Structural integrity and characteristics at lattice and nanometre levels of ZrN polycrystalline irradiated by 4 MeV Au ions

We report an as-hot-pressed zirconium nitride polycrystalline with its primary crystal structure maintained no change but lattice defects and features were introduced at nanometre-scale after being irradiated by 4 MeV Au 2+ with a total fluence of 5 × 10 16 /cm 2 . The variation of grey-level seen in backscattered electron images and electron backscattered diffraction maps directly evidenced the structure integrity of the polycrystalline ZrN is well maintained with no crystal structure change of ZrN. The irradiation depth had no relevance to crystal orientation, and Au deposition peaked at a depth of ∼0.58 μm with a near-Gaussian distribution. Within a depth < 0.58 μm, long dislocation lines were developed with a Burgers vector of [01¯] b /2 and density 3.2 × 10 14 1/m 2 ; beyond this depth, dislocation loops were formed with much higher density. In the ionization zone, cubic ZrO 2 crystallites precipitated in a size of ∼5 nm. The irradiation damage processes are discussed based on the observed features

Bioluminescence Imaging of DNA Synthetic Phase of Cell Cycle in Living Animals

<div><p>Bioluminescence reporter proteins have been widely used in the development of tools for monitoring biological events in living cells. Currently, some assays like flow cytometry analysis are available for studying DNA synthetic phase (S-phase) targeted anti-cancer drug activity <em>in vitro</em>; however, techniques for imaging of <em>in vivo</em> models remain limited. Cyclin A2 is known to promote S-phase entry in mammals. Its expression levels are low during G1-phase, but they increase at the onset of S-phase. Cyclin A2 is degraded during prometaphase by ubiquitin-dependent, proteasome-mediated proteolysis. In this study, we have developed a cyclin A2-luciferase (CYCA-Luc) fusion protein targeted for ubiquitin-proteasome dependent degradation, and have evaluated its utility in screening S-phase targeted anti-cancer drugs. Similar to endogenous cyclin A2, CYCA-Luc accumulates during S-phase and is degraded during G2/M-phase. Using Cdc20 siRNA we have demonstrated that Cdc20 can mediate CYCA-Luc degradation. Moreover, using noninvasive bioluminescent imaging, we demonstrated accumulation of CYCA-Luc in response to 10-hydroxycamptothecin (HCPT), an S-phase targeted anti-cancer drug, in human tumor cells <em>in vivo</em> and <em>in vitro</em>. Our results indicate that a CYCA-Luc fusion reporter system can be used to monitor S-phase of cell cycle, and evaluate pharmacological activity of anti-cancer drug HCPT in real time <em>in vitro</em> and <em>in vivo</em>, and is likely to provide an important tool for screening such drugs.</p> </div

CYCA-Luc accumulates in response to S-phase-specific drug.

<p>(<b>A</b>) Proportion of cells accumulated in S-phase following S-phase-specific drug (HCPT) treatment or M-phase-specific drug (PTX) treatment. U2OS-CYCA-Luc cells were analyzed for DNA content by FACS after propidium iodide staining, or were lysed, after treatment with PBS (20 h), HCPT (1 µg/mL; 48 h), or PTX (50 nM; 20 h). (<b>B, C</b>) Cell extracts were analyzed by immunoblotting (<b>B</b>) or assayed for luciferase activity (<b>C</b>). (<b>D</b>) After treatment with HCPT (0, 0.01, 0.1, 1 and 10 µg/mL) for 48 h, HeLa-CYCA-Luc cells were lysed, and cell lysates were assayed for luciferase activity. For normalization of CYCA-Luc activity or Luc, the signal (1 µg protein) for untreated cells was set to 1. This experiment was repeated three times (n = 3); error bars indicate standard error; *, p<0.05 compared with PBS.</p

CYCA-Luc accumulates in response to S-phase blockage, and is degraded via the ubiquitin-proteasome pathway in HeLa-CYCA-Luc cells.

<p>(<b>A</b>) After treatment with 5 µg/mL HCPT for 48 h, HeLa-CYCA-Luc cells were analyzed for DNA content by FACS after propidium iodide staining or were lysed. (<b>B, C</b>) Cell extracts were analyzed by immunoblotting (<b>B</b>) or assayed for luciferase activity (<b>C</b>). After treatment with 12 µmol MG132 for 15 h, HeLa-CYCA-Luc cells were lysed, and cell lysates were analyzed by immunoblotting (<b>D</b>) or assayed for luciferase activity (<b>E</b>). For normalization of luciferase or CYCA-Luc activity, the signal for untreated cells was set to 1. This experiment was repeated three times (n = 3). Error bars indicate standard error; *, p<0.05 compared with PBS.</p

CYCA-Luc mimics cyclin A2 with respect to regulation by the cell cycle.

<p>(<b>A</b>) U2OS-CYCA-Luc cells were arrested in S-phase by growth in 0.2 mM mimosine. At 0, 3, 6, 9, and 12 h after removal of mimosine, cells were analyzed for DNA content by FACS after propidium iodide staining, or were lysed. (<b>B, C</b>) Cell extracts were analyzed by immunoblotting (<b>B</b>) or assayed for luciferase activity (<b>C</b>). (<b>D, E</b>) Immunoblot analysis of U2OS-CYCA-Luc cells (<b>D</b>), and luciferase activity in U2OS-CYCA-Luc cells or U2OS-Luc cells (<b>E</b>) transfected with Cdc20 or scrambled (negative control) siRNA. For normalization of luciferase or CYCA-Luc activity, the signal for untreated cells was set to 1. This experiment was repeated three times (n = 3). Error bars indicate standard error; *, p<0.05 compared with PBS.</p

Luciferase activity or immunoblot analysis in U2OS cells following short-term transfection of the wild-type luciferase (Luc) and CYCA-Luc.

<p>(<b>A</b>) Schematic diagrams of cyclinA2-luciferase constructs. The recombinant plasmid pGL3-CYCA-Luc encoding CYCA-Luc fusion protein contained the cyclin A2 gene fused in-frame at the N termini of the luciferase gene. (<b>B, C</b>) U2OS cells were placed into wells of a 6 well plate and transfected using the Lipofectamine 2000 with equal concentrations of plasmid DNA encoding either wild-type luc or CYCA-Luc chimera; pcDNA3.1 plasmid was used as a negative control. Transfected cells were cultured for 48 h, lysed, and cell extracts were assayed for luciferase activity (<b>B</b>), or were immunoblotted with the indicated antibodies (<b>C</b>). For normalization of CYCA-Luc activity or Luc, the signal (1 µg protein) for control was set to 1. This experiment was repeated three times (n = 3); error bars indicate standard error; *, p<0.05 compared with control.</p

Monitoring S-phase-specific drug <i>in vivo</i> using bioluminescent imaging.

<p>(<b>A, C</b>) 1×10⁷ HeLa-CYCA-Luc cells or HeLa-Luc cells in 0.2 mL PBS were injected subcutaneously into each flank of BALB/C nude mice under anesthesia (isoflurane). (<b>A</b>) Left, Bioluminescent images were obtained 3 weeks later, when tumors of comparable size (∼5 mm) had formed bilaterally. Right, repeat images were obtained 48 h after intraperitoneal injection of HCPT (30 mg/kg). (<b>B</b>) Normalized fold induction of CYCA-Luc in mice treated with the indicated doses of HCPT (four mice per treatment group). (<b>C</b>) Normalized fold induction of Luc in mice treated with the indicated doses of HCPT (four mice per treatment group) and imaged as in <b>A</b>. (<b>B, C</b>) Fold induction was calculated as CYCA-Luc/Luc_{post-treatment} ÷ CYCA-Luc/Luc_pretreatment. Error bars indicate standard error; *, p<0.05 compared with PBS. (<b>D</b>) Representative immunohistochemical images of cyclin A2 and p27. Tumor sections were obtained 48 h after initial injection of 30 mg/kg HCPT (n = 4 per group). The representative images were taken at an original magnification of 200×.</p

Induction of CYCA-Luc by S-phase-specific drug <i>in vitro</i>.

<p>(<b>A</b>) HeLa-CYCA-Luc cells were serially diluted, placed into wells of a 96 well plate, and immediately imaged using the IVIS Lumina imaging system to obtain FLUX measurements (<b>left, images</b>). These data were averaged (n = 3) and used to generate a plot comparing total flux to cell number (<b>right, graph</b>). (<b>B, C</b>) HeLa-CYCA-Luc cells were placed into wells of a 96 well plate. Images were obtained after 48 h treatment with HCPT (0, 0.01, 0.1, 1, and 10 µg/mL) (<b>B</b>), or 20 h treatment with PTX (0, 5, 10, 50 and 100 nM) (<b>C</b>). Left, cellular images obtained after treatment with HCPT or PTX. Right, normalized fold induction of CYCA-Luc or Luc treated with the indicated doses of drugs. For normalization of luciferase or CYCA-Luc activity, the signal for untreated cells was set to 1. Quantitative data represent the mean ± standard error (n = 3 per group).</p

Fabrication of Ultrasensitive Field-Effect Transistor DNA Biosensors by a Directional Transfer Technique Based on CVD-Grown Graphene

Most graphene field-effect transistor (G-FET) biosensors are fabricated through a routine process, in which graphene is transferred onto a Si/SiO₂ substrate and then devices are subsequently produced by micromanufacture processes. However, such a fabrication approach can introduce contamination onto the graphene surface during the lithographic process, resulting in interference for the subsequent biosensing. In this work, we have developed a novel directional transfer technique to fabricate G-FET biosensors based on chemical-vapor-deposition- (CVD-) grown single-layer graphene (SLG) and applied this biosensor for the sensitive detection of DNA. A FET device with six individual array sensors was first fabricated, and SLG obtained by the CVD-growth method was transferred onto the sensor surface in a directional manner. Afterward, peptide nucleic acid (PNA) was covalently immobilized on the graphene surface, and DNA detection was realized by applying specific target DNA to the PNA-functionalized G-FET biosensor. The developed G-FET biosensor was able to detect target DNA at concentrations as low as 10 fM, which is 1 order of magnitude lower than those reported in a previous work. In addition, the biosensor was capable of distinguishing the complementary DNA from one-base-mismatched DNA and noncomplementary DNA. The directional transfer technique for the fabrication of G-FET biosensors is simple, and the as-constructed G-FET DNA biosensor shows ultrasensitivity and high specificity, indicating its potential application in disease diagnostics as a point-of-care tool

Minutes of the 122nd Meeting of the SPSC, Tuesday and Wednesday, 21-22 June, 2016

BUSCO assessment results of the transcriptome assembly. (TIFF 58 kb