Lignin-Polylactide Reverse Emulsions for Water and UV-Resistant Composite Films

We use lignin’s surface activity to stabilize reverse (water-in-oil) emulsions through interfacial interactions with an organic, continuous phase containing polylactide (PLA). The rheological features of the emulsions are easily tailored to develop biobased coatings on hydrophilic substrates (glass or paper). Significantly, upon drying, the bicomponent coatings are shown for their uniformity and mechanical integrity at lignin loadings as high as 95% (with PLA being the balance). Compared to a commercial sunscreen, the PLA-lignin film templated from the emulsion is notably more effective in the UV-A region. Hence, UV-blocking (lignin) and water resistance (PLA) properties are combined in a system of practical relevance. Meanwhile, the transparency of the film is maintained. This work offers a novel and facile approach to achieving homogeneous lignin-based composite coating, further advancing this biomacromolecule as a substitute for non-renewable counterparts

Effects of miR-29a/b on NPC cell growth and cell cycle.

<p>The results of MTT assays following transfection of pre-miR-29a/b and their inhibitors into S18 cells for the indicated 24 h, 48 h and 72 h post-transfection times. The values are the mean and SD optical density (OD) units. (B) miR-29b increased the proportion of S18 cells at the G1/S transition, whereas miR-29a did not have a similar effect. Cells were treated with pEGFP-miR-29a/b or anti-miR-29a/b transfection and control vector pEGPF-C. Cell cycle distributions were detected 20 h later. A representative result of 3 independent experiments is shown. In all experiments, the negative control was pre-miRNA negative control.</p

Integrated microRNA-gene network.

<p>(A) The miRNA-gene network shows the relationships between 9 miRNAs and 51 dysregulated genes. Only genes reported in BioGrid are shown in this network. Interaction among genes and regulation of miRNAs are indicated by arrows. Red nodes represent up-regulated genes in NPC, and blue nodes represent down-regulated genes. miRNAs with more than 2 targets are shown in the figure. (B) The particular network for miR-29a/b and their targets stems from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120969#pone.0120969.g001" target="_blank">Fig. 1A</a>.</p

Identification of SPARC and COL3A1 as miR-29a/b targets.

<p>(A) Conserved miR-29a/b binding sites in the 3′-UTRs of SPARC and COL3A1 are predicted by TargetScan; candidates were filtered using a hybridization free-energy threshold of −19.0 (kcal/mol).The sequences in green refer to miRNA, and the sequences in red refer to the 3′-UTR of mRNA. (B) A schematic diagram showing the 3′-UTR reporter constructs. The sequences of the wild type or mutant site in the 3′-UTR fragments are shown. MT: Nucleotide substitutions disrupting the miRNA-29a/b-binding sites were introduced in the 3′ UTRs of SPARC and COL3A1 cloned downstream of the Renilla luciferase gene. (C) Luciferase activities were measured 48 h after transfection with plasmids pEGFP-miR-29a/b and their mutants. The activities of Renilla luciferase were normalized to firefly luciferase activities, and the target validation data were confirmed in duplicate experiments. (D) Quantitative RT-PCR of SPARC and COL3A1 expression in S18 cells after transfection of miR-29a/b and their inhibitors for 48 h. *<i>p</i> < 0.05.</p

High SPARC expression correlates with shorter overall survival in NPC patients.

<p>(A) SPARC protein levels correlate with NPC aggressiveness. Representative tissue is stained with an antibody against NPC. Immunohistochemical stains show absent nuclear staining in normal samples (left, original magnification 100×), moderate and strong nuclear staining in NPC samples with low and high risk of metastasis (center right and right, respectively; original magnification 100×). Weak staining shown is a benign nasopharynx adjacent to NPC (center left, original magnification 100×). (B) Tissue analysis of SPARC expression for each tissue spot. The mean SPARC protein expression for the indicated NPC tissues is summarized using error bars with 95% confidence intervals, demonstrating a significantly lower score in NPC with a high risk of metastasis compared with non-malignant controls (Mann-Whitney test, two-tailed, <i>p</i> < 0.001). (C) The OS of patients with low SPARC expression levels was significantly higher than that of patients with high SPARC expression levels (log rank test, <i>p</i> = 0.04).</p

miR-29b is associated with specific risk groups and NPC patient survival.

<p>(A, B) Comparison of the miR-29a/b abundance in paired NPC tumors (42 NPC patients) and adjacent normal tissues (42 normal controls). The solid squares represent the relative expression level of miR-29a/b. The miR-29a/b abundance for each paired non-tumor and tumor tissues were separately shown in the left and right parts and connected by a dash line. (C, D) The expression levels of miR-29a/b in serum were quantified by real-time PCR in 83 patients with highly metastatic/invasive, 110 patients with low metastatic/non-invasive cancer and 65 healthy donors. (C) There was a small change in miR-29a expression in NPC patients and healthy donors, as well as patients with high risk for metastasis and the low-risk group. The formula used to calculate the relative Ct values was (ΔCt = assay Ct − control Ct). A higher ΔCt value indicates that the miRNA is less abundant in a sample. (D) miR-29b was significantly up-regulated in the NPC patients at high-risk for metastasis compared with the low-risk group. (E) Kaplan–Meier survival curves of NPC patients. No significant differences were observed in OS rates between patients with high and low miR-29a expression. (F) The 5-year overall survival rate of NPC patients with high serum miR-29b expression was significantly lower than that of those with low serum miR-29b expression (<i>p</i> < 0.001).</p

miR-29a/b target the SPARC/COL3A1 pathways in NPC cells.

<p>(A, B) S18 cells were transfected with 50 nmol of mimics-NC (control miRNA), mimics-miR-29a/b, anti-scramble (control anti-miRNA) and anti-miR-29a/b. The levels of miR-29a and miR-29b were assessed by qRT-PCR. Cell lysates were prepared for Western blotting with antibodies against SPARC and COL3A1, and GAPDH expression served as a loading control. Western blot figures are representative of at least three independent experiments. The value under each sample indicates the fold change of SPARC and COL3A1 protein levels relative to that of the control. (C) Western blot analysis of the expression level of SPARC and COL3A1 in NPC cells following treatment with vehicle, siRNA-SPARC and siRNA-COL3A1 for 24 h. The value under each sample indicates the fold changes of SPARC and COL3A1 protein levels relative to that of the control. Three independent experiments performed in triplicate. (D) A schematic model shows the function of miR-29a and miR-29b in NPC cell proliferation, migration and cell invasion. In response to miR-29a/b stimuli, the G1/S transition arrest triggers both a classical proliferative inhibition and an adapted metabolic switch. SPARC and COL3A1 protein levels inversely correlate with miR-29a and miR-29b expression in NPC cells, respectively. (i) The indirect effect of miR-29a to increase SPARC expression may be mediated by its unknown targets that affect cell survival, and subsequently SPARC could down-regulate the expression of COL3A1. (ii) COL3A1 is a direct target of miR-29b and affects the expression of the various ECM proteins, resulting in derepression of NPC cell migration and invasion.</p

Effects of miR-29a/b expression on NPC cell migration and invasion.

<p>(A, B) Transwell migration and invasion assays showing the effect of miR-29a/b overexpression or knockdown on the migrated and invasive activity of S18 cells transfected with pEGFP-miR-29 or anti-miR-29. The migrated and invasive NPC cells that grew on the lower surface were stained and counted manually using a microscope (original magnification 50×) at 24 h after reseeding. Representative images are shown in the left panel. The mean number of cells per visual field was determined in four randomly selected visual fields per chamber and the experiments were performed in triplicate (right panel). *<i>p</i> < 0.05.</p