Laboratory Investigation of Skid Resistance for Steel Slag Utilization as Chip Seal

Slag as waste material of steel-making process has similar characteristics with aggregate that has been widely used in pavement construction. The use of slag as chip seal aggregate to provide skid resistance needs to be analyzed. In this laboratory study, the chip seal samples are made using steel slag and natural aggregate. The bonding materials used are asphalt and epoxy resin. Skid resistance tests for all chip seal samples and also hot rolled sheet pavement without chip seal application are performed using the Portable British Pendulum Tester. The results show the variations of chip seal aggregate weight are inconsistent. The natural aggregate used as chip seal material could produce high skid resistance value of 10.3% higher than that using steel slag. Also the skid resistance of chip seal with the ALD 3 mm are not significantly different with that of ALD 6 mm. Similar results occur on the skid resistance of chip seals using epoxy resin and asphalt

Effect of 3 weeks of nanovector delivery (i.v).

<p>(A) <b>miR-181c expression in the Heart and in the Heart-derived Mitochondrial Fraction.</b> qPCR shows that miR-181c expression in the whole heart homogenate is almost 1.5 times higher in the plasmid miR-181c group compared to its sham group. We isolated mitochondrial miRNA enriched total RNA, using our published protocol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096820#pone.0096820-Ambros1" target="_blank">[5]</a>. We also monitored the quality and integrity of the isolated RNAs, as described previously<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096820#pone.0096820-Ambros1" target="_blank">[5]</a>. qPCR shows that miR-181c expression is almost 2.5 times higher in the miR-181c-treated group compared to its sham group. We used the mitochondrial gene product, 12S rRNA, as a normalization control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096820#pone.0096820-Ambros1" target="_blank">[5]</a>. *p<0.05 vs. sham (n = 6 for whole heart, and n = 4 for the mitochondrial fraction). (B) <b>Heart Weight.</b> We measured the wet weight of the whole heart and measured the corresponding tibial length of each rat. The ratio of heart weight to tibial length is not different among the groups, suggesting no sign of hypertrophy in the miR-181c-treated rats. (n = 12).</p

Primer Sequences for PCR and Sequencing.

<p>a) miR-181c.</p

Mitochondrial function.

<p>Effect of 3 weeks of nanovector treatment on isolated mitochondria. (A) O₂ consumption was measured after adding the substrate for complex IV, TMPD/ascorbate. Overexpression of miR-181c markedly increases respiration compared to the sham, after adding TMPD/Ascorbate. These data show the role of miR-181c on mitochondrial energy metabolism by altering complex IV. *P<0.05 vs sham, n = 6. (B) Rate of ROS generation from heart-mitochondria overexpressing miR-181c. Without any substrate the rate of ROS production is not different between the two treatment groups. But, using glutamate/malate (complex I) and succinate (complex II), ROS production is significantly higher in the miR-181c overexpression groups. *p<0.05 vs. sham, n = 4.</p

Echocardiography.

<p>(A) 2D M-mode and Doppler echocardiography was performed on non-anesthetized rats, before (top) and after (lower) the sham (left) or the miR-181c expression vector (right) treatment. (B) Percent fractional shortening, and (C) ejection fraction, were calculated using the software of the echocardiography instrument (n = 10).</p

Physical Properties of the rats during the Treatment Protocols.

<p>(A) <b>Blood Pressure.</b> We used a non-invasive, tail cuff method, to measure diastolic blood pressure (DBP) and systolic blood pressure (SBP). We did not observe any significant difference between the two groups, either in DBP or SBP. The red color indicates the plasmid miR-181c treated group, and the blue color indicates the sham group. n = 3. (B) <b>Body weight</b>. Rats were weighed during the treatment period, and we found that the weight gain of miR-181c-treated rat group is significantly lower than the sham group, up to day 11. This is not a toxic reaction, but it is due to the high activity of these animals. These animals are not diabetic (dipstick and blood glucose tests were performed). But after day 13, miR-181c-treated animals become lethargic. The red bar indicates the plasmid miR-181c treated group, and the blue bar indicates the sham group. n = 12.(C) <b>Forced Swimming Test.</b> miR-181c overexpression shows a significant difference in exercise capacity compared to the sham group after the 18th day of treatment. Together with the echocardiography data we optimized day 20 for assessment.</p

Effect of miR-181c on Mitochondrial Function.

<p>Heart-derived mitochondria were isolated from the two groups of rats after 3 weeks of treatment. (A) TMRE (tetramethylrhodamine methyl ester) fluorescence was measured from mitochondria supplied with glutamate/malate from two different treatment groups for 25 min, followed by the addition of the uncoupler, CCCP (Carbonyl cyanide m-chlorophenyl hydrazone). The miR-181c treatment group showed a higher level of TMRE intensity before the CCCP addition. n = 3. (B) Isolated mitochondria were incubated with Calcium Green 5N and fluorescence intensity was monitored after adding Ca²⁺. Following a Ca²⁺ pulse, fluorescence increases initially, but reverts back towards baseline as mitochondria take up Ca²⁺. When mitochondria take up enough Ca²⁺, the mPTP opens. This results in Ca²⁺ release and a decrease in fluorescence. (↑) indicates addition of 10 µM Ca²⁺. n = 3. (C) Western blot shows that miR-181c overexpression significantly increases the protein content of the mitochondrial calcium uniporter (MCU) both from total heart homogenate and mitochondrial fraction. VDAC was used as a loading control. The data were normalized to the sham group. *p<0.05 vs. sham (n = 4). (D) pyruvate dehydrogenase (PDH) and (E) α-ketoglutarate dehydrogenase (αKGDH) activities were significantly higher in the isolated mitochondria from the miR-181c treated hearts compared to sham, suggesting that the mitochondrial matrix Ca²⁺ concentration is increased in the miR-181c treated animals.</p

miR-181c Regulates the Mitochondrial Genome, Bioenergetics, and Propensity for Heart Failure <i>In Vivo</i>

<div><p>MicroRNAs (miRNAs) are small non-coding RNAs, which inhibit the stability and/or translation of a mRNA. miRNAs have been found to play a powerful role in various cardiovascular diseases. Recently, we have demonstrated that a microRNA (miR-181c) can be encoded in the nucleus, processed to the mature form in the cytosol, translocated into the mitochondria, and ultimately can regulate mitochondrial gene expression. However the <i>in vivo</i> impact of miR-181c is unknown. Here we report an <i>in-vivo</i> method for administration of miR-181c in rats, which leads to reduced exercise capacity and signs of heart failure, by targeting the 3′-end of mt-COX1 (cytochrome c oxidase subunit 1). We cloned miR-181c and packaged it in lipid-based nanoparticles for systemic delivery. The plasmid DNA complexed nanovector shows no apparent toxicity. We find that the mRNA levels of mitochondrial complex IV genes in the heart, but not any other mitochondrial genes, are significantly altered with miR-181c overexpression, suggesting selective mitochondrial complex IV remodeling due to miR-181c targeting mt-COX1. Isolated heart mitochondrial studies showed significantly altered O₂-consumption, ROS production, matrix calcium, and mitochondrial membrane potential in miR-181c-treated animals. For the first time, this study shows that miRNA delivered to the heart <i>in-vivo</i> can lead to cardiac dysfunction by regulating mitochondrial genes.</p></div

Histology of miR-181a1/b1^-/- (a/b KO) and control (WT).

<p><b>A</b>, Top row shows H&E staining, followed by Masson-Trichrome staining and Movat’s staining. 20X magnification was used to take the pictures. <b>B</b>, Quantification of collagen staining for all layers and vascular media between miR-181a1/b1^-/- (a/b KO) and control (WT) (n = 5–6). Whole vascular ring were used for analysis. Values are mean ±SEM, *p<0.05, **p<0.01, ***p<0.001.</p

TGF-β1 and Mut TGF- β1 3'-UTR Primers.

<p>TGF-β1 and Mut TGF- β1 3'-UTR Primers.</p