Heterogeneous Reaction of NO₂ on Al₂O₃: The Effect of Temperature on the Nitrite and Nitrate Formation

Although recent evidence suggests that the heterogeneous reaction of NO₂ on the surface of mineral aerosol plays an important role in the atmospheric chemistry, a fundamental understanding of how temperature influences the rate and extent of nitrate formation processes remains unclear. This work presents the first laboratory study of the effect of temperature on heterogeneous reaction of NO₂ on the surface of γ-Al₂O₃ in the temperature range of 250–318 K at ambient pressure. From the analysis of IR spectra, nitrite was found to be an intermediate product at temperatures between 250 and 318 K. It is proved by our experiments that nitrite would convert to the bidentate nitrate as the reaction proceeded. In addition, it is interesting to find that the rate of conversion increased with decreasing temperature. Along with nitrite decrease, the initial rate of nitrate formation increased while the rate of nitrate formation in the steady region decreased with decreasing temperature. The uptake coefficients at seasonal temperatures were determined for the first time and were found to be sensitive to temperature. Finally, atmospheric implications of the role of temperature on the heterogeneous reaction of NO₂ with mineral aerosol are discussed

Mitochondrial membrane potential, ROS production, arginase, and IDOs in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells.

<p>(A) Live wild type (HD1A) and <i>lal</i>-/- (HD1B) cells were stained with JC1 to measure the mitochondria membrane potential. JC1 fluorescent red staining represents a healthy membrane potential state, while JC1 fluorescent green staining represents a damaged membrane potential state. (B) The ROS levels in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells by flow cytometry analysis. Results are mean ± SD from three independent experiments (n = 3), **, p< 0.001. (C) The arginase activity in HD1A and HD1B were measured. The result is mean ± SD from three independent experiments (n = 3), **, p< 0.001. (D) The IDO1 and IDO2 expression levels were measured by Real-Time. The results are means ± SD from three independent experiments (n = 3), **, p< 0.001.</p

Immunosuppression on T cell proliferation and function by wild type (HD1A) and <i>lal</i>-/- (HD1B) cells.

<p>(A) CFSE labeled wild type CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies, and co-cultured with wild type (HD1A) or <i>lal</i>-/- (HD1B) cells (1:30). CD3 and CD28 antibody unstimulated CD4⁺ T cells served as a negative control. Results are mean ± SD from three independent experiments (n = 3), *, p< 0.05; **, p<0.01. (B) Secretion of T cell releasing IL-2, IL-4 and INF-γ in the above co-culture experiment was measured to assess the CD4⁺ T cell function. Results are mean ± SD from three independent experiments (n = 3), *, p< 0.05; **, p<0.01.</p

Increase of lysosome genesis and metabolic disorder in HD1B cells.

<p>(A) Western blot analysis of lysosome marker LAMP1 expression in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells; (B) Immunofluorescence staining of LAMP1 in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. Bars = 20 μm; (C) LysoTracker Red DND-99 staining of live wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. Bar = 25 μm. (<b>D</b>) Expression of CD36, Foxo3, Sirt1, CPT1, CPT2, CPT3 with the housekeeping gene β-Actin as internal control by Real-time PCR. The results are means ± SD from three independent experiments (n = 3), *, p< 0.05, **, p< 0.001.</p

Glucose transportation, glycolysis and TCA in HD1A and HD1B cells.

<p>(<b>A</b>) The glucose and pyruvate concentrations, and aconitase activity in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. The results are means ± SD from three independent experiments (n = 3), *, p< 0.05, **, p< 0.001. (<b>B</b>) Expression of <i>Glut1 to Glut13</i> with the housekeeping gene β-<i>Actin</i> as internal control by Real-time PCR. The results are means ± SD from three independent experiments (n = 3), *, p< 0.05, **, p< 0.001.</p

Lal-/- cells (HD1B) stimulated cancer cells growth.

<p>(A) Wild type (HD1A) and <i>lal</i>-/- (HD1B) cells were co-cultured with CFSE labeled LLC or B16 melanoma cells (5:1) for 3 days. The results are presented as percentage of CFSE positive cells increased from none co-culture base line. The results are mean ± SD from three independent experiments (n = 3), **, p< 0.01. (B) mTOR siRNA knockdown in <i>lal</i>-/- (HD1B) cells inhibited the stimulatory activity of cancer cell growth (LLC and B16 melanoma cells). The same cell ratio was used. The results are mean ± SD from three independent experiments (n = 3), **, p< 0.01; (C) B16 melanoma cells (2 X 10⁵) that were mixed with the same amount of wild type (HD1A) or <i>lal</i>-/- (HD1B) cells were left-side and right-side pair-injected subcutaneously into C57BL/6 or FVB/N mice. After 14 days, tumor volumes were measured using the formula: (length X width²) /2. The results are mean ± SD from 11–12 independent experiments (n = 11–12), p values are listed in comparison of tumor sizes between co-injection of wild type (HD1A) and <i>lal</i>-/- (HD1B) cells.</p

siRNA knockdown of mTOR inhibits immunosuppression on T cell proliferation and function of <i>lal</i>-/- (HD1B) cells.

<p>(A) Western blot analyses showed the mTOR protein expression level, phosphorylated mTOR, and phosphorylated S6 levels reduced in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells by mTOR siRNA knocking down. (B) T cell suppressive activity of HD1B was reduced upon mTOR knocking down by siRNA transfection. Wild type (HD1A) or <i>lal</i>-/- (HD1B) cells were pretreated with control (C) siRNA or mTOR (mTOR) siRNA. Treated cells were incubated with CFSE labeled wild type CD4⁺ T cells and stimulated with anti-CD3 and anti-CD28 antibodies. Results are mean ± SD from three independent experiments (n = 3), p< 0.05; (C) Secretion of T cell releasing IL-4 in the above co-culture experiment was measured to assess the CD4⁺ T cell function. Results are mean ± SD from three independent experiments (n = 3), p< 0.05.</p

The morphological change of mitochondria in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells.

<p>(A) MitoTracker Green FM staining of live wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. Bar = 25 μm. (B) Cell proliferation of wild type (HD1A) and <i>lal</i>-/- (HD1B) cells <i>in vitro</i>; (C) Western blot analyses of protein expression of Opa1 and phosphorylation at Ser616 of DRP1 in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. (D) Concentrations of glucose, pyruvate and the aconitase activity in HD1A and HD1B cells were measured. Results are means ± SD from three independent experiments (n = 3), *, p< 0.05.</p

Over-activation of the mTOR signal pathway in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells.

<p>(A) Western blot analyses of the phosphorylation level of mTOR downstream effector pS6 in wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. Both cells were treated with solvent (S) or with mTOR inhibitor rapamycin (R) or PP242 (P). (B) Flow cytometry analyses of the ROS levels of rapamycin or PP242 treated or untreated wild type (HD1A) and <i>lal</i>-/- (HD1B) cells. Results are mean ± SD from three independent experiments (n = 3), p< 0.001. (C) Mitochondria membrane potential was analyzed in rapamycin or PP242 treated or untreated wild type (HD1A) and <i>lal</i>-/- (HD1B) cells by JC1 staining. Treatment of mTOR inhibitors restored the mitochondria membrane potential in <i>lal</i>-/- (HD1B) cells. (D) Antioxidant reagent NAC or Tempol treated or untreated wild type (HD1A) and <i>lal</i>-/- (HD1B) cells were stained with JC1 to measure the mitochondria membrane potential. Treatment of antioxidants restored the mitochondria membrane potential in <i>lal</i>-/- (HD1B) cells.</p

Decreases of monomeric low-molecular-weight Ras GTPase superfamily in <i>lal−/−</i> bone marrow MDSCs.

<p>Decreases of monomeric low-molecular-weight Ras GTPase superfamily in <i>lal−/−</i> bone marrow MDSCs.</p