Surface Wetting-Driven Separation of Surfactant-Stabilized Water–Oil Emulsions

Four fluorocarbon polymers including polytetrafluoroethylene and polyvinylidene fluoride were coated on a stainless steel felt to separate emulsified water droplets from ultralow sulfur diesel (ULSD) fuels. The original fuel treated with clay to remove additives was additized again with four known surfactants including pentaerythrityoleate, (octadecadienoic acid) dipolymer, (octadecadienoic acid) tripolymer, and monoolein individually. The different surfactants adsorbed on the fuel–water interface reduce the interfacial intension with different intensities. The separation efficiency at various surfactant concentrations was used to evaluate the coalescence effect exerted by these coatings. It was found the separation was both surfactant- and coating-dependent. A fluoro-polyurethane coating (FC1) stood out to counteract the adverse effect of all the surfactants. Solid free energy was then measured using acid–base and Kaelble–Uy adhesion theories for all the coatings, but its correlation with coalescence was not found at all. Coating aging in surfactant-additized fuel on the coating’s water wettability was also examined to better understand how historical wetting affects separation. A tumbled model for fluorocarbons was identified that well-explained the continuous decline of the water contact angle on the FC1 coating in fuel. Subject to the challenge of the foreign environment, the fluoroalkyl chains of the polymer tilt to expose the carbonyl groups underneath, resulting in favored coalescence separation in the presence of surfactants

MicroRNA Modulation Induced by AICA Ribonucleotide in J1 Mouse ES Cells

<div><p>ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs) are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells). It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells). AICA ribonucleotide (AICAR) is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for AICAR in ES cells pluripotency maintenance and give insight for its usage in iPS cells generation.</p></div

Down-regulation of miR-134 is partly responsible for increased expression of pluripotency markers.

<p>(A) MiR-134 expression vector pCDH-mir134 and its negative control were transfected into J1 ES cells, and the expression of miR-134 was detected by real-time PCR. (B) The luciferase reporters psi-Nanog or psi-Sox2 were co-transfected with miRNA expression vectors pCDH-mir134, and empty vector pCDH-GFP without insertion was used as control. 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (C) Real-time PCR validation of Nanog, Oct4, Sox2, and Klf4 in J1 ES cells in the presence or absence of 1 mM AICAR for 24 h using the comparative Ct method. Gapdh was used to normalize template levels. (D) Western blot analysis of pluripotency markers Nanog, Oct4, Sox2 and Klf4 in J1 ES cells in the presence of 1,000 U/ml LIF and with or without 1 mM AICAR for 24 h. Cell lysates were extracted and analyzed by western blot. Relative expression levels were comparing to Gapdh. (E) MiR-134 expression vector pCDH-mir134 and its negative control were transfected into J1 ES cells. 48 h after transfection, Nanog, Sox2, Oct4, and Klf4 expression was detected by real-time PCR. Gapdh was used to normalize template levels. (F) MiR-134 expression vector pCDH-mir134 and its negative control were transfected into J1 ES cells. 12 h after transfection, AICAR or DMSO was added to the cell culture medium, and Nanog and Sox2 expression was detected by real-time PCR. (G) MiR-134 expression vector pCDH-mir134 and its negative control were transfected into J1 ES cells. 12 h after transfection, AICAR or DMSO was added to the cell culture medium, and alkaline phosphatase staining of J1 cells were performed 24 hours later. (WB: western blot; *: p<0.05; **: p<0.01).</p

Overview of sRNA high-throughput sequencing data.

<p>(A) A flowchart that shows stepwise data analysis process of small RNA reads from Solexa Hiseq sequencing. (B) Comparison of the known miRNA expression between DMSO- and AICAR-treated samples; the scatter plot shows differentially expressed miRNAs in the two samples. (C) Comparison of the predicted novel miRNA expression between DMSO- and AICAR-treated samples; the scatter plot shows differentially expressed miRNAs in the two samples.</p

AICAR modulates the expression of multiple miRNAs associated with ES cell pluripotency.

<p>(A) Representative miRNAs that were down-regulated in the presence of 1 mM AICAR; this finding was validated by real-time PCR. J1 ES cells were cultured in medium with serum and 1,000 U/mL LIF in the presence or absence of 1 mM AICAR for 24 h. Mature miRNAs were transcribed, and the relative expression level of miRNAs was determined by real-time PCR. Data are presented as the mean ± SD of three independent experiments. (B) Representative miRNAs that were up-regulated in the presence of 1 mM AICAR were validated by real-time PCR. (C) Real-time PCR validation of miR-290 cluster after AICAR treatment. (D) Real-time PCR detection of the targets of miR-290 cluster in the presence of 1 mM AICAR. (E) The expression of miR-138-5p and its target Trp53 was validated by real-time PCR. (F) The expression of miR-23a-3p and its targets was validated by real-time PCR. (G) The expression of miR-129-1 and miR-129-2 and their target Sox4 was validated by real-time PCR. (H) The expression of miR-434-3p and its target Ccnl1 was validated by real-time PCR. (*: p<0.05; **: p<0.01).</p

Several miRNAs targeted Myc and down-regulated its expression.

<p>(A) The expression of Myc and the predicted miRNAs was validated by real-time PCR in the presence of 1 mM AICAR. (B) Western blot analysis of Myc expression in J1 ES cells in the presence of 1,000 U/ml LIF and with or without 1 mM AICAR for 24 h. Cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (C/D/E/F/G) MiR-34a/34b/34c/340/135b expression vector pCDH-mir34a/34b/34c/340/135b and their negative control were transfected into J1 ES cells, and miR-34a/34b/34c/340/135b expression was detected by real-time PCR. (H) The schematic representation for construction of the firefly luciferase reporter vector. Myc 3′-UTR was inserted into psiCHECK-2 vector by XbaI and EcoRI sites. (I) Brief description of predicted miRNAs and their target sites on Myc 3′-UTR. (J) The luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 and 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (K) Brief description of mutated sites on Myc 3′-UTR. (L) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 24 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (M) The mutated luciferase reporters were co-transfected with miRNA expression vectors, and empty vector pCDH-GFP without insertion was used as control. 48 h after transfection, luciferase activity was detected using dual-luciferase reporter assay. (N) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, Myc expression was detected by real-time PCR. Gapdh was used to normalize template levels. (O) The indicated miRNA expression vectors were transfected into J1 ES cells. 48 h after transfection, cell lysates were extracted and analyzed by western blot. Relative expression level were comparing to Gapdh. (WB: western blot; *: p<0.05; **: p<0.01).</p