) and 12.1-fold (1 h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 5.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1 h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1 h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies

Celastrol treatment results in multiple defects in mature lineages.

<p>(A) Representative FACS analysis of mature lineages from peripheral blood of control (left) and celastrol treated-mice (right). Mice received four consecutive daily intraperitoneal injections and peripheral blood cells were harvested the day following the last injection, processed and analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#s2" target="_blank">Materials and Methods</a>. The percentages shown correspond to raw data numbers. Data shown are representative of 10 mice. (B) Representative FACS analysis of total B cells (gated as LIVE/DEAD⁺, CD5⁻, CD19⁺, and analyzed for IgD and IgM expression) from bone marrow, spleen and peritoneal cavity of control (left) and celastrol treated-mice (right) (n = 10). FACS-analysis of the different sub-populations of B lymphoid progenitors was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Ghosn1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Yang2" target="_blank">[20]</a>. MZ B cells: Marginal Zone B cells. (C) Representative FACS analysis of total red cells (gated as LIVE/DEAD⁺, CD5⁻, CD19⁻, CD11b⁻, Gr-1⁻, SSC-A^low and analyzed for CD71 and Ter119 expressions) from bone marrow, spleen and peritoneal cavity of control (left) and celastrol treated-mice (right) (n = 10). FACS-analysis of the different sub-populations of red cell progenitors was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Cao1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Liu1" target="_blank">[22]</a>.</p

Changes in peripheral blood parameters and cellularity in celastrol treated-BALB/c mice.

<p>(A) Peripheral blood parameters in celastrol treated-mice. Mice received four consecutive daily IP injections of DMSO 5% or various concentrations of celastrol (0.01, 0.1, 1 or 5 mg/kg/day). Peripheral blood was harvested the day following the last injection. Values are percent of control. RBC: red blood cells; HGB: Hemoglobin; HCT: hematocrit; WBC: white blood cells. Mean ± SEM, n = 10. (B) Cellularity in control and celastrol treated-mice. Mice received four consecutive daily IP injections of DMSO 5% or celastrol (5 mg/kg). Cells from bone marrow (1 femur+1 tibia), spleen and peritoneal cavity were harvested the day following the last injection. Mean ± SEM, n = 10.</p

Celastrol treatment results in multiple defects in BM progenitors.

<p>Representative FACS analysis of Megakaryocyte-Erythrocyte Progenitors (MEP), Common Myeloid Progenitors (CMP), Granulocyte-Monocyte Progenitors (GMP), LSK CD34⁻ (Lin⁻ Sca-1⁺ c-Kit⁺ CD34⁻) cells and Common Lymphoid Progenitors (CLP) from bone marrow of control (left) and celastrol treated-mice (right) (n = 10). Cells were harvested from animals treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone-0035733-g002" target="_blank">Figure 2</a> and percentages shown correspond to raw data numbers. FACS-analysis of the different sub-populations of multipotent progenitors was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035733#pone.0035733-Kusy1" target="_blank">[23]</a>.</p