Label-free emission assay of mercuric ions using DNA duplexes of poly(dT)

In this study, an assay to quantify the presence of mercuric ions and methyl mercury by double-stranded DNA containing a poly(dT) sequence was developed using a light switch compound, Ru(phen)(2)(dppz)(2+) (1), which is known to intercalate into double-stranded DNA. Upon treatment with mercuric ions, the metal-to-ligand charge transfer (MLCT) emission derived from the intercalation of 1 was reduced due to the formation of DNA duplexes containing T-Hg(2+)-T base pairs by the dehybridization of poly(dT)-poly(dA) duplexes at room temperature. As the concentration of Hg(2+) was increased, the emission of 1 gradually decreased. This label-free method had a detection limit of 5 nM. Other metal ions, such as K(+), Ag(+), Ca(2+), Mg(2+), Zn(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Cd(2+), Cr(3+), Fe(3+), had no significant effect on reducing emission. This emission method can differentiate matched and mismatched poly(dT) sequences based on the dehybridization rate of dsDNA and the rate decreased in the order of T(10)C·A(11)～ T(10)A·A(11) > T(10)G·A(11) > T(11)·A(11).     

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.     

TEMPO-Assisted Free Radical-Initiated Peptide Sequencing Mass Spectrometry (FRIPS MS) in Q-TOF and Orbitrap Mass Spectrometers: Single-Step Peptide Backbone Dissociations in Positive Ion Mode

The present study demonstrates that one-step peptide backbone fragmentations can be achieved using the TEMPO [2-(2,2,6,6-tetramethyl piperidine-1-oxyl)]-assisted free radical-initiated peptide sequencing (FRIPS) mass spectrometry in a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer and a Q-Exactive Orbitrap instrument in positive ion mode, in contrast to two-step peptide fragmentation in an ion-trap mass spectrometer (reference Anal. Chem. 85, 7044–7051 (30)). In the hybrid Q-TOF and Q-Exactive instruments, higher collisional energies can be applied to the target peptides, compared with the low collisional energies applied by the ion-trap instrument. The higher energy deposition and the additional multiple collisions in the collision cell in both instruments appear to result in one-step peptide backbone dissociations in positive ion mode. This new finding clearly demonstrates that the TEMPO-assisted FRIPS approach is a very useful tool in peptide mass spectrometry research.

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Platinum-free tungsten carbides as an efficient counter electrode for dye sensitized solar cells

Mesoporous tungsten carbides displayed an excellent solar conversion efficiency (7.01%) as a counter electrode for dye sensitized solar cells under 100 mW cm(-2), AM 1.5G illumination, which corresponded to ca. 85% of the efficiency of the conventional platinum electrode.     

2,3,7‐Triazabicyclo[3.3.0]octenes Prepared by Tandem Cascade Reaction of Allyl Azides and Olefinic Dipolarophiles

Tandem cascade reactions of allylazides and olefinic dipolarophiles to give cis‐fused 2,3,7‐triazabicyclo [3.3.0]octenes ( 5, 6 or 7 ) are reported. Therein, an intermolecular dipolar cycloaddition of azide and alkene gave a triazoline which was followed by isomerization of the triazoline to a diazoester ( 4 ) and then an intramolecular dipolar cycloaddition from the diazo functional group and the double bond in 4 to give 5 . Compound 5 may further more undergo a Michael addition to give 7‐substituted‐ 2,3,7‐ triazabicyclo [3.3.0]oct‐2‐ene ( 6 ) or a tautomerization to give 2,3,7‐triazabicyclo[3.3.0]oct‐3‐ene ( 7 ). The reaction may be manipulated to stop at a particular stage by adopting a suit able solvent or an appropriate temperature.     

Novel crystalline supramolecular assemblies of amorphous polypyrrole nanoparticles through surfactant templating

A lamellar-structured crystalline polypyrrole (PPy) supramolecular assembly was prepared by surfactant templating, and the regularly linked amorphous PPy nanoparticles with tunable window sizes could play the role of crystalline lattices in the supramolecular assembly.     

A cationic cysteine-hydrazide as an enrichment tool for the mass spectrometric characterization of bacterial free oligosaccharides

In Campylobacterales and related ε-proteobacteria with N-linked glycosylation (NLG) pathways, free oligosaccharides (fOS) are released into the periplasmic space from lipid-linked precursors by the bacterial oligosaccharyltransferase (PglB). This hydrolysis results in the same molecular structure as the oligosaccharide that is transferred to a protein to be glycosylated. This allowed for the general elucidation of the fOS-branched structures and monosaccharides from a number of species using standard enrichment and mass spectrometry methods. To aid characterization of fOS, hydrazide chemistry has often been used for chemical modification of the reducing part of oligosaccharides resulting in better selectivity and sensitivity in mass spectrometry; however, the removal of the unreacted reagents used for the modification often causes the loss of the sample. Here, we develop a more robust method for fOS purification and characterize glycostructures using complementary tandem mass spectrometry (MS/MS) analysis. A cationic cysteine hydrazide derivative was synthesized to selectively isolate fOS from periplasmic fractions of bacteria. The cysteine hydrazide nicotinamide (Cyhn) probe possesses both thiol and cationic moieties. The former enables reversible conjugation to a thiol-activated solid support, while the latter improves the ionization signal during MS analysis. This enrichment was validated on the well-studied Campylobacter jejuni by identifying fOS from the periplasmic extracts. Using complementary MS/MS analysis, we approximated data of a known structure of the fOS from Campylobacter concisus. This versatile enrichment technique allows for the exploration of a diversity of protein glycosylation pathways.     

Monolayer Co3O4 Inverse Opals as Multifunctional Sensors for Volatile Organic Compounds

Monolayers of periodic porous Co₃O₄ inverse opal (IO) thin films for gas‐sensor applications were prepared by transferring cobalt‐solution‐dipped polystyrene (PS) monolayers onto sensor substrates and subsequent removal of the PS template by heat treatment. Monolayer Co₃O₄ IO thin films having periodic pores (d≈500 nm) showed a high response of 112.9 to 5 ppm C₂H₅OH at 200 °C with low cross‐responses to other interfering gases. Moreover, the selective detection of xylene and methyl benzenes (xylene+toluene) could be achieved simply by tuning the sensor temperature to 250 and 275 °C, respectively, so that multiple gases can be detected with a single chemiresistor. Unprecedentedly high ethanol response and temperature‐modulated control of selectivity with respect to ethanol, xylene, and methyl benzenes were attributed to the highly chemiresistive IO nanoarchitecture and to the tuned catalytic promotion of different gas‐sensing reactions, respectively. These well‐ordered porous nanostructures could have potential in the field of high‐performance gas sensors based on p‐type oxide semiconductors.     

Tracking bacterial infection of macrophages using a novel red-emission pH sensor

The relationship between bacteria and host phagocytic cells is key to the induction of immunity. To visualize and monitor bacterial infection, we developed a novel bacterial membrane permeable pH sensor for the noninvasive monitoring of bacterial entry into murine macrophages. The pH sensor was constructed using 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) as an electron-withdrawing group and aniline as an electron-donating group. A piperazine moiety was used as the pH-sensitive group. Because of the strong electron-donating and -withdrawing units conjugated in the sensing moiety M, the fluorophore emitted in the red spectral window, away from the autofluorescence regions of the bacteria. Following the engulfment of sensor-labeled bacteria by macrophages and their subsequent merger with host lysosomes, the resulting low-pH environment enhances the fluorescence intensity of the pH sensors inside the bacteria. Time-lapse analysis of the fluorescent intensity suggested significant heterogeneity of bacterial uptake among macrophages. In addition, qRT-PCR analysis of the bacterial 16 S rRNA gene expression within single macrophage cells suggested that the 16 S rRNA of the bacteria was still intact 120 min after they had been engulfed by macrophages. A toxicity assay showed that the pH sensor has no cytotoxicity towards either E. coli or murine macrophages. The sensor shows good repeatability, a long lifetime, and a fast response to pH changes, and can be used for a variety of bacteria.