Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.     

Chromosome targeting at short polypurine sites by cationic triplex-forming oligonucleotides

Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.     

Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 x 10(-)5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.     

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.     

Induction of DNA strand breaks by trihalomethanes in primary human lung epithelial cells

Trihalomethanes (THMs) are disinfection by-products and suspected human carcinogens present in chlorinated drinking water. Previous studies have shown that many THMs induce sister chromatid exchanges and DNA strand breaks in human peripheral blood lymphocytes in vitro. Exposure to THMs occurs through oral, dermal, or inhalation routes, with the lung being a target of exposure by the latter route, although not a target for rodent carcinogenicity. Thus, to examine the genotoxicity of THMs in this tissue, we used the comet assay to examine the DNA damaging ability of five THMs in primary human lung epithelial cells. Cells were collected by scraping the large airways of four volunteers with a cytology brush and then passaging the cells no more than three times in order to have sufficient numbers for the experiments. Cells were exposed for 3h to 10, 100, or 1000 microM CHCl(3), CHCl(2)Br, CHClBr(2), or CHBr(3); CH(2)Cl(2) was also used as a model dihalomethane for comparison to the THMs. The compounds ranked as follows for DNA damaging ability: CHCl(2)Br>CHBr(3)>CHCl(3) approximately equal CH(2)Cl(2); CHClBr(2) was negative. Considerable inter-individual variation was observed. For example, CHCl(3) was genotoxic in only two subjects, and the interaction between dose and donor was highly significant (P<0.001). The same variation was observed for CHCl(2)Br, which was positive only in the two subjects in which CHCl(3) was negative. This variation was not due to the GSTT1-1 genotype of the subjects. Although two subjects were GSTT1-1(+), and two were GSTT1-1(-), no cultured cells with a GSTT1-1(+) genotype had detectable GSTT1-1 enzymatic activity nor did any frozen epithelial cells that had not been cultured. However, GSTT1-1 enzymatic activity was detected in fresh (neither frozen nor cultured) lung cells. These results show that freezing or culturing causes lung cells to lose GSTT1-1 activity and that factors other than GSTT1-1 activity play a role in the variable responses of these human cells to the genotoxicity of the halomethanes studied here.     

Biodecolourization of azo and triphenylmethane dyes by <Emphasis Type="Italic">Dichomitus squalens</Emphasis> and <Emphasis Type="Italic">Phlebia</Emphasis> spp

Nine white-rot fungal strains were screened for biodecolourization of brilliant green, cresol red, crystal violet, congo red and orange II. Dichomitus squalens, Phlebia fascicularia and P. floridensis decolourized all of the dyes on solid agar medium and possessed better decolourization ability than Phanerochaete chrysosporium when tested in nitrogen-limited broth medium. Journal of Industrial Microbiology & Biotechnology (2002) 28, 201–203 DOI: 10.1038/sj/jim/7000222 Received 12 July 2001/ Accepted in revised form 22 October 2001     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

Purification and characterization of a novel UV lesion-specific DNA glycosylase/AP lyase from Bacillus sphaericus

The purification and characterization of a pyrimidine dimer-specific glycosylase/AP lyase from Bacillus sphaericus (Bsp-pdg) are reported. Bsp-pdg is highly specific for DNA containing the cis-syn cyclobutane pyrimidine dimer, displaying no detectable activity on oligonucleotides with trans-syn I, trans-syn II, (6-4), or Dewar photoproducts. Like other glycosylase/AP lyases that sequentially cleave the N--glycosyl bond of the 5' pyrimidine of a cyclobutane pyrimidine dimer, and the phosphodiester backbone, this enzyme appears to utilize a primary amine as the attacking nucleophile. The formation of a covalent enzyme-DNA imino intermediate is evidenced by the ability to trap this protein-DNA complex by reduction with sodium borohydride. Also consistent with its AP lyase activity, Bsp-pdg was shown to incise an AP site-containing oligonucleotide, yielding beta- and delta-elimination products. N-terminal amino acid sequence analysis of this 26 kDa protein revealed little amino acid homology to any previously reported protein. This is the first report of a glycosylase/AP lyase enzyme from Bacillus sphaericus that is specific for cis-syn pyrimidine dimers.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.