Catalysts for oxygen reduction reaction (ORR) are crucial in fuel cells. Developing metal-free catalyst with high activity at low-cost and high-volume production remains a great challenge. Here, we report a novel type of nitrogen-doped nanoporous carbon nanosheets derived from a conveniently available and accessible plant, Typha orientalis. The nanosheets have high surface area (the highest surface area can be 898 m 2 g À1 ), abundant micropores and high content of nitrogen (highest content of 9.1 at.%). The typical product exhibits an unexpected, surprisingly high ORR activity. In alkaline media, it exhibits similar catalytic activity but superior tolerance to methanol as compared to commercial 20% Pt/C. In acidic media as well, it shows excellent catalytic ability, stability and tolerance to methanol. This low-cost, simple and readily scalable approach provides a straightforward route to synthesize excellent electrocatalysts directly from biomass, which may find broad applications in the fields of supercapacitors, sensors, and gas uptake. Broader contextElectrocatalysts for the oxygen reduction reaction (ORR) are crucial in fuel cells and other electrochemical devices. It is highly challenging but extremely desirable to develop inexpensive metal-free catalysts, with high activity and with the capability of high-volume production of high yields for fuel cells. In this work, we have reported a novel type of nitrogen-doped nanoporous carbon nanosheets, which has been derived for the rst time from a conveniently available and accessible plant Typha orientalis. The materials have a high surface area, abundant micropores and a high content of nitrogen. A typical product exhibits an unexpected, surprisingly high ORR activity. In alkaline media, it exhibits similar catalytic activity but superior tolerance to methanol than commercial 20% Pt/C. The high activities can be ascribed to the large surface area with abundant micropores, the high content of pyridine and pyrrolic-like nitrogen atoms within the materials and the 3D interpenetrated network structure produced by numerous carbon nanosheets. The products were prepared using the plant Typha orientalis as carbon source and without using any organic solvent. The synthesis strategy is simple, low-cost, and can be easily scaled up for production.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T ⅐ A-to-A ⅐ T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.Oligonucleotides can bind to duplex DNA and form triple helices in a sequence-specific manner (2,3,5,12,25,39). Progress in elucidating the third-strand binding code has raised the possibility of developing nucleic acids as sequence-specific reagents for research and possibly clinical applications. Oligonucleotide-mediated triplex formation has been shown to prevent transcription factor binding to promoter sites and to block mRNA synthesis in vitro and in vivo (4,9,11,17,18,21,26,29,33,41). Such inhibition of expression, however, is transient, depending on the sustained presence of the oligonucleotides. It also depends on the stability of the triple helix, which can be disrupted by transcription initiated at nearby sites (37). To overcome these problems, methods to prolong oligonucleotide-duplex interactions using DNA intercalating or cross-linking agents have been explored in experiments to block transcription initiation or elongation (17,18,39,40).Instead of using triplex formation to transiently block gene expression, however, we reasoned that it would be advantageous to use triple helix formation to target m...

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.

Although much is known about the mechanisms of signal-mediated protein and RNA nuclear import and export, little is understood concerning the nuclear import of plasmid DNA. Plasmids between 4.2 and 14.4 kilobases were specifically labeled using a fluoresceinconjugated peptide nucleic acid clamp. The resulting substrates were capable of gene expression and nuclear localization in microinjected cells in the absence of cell division. To elucidate the requirements for plasmid nuclear import, a digitonin-permeabilized cell system was adapted to follow the nuclear localization of plasmids. Nuclear import of labeled plasmid was time-and energydependent, was inhibited by the lectin wheat germ agglutinin, and showed an absolute requirement for cytoplasmic extract. Addition of nuclear extract alone did not support plasmid nuclear import but in combination with cytoplasm stimulated plasmid nuclear localization. Whereas addition of purified importin ␣, importin ␤, and RAN was sufficient to support protein nuclear import, plasmid nuclear import also required the addition of nuclear extract. Finally, nuclear import of plasmid DNA was sequence-specific, requiring a region of the SV40 early promoter and enhancer. Taken together, these results confirm and extend our findings in microinjected cells and support a protein-mediated mechanism for plasmid nuclear import.The nuclear envelope presents an effective barrier between the nuclear and cytoplasmic compartments of the cell. Although it is impermeant to large non-nuclear molecules, a multitude of macromolecules must enter and exit the nucleus across this envelope every second in order for the cell to live. All macromolecular exchange between the nucleus and the cytoplasm studied to date occurs through the nuclear pore complex (NPC), 1 is signal-dependent, and utilizes a series of receptor proteins (for a review see Ref. 1). In the case of proteins destined for the nucleus, the nuclear localization signal (NLS) interacts with one of a growing number of importin family members to target the complex to the NPC. In the classical case of NLS-containing proteins, the protein binds to importin ␣, the NLS "receptor," which in turn interacts with importin ␤. Once at the NPC, the complex interacts with the small GTP-binding protein RAN in its GDP-bound state and its accessory factor NTF2 while being translocated across the NPC. After translocation into the nucleus, the complex disassembles because of the conversion of RAN-GDP to RAN-GTP by exchange or replacement and the importins return to the cytoplasm (2). Similar scenarios of receptor proteins interacting with signals to mediate translocation across the nuclear envelope also occur for the nuclear export of proteins (e.g. exportin and the nuclear export signal) and viral mRNAs (Crm1p, HIV Rev, and the Rev response element), and the nuclear import of small nuclear RNAs that contain both protein-encoded NLSs and the trimethylguanosine cap as import signals (1, 3).We have recently shown that the nuclear import of plasmid DNA (pDNA) also u...