NHEJ efficiency declines with age in R26NHEJ mice.

<p>(<b>A</b>) Astrocytes and fibroblasts from heart, kidney, lung, and skin were isolated from 5 young and 5 old mice. After 2 passages, cells were transfected with 5 µg I-SceI for DSB induction and 0.025 µg DsRed to normalize the transfection efficiency. Three days later, cells were analyzed by FACS and NHEJ efficiency was calculated as the ratio of GFP⁺/DsRed⁺ cells. For each treatment, 20,000 cells were counted. At least 4 transfections were performed on cells from each mouse and the average NHEJ efficiency from young and old mice was plotted for the individual cell types analyzed. Error bars show s.e.m. (*<i>p</i><0.05, **<i>p</i><0.005, <i>t</i>-test). The data for individual mice is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004511#pgen.1004511.s002" target="_blank">Figure S2</a>. (<b>B</b>) Transcription from ROSA26 locus containing the knocked-in NHEJ reporter does not change with age. Total RNA was extracted from the cells of young and old R26NHEJ mice. qRT-PCR was performed using primers homologous to the first exon of GFP and actin primers as the internal control. The experiment was repeated three times and error bars indicate s.d. (<b>C</b>) There is no significant difference in the I-SceI expression between cells from young and old mice. Total proteins were extracted from young and old fibroblasts and astrocytes and the I-SceI levels were analyzed by Western blot with antibodies to the HA-tag. β-actin was used as a loading control. The experiment was repeated three times and a representative image is shown.</p

Analysis of deletions and insertions at NHEJ junctions in cells from young and old mice.

<p>A total of 300 independent junctions, 30 young and 30 old, for each cell type, were analyzed. The complete list of junction sequences is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004511#pgen.1004511.s004" target="_blank">Table S1</a>. (<b>A</b>) Average deletion size decreases with age in heart fibroblasts and increases in lung and skin fibroblasts. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test). (<b>B</b>) Large deletions are more frequently found in old lung and skin fibroblasts and in young heart fibroblasts. The graph shows percentage of NHEJ clones containing deletions larger than 500 bp. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test). (<b>C</b>) Average size of insertions increases in astrocytes and decreases in kidney and lung fibroblasts. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test). (<b>D</b>) The frequency of insertions decreases with age in kidney and lung fibroblasts. Asterisk indicates significant difference between young and old mice (<i>p</i><0.05, <i>t</i>-test).</p

Generation of R26NHEJ knock-in mouse model.

<p>(<b>A</b>) The pROSA26PA-NHEJ vector containing NHEJ reporter construct targeted to the ROSA26 genomic sequence. The construct consists of GFP exons separated by the Pem1 intron, interrupted by the killer exon Ad2. Flanking Ad2 are unique I-SceI recognition sites for inducing DSB. Successful NHEJ repair leads to the reconstitution of GFP. Two <i>loxP</i> sites in direct orientation flanking Neomycin/Kanamycin genes (Neo/Kana) and Bacterial origin of Replication (OriC) are located downstream. This vector was targeted by homologous recombination into the endogenous ROSA26 locus of C57BL/6 mouse ES cells. (<b>B</b>) NHEJ construct integrated into ROSA26 locus in the mouse genome. (<b>C</b>) DNA from G418-resistant ES cell clones was digested with <i>Bam</i>HI and hybridized to a GFP probe (indicated in B). (<b>D</b>) Founder mice were genotyped using PCR primers indicated in (B). The positive control lanes contain PCR reactions with genomic DNA from ES cell clones shown in (C) as a template. Negative control lane contains PCR reactions with DNA from a wild-type C57BL/6 mouse as template and is followed by a No template control.</p

Incorporating α‑Al₂O₃ Nanodots into Expanded Graphite Anodes toward Stable Fast Charging for Lithium-Ion Batteries

High-power lithium-ion batteries place high demands on the fast charging ability of electrode materials, while for the current graphite anode, it suffers from anisotropic and sluggish Li+ transport due to its small interlayer spacing. In addition, the large polarization at low lithiation potential at a high rate leads to Li+ deposition and side reactions of Li with the electrolyte. In this work, α-Al2O3 nanodots incorporated into aggregates of thin-layer graphite have been developed by facile high-energy ball milling of graphite and layer-structured pseudo-boehmite. By optimization, the ball-milled graphite/Al2O3 (BG/Al2O3) manifests a high reversible capacity of 344 mAh g–1 higher than the 98.7 mAh g–1 of graphite after 500 cycles at 1 A g–1 (∼2.7C) and 200 mAh g–1 higher than the 59.6 mAh g–1 of raw graphite at 3 A g–1 after 500 cycles. The wrinkled edges and expanded interlayer spacing generated by high-energy ball milling optimize the Li+ transport and accelerate reaction kinetics, contributing high pseudocapacitance and enabling fast charging ability. The α-Al2O3 nanodots can decrease the side reactions between the electrolyte and graphite electrode, contributing high cyclic stability. This study lays a foundation for the one-step mechanical force chemistry method to prepare highly stable fast-charging graphite anode materials for lithium-ion batteries

Damage-Free Removal of Residual Carbon in a Dielectric Barrier Discharge (DBD) Plasma for Carbothermal-Synthesized Materials

In this work, we demonstrate damage-free removal of residual carbon in a dielectric barrier discharge (DBD) plasma for carbothermal-synthesized materials, such as CaAlSiN₃:Eu²⁺ phosphors, SnSb alloy anode materials, and TiN ceramic powders. The efficiency of residual carbon removal and the damaging effects of the plasma for treated materials are investigated in detail, with carbothermal-synthesized CaAlSiN₃:Eu²⁺ phosphors being used as an example. Results show that the residual carbon in carbothermal-synthesized CaAlSiN₃:Eu²⁺ phosphors could be removed effectively within a DBD plasma generator, resulting in the significant improvement of luminescent properties. The damage-free character of this DBD plasma decarburization process to phosphors is revealed, showing amazing superiority over the traditional high-temperature decarburization route. These results offer an attractive strategy for the removal of residual carbon for various carbothermal-synthesized materials with finely controlled compositions

Novel g‑C₃N₄/CoO Nanocomposites with Significantly Enhanced Visible-Light Photocatalytic Activity for H₂ Evolution

Novel g-C₃N₄/CoO nanocomposite application for photocatalytic H₂ evolution were designed and fabricated for the first time in this work. The structure and morphology of g-C₃N₄/CoO were investigated by a wide range of characterization methods. The obtained g-C₃N₄/CoO composites exhibited more-efficient utilization of solar energy than pure g-C₃N₄ did, resulting in higher photocatalytic activity for H₂ evolution. The optimum photoactivity in H₂ evolution under visible-light irradiation for g-C₃N₄/CoO composites with a CoO mass content of 0.5 wt % (651.3 μmol h^–1 g^–1) was up to 3 times as high as that of pure g-C₃N₄ (220.16 μmol h^–1 g^–1). The remarkably increased photocatalytic performance of g-C₃N₄/CoO composites was mainly attributed to the synergistic effect of the junction or interface formed between g-C₃N₄ and CoO

Nitrogen-Deficient Graphitic Carbon Nitride with Enhanced Performance for Lithium Ion Battery Anodes

Graphitic carbon nitride (g-C₃N₄) behaving as a layered feature with graphite was indexed as a high-content nitrogen-doping carbon material, attracting increasing attention for application in energy storage devices. However, poor conductivity and resulting serious irreversible capacity loss were pronounced for g-C₃N₄ material due to its high nitrogen content. In this work, magnesiothermic denitriding technology is demonstrated to reduce the nitrogen content of g-C₃N₄ (especially graphitic nitrogen) for enhanced lithium storage properties as lithium ion battery anodes. The obtained nitrogen-deficient g-C₃N₄ (ND-g-C₃N₄) exhibits a thinner and more porous structure composed of an abundance of relatively low nitrogen doping wrinkled graphene nanosheets. A highly reversible lithium storage capacity of 2753 mAh/g was obtained after the 300th cycle with an enhanced cycling stability and rate capability. The presented nitrogen-deficient g-C₃N₄ with outstanding electrochemical performances may unambiguously promote the application of g-C₃N₄ materials in energy-storage devices

Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

<div><p>Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the <i>MDR1</i> gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR. </p> </div

Generation of K562/ADM cells.

<p>(A) K562/ADM cells were established after exposure of K562 cells to ADM. MDR1 expression in parental and drug-treated K562 cells was normalized to the GAPDH housekeeping gene. (B) FACS analysis of K562 and K562/ADM cells stained with P-gp antibody. (C) MTT assay shows survival rate of K562 and K562/ADM cells under VBL treatment. The ratio of OD₄₉₀ of VBL-treated cells <i>versus</i> control cells was used as the measure of survival rate. At least three independent experiments were performed and error bars represent standard deviation (S.D.). (D) FACS analysis of Rh123 accumulation in K562 and K562/ADM cells.</p

Expression of primary transcripts in the miR cluster on chromosome 14q32.31.

<p>(A) Schematic representation of known EST sites and selected miRs shown in UCSC genome browser (Human Feb. 2009 (GRCh37/hg19)) at chromosome region 14q32.31. EST primer sets A to E are indicated in triangles below each EST. Transcription start sites sourced from FANTOM CAGE tags are shown as arrows on the top of the 14q32.31 region. (B) Expression of selected miRs (miR-494, miR-495, miR-376c, miR-381 and miR-655) in K562 and K562/ADM cells was validated by stem-loop real-time PCR. The Ct values of these miRs were normalized to that of miR-425 whose level of expression was similar in the two cell lines. The relative expression of miR-494, miR-495, miR-376c, miR-381, miR-655 and miR-16 in K562/ADM cells was calculated. The results are shown as the mean ± SD of three independent experiments. (C) PCR products were resolved on a 2% agarose gel. (D) Expression of the primary transcripts in both K562 and K562/ADM was determined by RT-PCR using primers designed against ESTs A to E. The data were normalized to GAPDH. The results are shown as the mean ± SD of three independent experiments. Quantification of the change of expression level in K562/ADM cells relative to K562 cells is shown on the bottom. (E) Expression of three human RNA nuclease genes, Drosha, DGCR8 and Dicer, in both K562 and K562/ADM cells, was determined by real-time PCR. The expression values were normalized to GAPDH, and are shown as the mean ± SD of three independent experiments.</p