One-Step Synthesis of Gasoline Octane Booster and Diesel Fuel from Glycerol and <i>tert</i>-Butyl Alcohol

One-step synthesis of an octane booster for gasoline and a diesel fuel was performed using glycerol and <i>tert</i>-butyl alcohol (TBA) as raw materials and the solid acid Amberlyst-70 (A-70) as a catalyst. In contrast to the conventional etherification of glycerol with TBA operated at a low temperature resulting in a homogeneous product mixture, our study revealed that the same process at elevated reaction temperatures would automatically separate the products into an organic phase and an aqueous phase. The components of the former consisted of unconverted TBA, isobutylene (IB), 2,2,4-trimethyl-1-pentene, 2,4,4-trimethyl-2-pentene (DIB), 2,2,4,6-pentamethyl-3-heptene, 2,2,6,6-tetramethyl-4-methyleneheptane (TIB), and oxygen-limited hydrocarbons (HCs), which are constituents that are different from the product distribution of the conventional reaction. The latter was composed of water, unconverted TBA, and glycerol. After a simple laboratory distillation of the organic phase product mixture, the distillate obtained by a simple distillation maintained at 225 °C in the still could be used as an octane booster for gasoline, while the residue could be adopted as diesel fuel

Induction of Thoracic Aortic Remodeling by Endothelial-Specific Deletion of MicroRNA-21 in Mice

<div><p>MicroRNAs (miRs) are known to have an important role in modulating vascular biology. MiR21 was found to be involved in the pathogenesis of proliferative vascular disease. The role of miR21 in endothelial cells (ECs) has well studied in vitro, but the study in vivo remains to be elucidated. In this study, miR21 endothelial-specific knockout mice were generated by Cre/LoxP system. Compared with wild-type mice, the miR21 deletion in ECs resulted in structural and functional remodeling of aorta significantly, such as diastolic pressure dropping, maximal tension depression, endothelium-dependent relaxation impairment, an increase of opening angles and wall-thickness/inner diameter ratio, and compliance decrease, in the miR21 endothelial-specific knockout mice. Furthermore, the miR21 deletion in ECs induced down-regulation of collagen I, collagen III and elastin mRNA and proteins, as well as up-regulation of Smad7 and down-regulation of Smad2/5 in the aorta of miR21 endothelial-specific knockout mice. CTGF and downstream MMP/TIMP changes were also identified to mediate vascular remodeling. The results showed that miR21 is identified as a critical molecule to modulate vascular remodeling, which will help to understand the role of miR21 in vascular biology and the pathogenesis of vascular diseases.</p> </div

List of primers used for genotyping and real-time PCR.

<p>List of primers used for genotyping and real-time PCR.</p

Generation of the miR21 endothelial-specific knockout mice.

<p>(<b>A</b>) Diagram for miR21 exon1-floxed targeting construct and location of primers used for genotyping of targeted locus and FLP, Cre-mediated excised targeted locus. The exon1 of miR21 and 3.2 kb, 5 kb genomic fragments flanking exon1 (left and right arm) were PCR-amplified and subcloned into a targeting vector such that exon1 of miR21 was followed by a neo cassette, and exon1 was flanked by two loxP sites, neo was flanked by two FRT sites. This targeting construct was used to generate miR21 flox/flox mice, which were crossed with FLP mice to excised neo mice, crossed with Tek-Cre mice to produce an endothelial-specific miR21 knockout. Sequences of primers are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059002#pone-0059002-t001" target="_blank">Table 1</a>. (<b>B</b>) Analysis of the targeting construction for the miR21 locus. Tail DNA was genotyped by PCR using P1/P2, P3/P4. The 3.2 kb and 5 kb band amplified by P1/P2, P3/P4 primer pairs indicated presence of the unexcised targeted alleles. (<b>C</b>) Analysis of the expression of FLP and Cre recombinases. PCR conditions were described in the Jackson Laboratories genotyping protocol. PCR primers are also showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059002#pone-0059002-t001" target="_blank">Table 1</a>. (<b>D</b>) Merging of the DAPI and Tek-Cre immunoreactivity in the thoracic aorta of miR21 flox/flox, Tek-Cre mice. ECs were clearly stained. Bar = 25 µm. (<b>E</b>) The cF1 and cR1 primer pair identified the targeted miR21 flox/flox allele with a 925 bp product; while they amplified the wild-type allele into a 713 bp band. The cF1 and cR1 primer pair amplified the Cre-excised miR21 flox/flox locus into a 427 bp product. Thus, PCR reaction using cF1 and cR1 primer pair distinguished among miR21 flox/flox, Tek-Cre, miR21 flox/+, Tek-Cre, and miR21 flox/flox, wild type, respectively. All three PCR products (1, 2, 3) were verified by sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059002#pone.0059002.s001" target="_blank">Figure S1</a>).</p

Genes , primer sequences for quantitative RT-PCR analysis.

<p>Genes , primer sequences for quantitative RT-PCR analysis.</p

Identification of the miR21 deletion in ECs.

<p>(<b>A</b>) Aortic ECs were isolated from the miR21 endothelial-specific KO mice and cultured in growth medium. In situ hybridization(ISH) was performed to determine the expression of miR21 (Green). Results in miR21 flox/flox, Tek-Cre group (miR21 endothelial-specific KO group) showed that there was not miR21 in EC of knockout mice. The DAPI-stained nuclei are blue. Bar = 100 µm. (<b>B</b>) ECs were immunostained with Cre, vWF antibody, indicating that these ECs expressed Cre recombinase. The nuclei were stained by DAPI (Blue). Bar = 100 µm. (<b>C</b>) Analysis of excision of miR21 on DNA level in ECs and the whole aorta. It showed that miR21-floxed alleles were almost excised in ECs. (<b>D</b>) Real-time PCR was performed to determine the level of miR21 from each group described in A. The miR21 expression level of endothelial-specific KO group was significantly decreased. (<b>E</b>) Semi-quantitative RT-PCR analysis showed by mixture of miR21 and U6 primers, the expression of miR21 in the miR21 endothelial-specific KO group was significantly lower than that in the control. These two PCR products were verified by sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059002#pone.0059002.s002" target="_blank">Figure S2</a>). Values shown are mean±<i>SD.</i></p

The altered aortic function of the miR21 endothelial-specific KO mice.

<p>(<b>A</b>) The response of maximal tension of aortic strips to 10⁻⁶M NE reduced significantly in the miR21 endothelial-specific deletion mice. <i>n</i> = 10, *<i>p</i><0.05. (<b>B</b>) The amplitude of ACh-induced endothelium-dependent relaxations was significantly altered in aortic strips from the miR21 KO group compared with the control, when expressed in percent of NE-induced contraction. <i>n</i> = 6 for each group, *<i>p</i><0.05. (<b>C</b>) In both the miR21 KO mice and the control, ACh-induced relaxations were abolished upon eNOS inhibition with L-NAME. <i>n</i> = 6 for each group. (<b>D</b>) The endothelium-dependent relaxations were significantly lowered by 40–50% in aortic strips from the miR21 KO mice. <i>n</i> = 6, *<i>p</i><0.05. (<b>E</b>) There was no significant difference in the endothelium-independent relaxations to SNP between the miR21 KO mice and the control. <i>n</i> = 6 for each group. Values shown are mean±<i>SD</i>.</p

Morphomety of the thoracic aorta of miR21 flox/flox and miR21 flox/flox, Tek-Cre mice.

<p>Values are mean <i>(SD)</i>, <i>n</i> = 5 for each group, * <i>p</i><0.05, miR21 flox/flox vs. miR21 flox/flox, Tek-Cre by Student's unpaired <i>t</i> test.</p

Aortic morphometry, micro-structure and contents of elastin and collagen, aortic stiffness and pressure (P)-volume (V) relationship.

<p>(A) Representative Weigert stained aortic sections showing elastin changes between the knockout group and the control group. Bar = 25 µm. (B) Representative Van Gieson stained aortic sections showing collagen changes between genotypes. Bar = 25 µm. (C) The medial area fraction of elastin content was quantitated, and significantly decreased in the miR21 endothelial-specific KO mice compared with control mice. <i>n</i> = 5 for each group, *<i>p</i><0.05. (D) There were no significant changes in collagen content observed in the area percent. <i>n</i> = 5 for each group. (E) Elastin contents was evaluated by a Fastin elastin assay and found decreased in the KO group. <i>n</i> = 4 for each group, *<i>p</i><0.05. (F) Collagen contents in thoracic aorta was evaluated by a Sircol collagen assay and found reduced in the KO group. <i>n</i> = 4 for each group, *<i>p</i><0.05. (G) Aortic stiffness was approximated by calculating the ratio of area percentage between collagen (D) to elastin (C), which showed an elevated trend. *<i>p</i><0.05. (H) P-V relation of aorta between the control and KO group. P-V relation curve of control is shifted downward to the right curve of the KO group, which showed decreased volume and increased pressure by decreasing compliance. <i>n</i> = 6 for each group. Values shown are mean±<i>SD</i>.</p

In vivo blood pressure and change of the opening angle of the arteries.

<p>(<b>A</b>) There were no difference in systolic pressure between the knockout group and the control group, but diastolic pressure was significantly reduced in the miR21 endothelial-specific KO mice. <i>n</i> = 6 for each group, *<i>p</i><0.01. (<b>B</b>) No difference in heart rate was observed between the miR21 endothelial-specific KO group and control group. (<b>C</b>) Mean blood pressure was also decreased significantly in these two groups. <i>n</i> = 6 for each group, *<i>p</i><0.05. (<b>D</b>) The longitudinal variation of the opening angle for four segments above diaphragm. The opening angle of aortic rings was significantly different. (<b>E</b>) The mean values of this four segments between the miR21 endothelial-specific KO group and control group were also significant different. <i>n</i> = 6 for each group, *<i>p</i><0.05. Values shown are mean±<i>SD</i>.</p