Nanoparticles and thin films of silver from complexes of derivatives of N-(diisopropylthiophosphoryl) thioureas

The derivatives of JV-(diisopropylthiophosphoryl)thiourea RC(S)NHP(S)(OiPr)2 (R = C5H11N, C5H6N2 or C10H7NH 2) followed by their complexation with silver are reported. All complexes are decomposed in hot hexadecylamine (HDA) to give HDA-capped silver nanoparticles. The absorption spectra of the HDA-capped silver nanoparticles exhibit surface plasmon resonance (SPR) absorption in the 400-420 nm region. Transmission electron microscopy (TEM) images of all particles are close to spherical in shape; with sizes ranging from 17 to 20 nm. The X-ray diffraction (XRD) patterns of the silver nanoparticles obtained from all three complexes could be indexed to face centered cubic silver. Scanning electron microscopy (SEM) image confirmed the spherical shape of the particles. The silver complex of 1-naphthylamine was also used to deposit thin films of silver by the aerosol-assisted chemical vapor deposition (AACVD). © 2009 American Chemical Society

Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets

<p>Abstract</p> <p>Background:</p> <p><it>Mycobacterium tuberculosis </it>continues to be a major pathogen in the third world, killing almost 2 million people a year by the most recent estimates. Even in industrialized countries, the emergence of multi-drug resistant (MDR) strains of tuberculosis hails the need to develop additional medications for treatment. Many of the drugs used for treatment of tuberculosis target metabolic enzymes. Genome-scale models can be used for analysis, discovery, and as hypothesis generating tools, which will hopefully assist the rational drug development process. These models need to be able to assimilate data from large datasets and analyze them.</p> <p>Results:</p> <p>We completed a bottom up reconstruction of the metabolic network of <it>Mycobacterium tuberculosis </it>H37Rv. This functional <it>in silico </it>bacterium, <it>iNJ</it>661, contains 661 genes and 939 reactions and can produce many of the complex compounds characteristic to tuberculosis, such as mycolic acids and mycocerosates. We grew this bacterium <it>in silico </it>on various media, analyzed the model in the context of multiple high-throughput data sets, and finally we analyzed the network in an 'unbiased' manner by calculating the Hard Coupled Reaction (HCR) sets, groups of reactions that are forced to operate in unison due to mass conservation and connectivity constraints.</p> <p>Conclusion:</p> <p>Although we observed growth rates comparable to experimental observations (doubling times ranging from about 12 to 24 hours) in different media, comparisons of gene essentiality with experimental data were less encouraging (generally about 55%). The reasons for the often conflicting results were multi-fold, including gene expression variability under different conditions and lack of complete biological knowledge. Some of the inconsistencies between <it>in vitro </it>and <it>in silico </it>or <it>in vivo </it>and <it>in silico </it>results highlight specific loci that are worth further experimental investigations. Finally, by considering the HCR sets in the context of known drug targets for tuberculosis treatment we proposed new alternative, but equivalent drug targets.</p

Genome-Scale Identification Method Applied to Find Cryptic Aminoglycoside Resistance Genes in Pseudomonas aeruginosa

BACKGROUND:The ability of bacteria to rapidly evolve resistance to antibiotics is a critical public health problem. Resistance leads to increased disease severity and death rates, as well as imposes pressure towards the discovery and development of new antibiotic therapies. Improving understanding of the evolution and genetic basis of resistance is a fundamental goal in the field of microbiology. RESULTS:We have applied a new genomic method, Scalar Analysis of Library Enrichments (SCALEs), to identify genomic regions that, given increased copy number, may lead to aminoglycoside resistance in Pseudomonas aeruginosa at the genome scale. We report the result of selections on highly representative genomic libraries for three different aminoglycoside antibiotics (amikacin, gentamicin, and tobramycin). At the genome-scale, we show significant (p<0.05) overlap in genes identified for each aminoglycoside evaluated. Among the genomic segments identified, we confirmed increased resistance associated with an increased copy number of several genomic regions, including the ORF of PA5471, recently implicated in MexXY efflux pump related aminoglycoside resistance, PA4943-PA4946 (encoding a probable GTP-binding protein, a predicted host factor I protein, a delta 2-isopentenylpyrophosphate transferase, and DNA mismatch repair protein mutL), PA0960-PA0963 (encoding hypothetical proteins, a probable cold shock protein, a probable DNA-binding stress protein, and aspartyl-tRNA synthetase), a segment of PA4967 (encoding a topoisomerase IV subunit B), as well as a chimeric clone containing two inserts including the ORFs PA0547 and PA2326 (encoding a probable transcriptional regulator and a probable hypothetical protein, respectively). CONCLUSIONS:The studies reported here demonstrate the application of new a genomic method, SCALEs, which can be used to improve understanding of the evolution of antibiotic resistance in P. aeruginosa. In our demonstration studies, we identified a significant number of genomic regions that increased resistance to multiple aminoglycosides. We identified genetic regions that include open reading frames that encode for products from many functional categories, including genes related to O-antigen synthesis, DNA repair, and transcriptional and translational processes

Mycobacterial dihydrofolate reductase inhibitors identified using chemogenomic methods and in vitro validation.

The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit

Structural Insights into the Quinolone Resistance Mechanism of Mycobacterium tuberculosis DNA Gyrase

Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA

Identification of a small molecule with activity against drug-resistant and persistent tuberculosis

A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-beta-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents

Use of evaporative cooling to improve ' Rosemarie' and ' Forelle' pear fruit blush colour and quality

The original publication is available at http://www.actahort.org/books/671/671_12.htmIn Pyrus communis ‘Rosemarie’, a potentially lucrative blushed pear cultivar, poor fruit colour has been ascribed to pre-harvest red colour loss during periods of high temperature. High temperatures combined with high irradiances also lead to sunburn on pome fruit. The use of pulsed overhead evaporative cooling (EC) to improve red colour and reduce sunburn in two blushed pear cultivars was evaluated over four seasons (2000/01 to 2003/04) in Stellenbosch, South Africa. ‘Rosemarie’ fruit blush was usually but not always improved under EC, with a late application (starting two weeks before harvest) yielding the best improvement. Peel anthocyanin concentrations were increased under EC. Incidence of sunburn was reduced during some years, but increased in EC-treated fruit during 2003/04 due to system failure on a hot day. EC initiated early in fruit development initially led to larger fruit with a lower TSS concentration and firmness, but this effect was gradually eliminated following reductions in EC water use. EC had no effect on ‘Forelle’ pear fruit colour or mass, but reduced firmness and TSS when started early in the season. Sunburn was only recorded in EC-treated fruit during 2002/03 due to system failure on a warm day. Though EC could be used to improve ‘Rosemarie’ fruit colour in warm production areas, its effect was relatively small compared to colour change in response to fluctuating temperature. The increased risk of sunburn during system ‘downtime’ highlights the requirement for a highly reliable system