Graphene Platform Used for Electrochemically Discriminating DNA Triplex

Triplex DNA has received great attention as new molecular biology tools and therapeutic agents due to their possible novel functions in biology systems. Therefore, it is important to distinguish triplex from among different forms of DNA, such as single-stranded and double-stranded DNA. In this report, several electrochemical techniques, cyclic voltammetry, electrochemical impedance spectroscopy, different pulse voltammetry, and electrochemiluminescence were used for distinguishing this unique structure among different DNA formations by using functionalized graphene/Nafion–Ru­(bpy)₃²⁺ (bpy = 2, 2′-bipyridine) modified glass carbon electrode. The different interactions between nucleotides and graphene surface and Ru­(bpy)₃²⁺ mediated guanine oxidation produced quite different electrochemical responses. Guanine bases are hidden inside the folded triplex DNAs, which are much less susceptible to be oxidized by Ru­(bpy)₃³⁺ produced on electrodes. Furthermore, the effect of guanine bases stacking in triplex also influences the electrochemical behaviors. By changing the different position and distance of guanine bases in DNA sequences, we found that the conjoint way of several guanines strongly influenced the catalytic electrochemical responses on graphene surface. Our results provide new insight into determination of less stable protonated triplex formation by using graphene-based rapid, low-cost and sensitive electrochemical techniques

Accelerating the Peroxidase-Like Activity of Gold Nanoclusters at Neutral pH for Colorimetric Detection of Heparin and Heparinase Activity

The peroxidase-like catalytic activity of gold nanoclusters (Au-NCs) is quite low around physiological pH, which greatly limits their biological applications. Herein, we found heparin can greatly accelerate the peroxidase-like activity of Au-NCs at neutral pH. The catalytic activity of Au-NCs toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H₂O₂ was 25-fold increased in the presence of heparin at pH 7. The addition of heparin not only accelerated the initial catalytic rate of Au-NCs but also prevented the Au-NCs from catalyst deactivation. This allows the sensitive colorimetric detection of heparin at neutral pH. In the presence of heparinase, heparin was hydrolyzed into small fragments, weakening the enhancement effect of catalytic activity. On the basis of this phenomenon, the colorimetric determination of heparinase in the range from 0.1 to 3 μg·mL^–1 was developed with a detection limit of 0.06 μg·mL^–1. Finally, the detection of heparin and heparinase activity in diluted serum samples was also demonstrated

Transient expression of AaCASPS16 induced apoptosis in C6/36 cells.

<p>Plasmids expressing C-terminally Flag-tagged AaCASPS16-WT, AaCASPS16-C300A and GFP were transfected into C6/36 cells separately. Caspase inhibitor z-VAD-FMK was added at 2 h before transfection of pIE-AaCASPS16, and proteasome inhibitor MG132 was added at 8 h before cells were harvested. Mock treated cells, GFP expressed C6/36 cells, and AaCASPS16-C300A expressed C6/36 cells were used as controls. At 24 h post transfection, cells were subjected to the following analyses: <b>(A)</b> Photographs of cells were taken under microscope (Scale bar indicated 50 μm). <b>(B)</b> Cell lysates were prepared and were incubated with Ac-DEVD-AFC and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. <b>(C)</b> Cell lysates were subjected to immunoblotting analysis using antibody against Flag and β-tubulin. The data in (B) were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, ** <i>P</i> < 0.01. NS: not significant.</p

Expression profile of <i>Aacasps16</i> in developmental and adult stages.

<p>Total RNAs were prepared from 1st to 4th instar larvae, pupae, female and male adults and subjected to qRT-PCR analysis. The vertical axis represents the relative expression of <i>Aacasps16</i> in different developmental stages or different genders. Pupae samples were designated as the standard and set to 1. The data were presented with the SD from three independent experiments. ND: not detected.</p

AaCASPS16 was processed in apoptosis triggered by UV and Act D treatment.

<p>C6/36 cells were treated with Act D (1.0 μg/mL) or UV treatment (200 μJ/cm²) separately and at 24 h post treatment, cells were subjected to the following analyses: <b>(A)</b> Cell pictures were taken under microscope (Scale bar indicated 50 μm). <b>(B)</b> Cell lysates were prepared and incubated with Ac-DEVD-AFC and subjected to caspase activity assay. Caspase activity was indicated as the changes in relative fluorescence units (RFU) per minute. <b>(C)</b> Cell lysates were analyzed by immunoblotting using antibody against AaCASPS16 and β-actin. A short vertical black line was used to indicate where lanes were removed and separate parts of the same Western blot image were joined together. The data in (B) were presented with the SD from three independent experiments, and statistical significance was calculated by <i>t</i> test, **<i>P</i> < 0.01.</p

The sequence of AaCASPS16.

<p>Predicted amino acid sequence of AaCASPS16 was shown in alignment with Strica homologs from <i>Aedes aegypti</i> (AeCASPS15, AeCASPS16, AeCASPS17 and AeCASPS21) and the caspases from <i>Drosophila melanogaster</i> (Dronc, Dredd, Drice, Decay, Dcp-1, Strica and Damm). The amino acid residues identical among 12 caspases are indicated by white letters within black boxes, the amino acid residues identical among 9 caspases are indicated by black letters within dark gray boxes, the amino acid residues identical among 6 caspases are indicated by black letters within medium gray boxes, and the amino acid residues identical among 3 caspases are indicated by black letters within light gray boxes. The alignment was performed using DNAMAN 7.0. Secondary structures were predicted using JPred3. Underline: catalytic center, black arrow: predicted cleavage site.</p

AaCASPS16 underwent autocatalytic cleavage when expressed from <i>E</i>. <i>coli</i>.

<p>(A) C-terminally His-tagged AaCASPS16-WT (lane 3) and the putative catalytic site mutant C300A (lane 4) were expressed from <i>E</i>. <i>coli</i> and detected by immunoblotting using an antibody against the His-tag following SDS-PAGE. The migration of full-length AaCASPS16 and the cleaved subunits were indicated. BL21 only (lane 1) and the uninduced WT preparations (lane 2) were used as control. A short vertical black line was used to indicate where lanes were removed and separate parts of the same Western blot image were joined together. (B) The schematic cartoon showing the molecular masses of the His-tagged full length AaCASPS16 with potential cleavage sites and the cleaved subunits.</p

Phosphorylation sites prediction in the prodomain of AaCASPS16.

<p>Phosphorylation sites in the AaCASPS16 prodomain were predicted at the GPS web server using the default parameters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157846#pone.0157846.ref025" target="_blank">25</a>]. (A) The numbers and positions of the potential phosphorylation sites were indicated. (B) Distribution of different potential kinase groups was calculated.</p

Phylogenetic analyses of AaCASPS16 with selected insect caspases.

<p>The predicted amino acid sequence of AaCASPS16 was aligned with 17 selected insect caspases, and a phylogenetic tree was constructed in MEGA 5.0 using the neighbor-joining method. AaCASPS16 was indicated by black dot. Accession numbers in GenBank and VectorBase of sequences are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157846#pone.0157846.s003" target="_blank">S1 Table</a>.</p