Comparison of nanoparticle generation by two plasma techniques: Dielectric barrier discharge and spark discharge

<p>Dielectric barrier discharge (DBD) and spark discharge, two versatile atmospheric pressure plasma-based techniques, have been employed to generate nanoparticles. This study compares the characteristics of metal nanoparticles generated by a DBD reactor and a spark discharge generator with argon as the working gas. The gas temperature in the discharge region of the DBD reactor remained near room temperature, while that of the spark reactor varied from 470 to 1120 K and generally increased with increasing applied voltage amplitude in the range of 2–10 kV and driving frequency in the range of 1–10 kHz. Comparing to spark-generated nanoparticles under the same voltage, frequency, and flow rate, DBD-generated nanoparticles have smaller sizes, better monodispersity, and lower number concentrations. The number concentration of DBD-generated particles decreases significantly under high working voltage and frequency, while the number concentration of spark-generated particles increases with increasing working voltage. Under continuous operations over several hours, the DBD reactor has better temporal stability in generating nanoparticles than the spark generator.</p> <p>© 2017 American Association for Aerosol Research</p

Zwitterion-Immobilized Imprinted Polymers for Promoting the Crystallization of Proteins

Zwitterion additives have been used in protein crystallization to prevent the appearance of crystal clusters. Herein, we have developed a novel approach for the immobilization of zwitterion onto molecularly imprinted polymers (MIPs) to yield high-quality single protein crystals. For lysozyme, trypsin, catalase, proteinase K, concanavalin A-type IV, and thaumatin, simply adding the selected zwitterion (3-(methacryloylamino)­propyl)-dimethyl­(3-sulfopropyl) ammonium hydroxide) into the free solution, the crystallization was improved. When further using the zwitterion-immobilized molecularly imprinted polymers (ziMIPs) developed in the current study, the formation of higher quality crystals was facilitated in a shorter time compared with regular MIPs and traditional crystallization trials. Most notably, concanavalin A-type IV, which has nonunique ordered assembly, gave only the form IV structure with higher resolution in the presence of ziMIPs, justifying the superior function of ziMIPs for the ordered assembly of protein molecules. Thus, the ziMIPs could be widely used in protein crystallization

Structure and biochemical characterizations of (−)-ABA.

<p>Structures of (+)-ABA (<b>A</b>) and <b>(−)</b>-ABA (<b>B</b>). (<b>C</b>) <b>(−)</b>-ABA mediated inhibition of HAB1 phosphatase activity by PYLs. The concentration for each PYLs protein was 5.0 µM and for HAB1 was 3.0 µM. All experiments were repeated three times (n = 3) and error bars represented s.d. The condition measuring the phosphatase activity of HAB1 was same below unless noted.</p

The stereospecificity of PYLs to (+)-ABA or (−)-ABA.

<p>(<b>A</b>) The different inhibitory efficiency of <b>(−)</b>-ABA by PYL3, PYL5 or PYL9. The concentrations of PYLs, HAB1 and <b>(−)</b>-ABA were 1 µM, 0.5 µM and 10 µM, respectively. (<b>B</b>) Superposition of apo-PYL5, PYL3-<b>(−)</b>-ABA and PYL9-(+)-ABA indicated that the major variant residues underlain the favour of PYL binding <b>(−)</b>-ABA. Two bulk side chains of I112 and L165 in PYL9 seriously collided to 7′ and 8′ methyl groups of <b>(−)</b>-ABA, respectively. The stereo constraints were vanished in PYL5 because of two corresponding small side chains (also seen Fig.S1). (<b>C</b>). The mutation V66I in PYL9 would give a strong coordination with 8′ and 9′ methyl groups in <b>(−)</b>-ABA through a strong hydrophobic network with surrounding residue V85. (<b>D</b>) PYL9 and PYL3 mutants were engineered to gain and cripple the binding of <b>(−)</b>-ABA, respectively.</p

Structural characterizations and bioactivity of PYL9-(+)-ABA.

<p>(<b>A</b>) Two protomers of PYL9-(+)-ABA in each asymmetric unit. 2<i>F_o</i>−<i>F_c</i> electron density map of (+)-ABA at 1.0σ. (<b>B</b>) One protomer of PYL9-(+)-ABA with five cysteine residues (green) and a disulphide formed between C34 and C159. (<b>C</b>) The monomeric state of PYL9 in solution was confirmed by the sedimentation velocity. The sample purity for sedimentation velocity experiments was detected by SDS-PAGE and then Coomassie Brilliant Blue staining in the subplot. (<b>D</b>) The C159 was the key residue in disulphide bond, while the C29 competed with the C34 to form the disulphide bond. PYL9 and its mutants were under different oxidation-reduction conditions for SDS-PAGE. 1% βme or 1% βme +100 mM DTT was used as reducing agents to disrupt the disulphide bond. Only C159S mutant was not affected by the reducing agents (black arrow).</p

Data collection and refinement statistics of PYLs and complexes.^*

†<p>Statistics for highest resolution shell. ^*Three crystal experiments for each structure.</p>2<p>Residues in favored, generously allowed and disallowed regions of the Ramachandran plot.</p

Characterizations of PYL5 and PYL3-(−)-ABA.

<p>(A). Chain A together with L2 of chain B and L4 of chain C displayed a whole protomer of apo-PYL5 (also seen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067477#pone.0067477.s004" target="_blank">Fig. S4C</a>). (<b>B</b>) Four PYL3-<b>(−)</b>-ABA molecules in an asymmetric unit, organized as two trans-dimers. (<b>C</b>) Close-up view of <b>(−)</b>-ABA (yellow) in the binding pocket. 2<i>F_o</i> − <i>F_c</i> electron density map of <b>(−)</b>-ABA at 1.0σ. (<b>D</b>) Superposition of PYL3-<b>(−)</b>-ABA and PYL3-(+)-ABA (PDB: 4DSC). There were partially rotation and shift between the rings in both ABA. <b>(−)</b>-ABA was constrained in a hydrophobic cavity with little flexibility in PYL3. (<b>E</b>) The binding affinity of <b>(−)</b>-ABA to PYL3, assessed by ITC assays, was less than that of (+)-ABA or (±)-ABA, (also seen <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067477#pone.0067477.s005" target="_blank">Fig. S5D,E</a>). (<b>F</b>) PP2C activity (upper panel) and GST-mediated pulldown of PYL3 mutants protein in the presence of <b>(−)</b>-ABA (lower panel). GST-HAB1 and PYL3, highlighted by red arrows, were visualized by Coomassie Brilliant Blue staining.</p