PDZ is necessary to accomplish an efficient nuclear export of L-PRX.

<p>(A) Structures of L-PRX and its PDZ-deleted mutants (DPDZ-L-PRX). Amino acid residues flanking each domain are numbered on top of the diagram. (B) Subcellular localization of EGFP-tagged L-PRX and its PDZ-deleted mutant (DPDZ-L-PRX). RSC96 cells were transfected with the plasmids encoding EGFP-tagged full-length L-PRX and its DPDZ-L-PRX. Scale bar = 25 μm. (C) GFP fusion constructs detected by Western blotting to ensure that the visualized proteins are not free GFP, or GFP fused to a truncated protein.</p

Cytoplasmic localization of a normal nuclear cyclin A1 by fusing with the PDZ domain of L-periaxin.

<p>(A) Schematic representation of cyclin A1 and its chimeric proteins fused with the PDZ domain of L-periaxin. N-terminal hatched boxes indicate EGFP-tagged peptides. The plain numbers on top of the boxes indicate the amino acid residue number of cyclin A1. Italic numbers correspond to the residues flanking the PDZ-domain of L-periaxin. The subcellular localization of each protein, when expressed in RSC96 cells, is indicated on the right. N, nucleus, C, cytoplasm. (B) Fluorescence analysis of RSC96 cells transfected with the plasmids encoding EGFP, EGFP-cyclin A1, and EGFP-PDZ-cyclin A1. Scale bar = 25 μm. (C) GFP fusion proteins are stable in the cell which is shown by Western blotting.</p

Nuclear accumulation of L-periaxin by mutating Leu₈₃ and Leu₈₅ to Gln.

<p>(A) Structures of L-PRX, PDZ-cyclin A1, and their mutants. Gray boxes indicate the amino acid residues replaced with Gln in the PDZ domain. (B) RSC96 cells were transfected with the plasmids encoding EGFP-tagged L-PRX, PDZ-cyclin A1, and their mutants. Scale bar = 25 μm. (C) The cytoplasm and nucleus were separated and determined the distribution of protein in the two fractions by Western blotting. β-actin and Histone served as an internal control for cytosol and nucleus, respectively. (D) Quantitative analysis of the distribution of EGFP fusion proteins in the two fractions by normalized to the internal control level. *P<0.05.</p

Nuclear export of L-periaxin is inhibited by LMB.

<p>(A) RSC96 cells were transfected with the plasmids encoding EGFP-tagged L-PRX and PDZ-cyclin A1. The cells in the second and fourth panels were treated with 37 nmol/L LMB for 1 h after 7 h post-transfection. EGFP-L-PRX and EGFP-PDZ-cyclin A1 localization was observed by Delta Vision. Scale bar = 25 μm. (B) The cytosol and nucleus were separated and determined the distribution of protein in the two fractions by Western blotting. β-actin and Histone served as an internal control for cytosol and nucleus, respectively. (C) Quantitative analysis of the distribution of EGFP fusion proteins in the two fractions by normalized to the internal control level. *P<0.05.</p

Alignment of potential NES sequences of L-periaxin with previously characterized leucine-rich NESs.

<p>NES sequences in mitogen-activated protein kinase kinase (MAPKK), zyxin, HIV.Rev, c-Abl, and LIM-kinase 1 are obtained from previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091953#pone.0091953-Yang1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091953#pone.0091953-Fischer1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091953#pone.0091953-FukudaM1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091953#pone.0091953-NixDA1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091953#pone.0091953-Taagepera1" target="_blank">[32]</a>. Residue numbers are indicated in parentheses. Consensus sequence is shown at the bottom. Ψ indicates hydrophobic residues, which include leucine, isoleucine, valine, phenylalanine, and methionine.</p

Activated Carbons Derived from Hydrothermally Carbonized Sucrose: Remarkable Adsorbents for Adsorptive Desulfurization

Activated carbons derived from hydrothermal carbonization of sucrose and subsequent KOH activation have been prepared and tested for the adsorptive removal of refractory thiophenic compounds. Textural and chemical properties of the carbons and their corresponding impacts on adsorption rates and capacities were discussed in detail. The optimum carbon possessed high adsorption capacity (41.5 mgS/g for 300 ppmwS model oil), fast adsorption rate (97% saturated within 5 min) as well as relatively good selectivity for the adsorption of thiophenic compounds due to the abundant small micropores, suitable mesopore fraction and various oxygen functionalities present in the carbon. Combined with the economic and environmental merits of the preparation procedure, the sucrose-derived activated carbons are promising candidates for potential practical applications

Selective Fluorescence Detection of Cysteine over Homocysteine and Glutathione Based on a Cysteine-Triggered Dual Michael Addition/Retro-aza-aldol Cascade Reaction

In this work, a cysteine (Cys)-triggered dual Michael addition/retro-aza-aldol cascade reaction has been exploited and utilized to construct a fluorescent probe for Cys for the first time. The resulting fluorescent probe 8-alkynylBodipy <b>1</b> contains an activated alkynyl unit as Michael receptor and a Bodipy dye as fluorescence reporter and can highly selectively detect Cys over homocysteine (Hcy)/glutathione (GSH) as well as other amino acids with a significant fluorescence off–on response (∼4500-fold) and an ultralow detection limit (0.38 nM). The high selectivity of <b>1</b> for Cys could be attributed to a kinetically favored five-membered cyclic intermediate produced by the dual Michael addition of Cys with the activated alkynyl unit of <b>1</b>. The big fluorescence off–on response is due to the subsequent retro-aza-aldol reaction of the five-membered cyclic intermediate that results in the release of a highly fluorescent 8-methylBodipy dye <b>2</b>. The probe has been successfully used to detect and image Cys in serum and cells, respectively

Sirtuin Deacetylation Mechanism and Catalytic Role of the Dynamic Cofactor Binding Loop

Sirtuins constitute a novel family of protein deacetylases and play critical roles in epigenetics, cell death, and metabolism. In spite of numerous experimental studies, the key and most complicated stage of its NAD⁺-dependent catalytic mechanism remains to be elusive. Herein, by employing Born–Oppenheimer ab initio quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations, a state-of-the-art computational approach to study enzyme reactions, we have characterized the complete deacetylation mechanism for a sirtuin enzyme, determined its multistep free-energy reaction profile, and elucidated essential catalytic roles of the conserved dynamic cofactor binding loop. These new detailed mechanistic insights could facilitate the design of novel mechanism-based sirtuin modulators

Sulfone-Rhodamines: A New Class of Near-Infrared Fluorescent Dyes for Bioimaging

Given the wavelength dependence of tissue transparency and the requirement for sufficiently low background autofluorescence, the development of fluorescent dyes with excitation and emission maxima beyond 700 nm is highly desired, but it is a challenging task. Herein, a new class of fluorescent dyes, named sulfone-rhodamines (SO₂Rs), was developed on the basis of the one-atom replacement of the rhodamine 10-position O atom by a sulfone group. Such a modification makes their absorption and emission maxima surprisingly reach up to 700–710 and 728–752 nm, respectively, much longer than their O-, C-, and Si-rhodamine analogs, due to the unusual d*−π* conjugation. Among these dyes, <b>SO</b>_<b>2</b><b>R4</b> and <b>SO</b>_<b>2</b><b>R5</b>, bearing disubstituted <i>meso</i>-phenyl groups, show the greatest potentials for bioimaging applications in view of their wide pH range of application, high photostability, and big extinction coefficients and fluorescence quantum yields. They could quickly penetrate cells to give stable NIR fluorescence, even after continuous irradiation by a semiconductor laser, making them suitable candidates for time-lapse and long-term bioimaging applications. Moreover, they could specifically localize in lysosomes independent of alkylmorpholine targeted group, thus avoiding the problematic alkalization effect suffered by most LysoTrackers. Further imaging assays of frozen slices of rat kidney reveal that their tissue imaging depth is suprior to the widely used NIR labeling agent Cy5.5

A Mitochondria-Targetable Fluorescent Probe for Dual-Channel NO Imaging Assisted by Intracellular Cysteine and Glutathione

A mitochondria-specific fluorescent probe for NO (1) was synthesized by the direct conjugation of a pyronin dye with one of the amino groups of o-phenylenediamino (OPD). The probe could selectively detect NO over dehydroascorbic acid (DHA), ascorbic acid (AA), and methylglyoxal (MGO) as well as the reactive oxygen/nitrogen species (ROS/RNS) with the significant off–on response due to the production of a red-emission triazole 2. In the presence of cysteine/glutathione (Cys/GSH), 2 could be further transformed into a green-emission aminopyronin 4 and a red-emission thiopyronin 5, respectively. Assisted by intracellular Cys and GSH, the probe demonstrated its potential to monitor mitochondrial NO in a dual-channel mode