Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

Observation of Combined Effect of Temperature and Pressure on Cubic to Hexagonal Phase Transformation in ZnS at the Nanoscale

The temperature of allotropic phase transformation in ZnS (cubic to wurtzite) changes with pressure and particle size. In this paper we have explored the interrelation among these through a detailed study of ZnS powders obtained by a temperature-controlled high energy milling process. By employing the combined effect of temperature and pressure in an indigenously built cryomill, we have demonstrated a large-scale, low-temperature synthesis of wurtzite ZnS nanoparticles. The synthesized products have been characterized for their phase and microstructure by the use of X-ray diffraction and transmission electron microscopic techniques. Further, it has been demonstrated that the synthesized materials exhibit photoluminescence emissions in the UV-visible region with an unusual doublet pattern due to the presence of both cubic and hexagonal wurtzite domains in the same particles. By further fine-tuning the processing conditions, it may be possible to achieve controlled defect related photoluminescence emissions from the ZnS nanoparticles.

Prediction of Multi-Target Networks of Neuroprotective Compounds with Entropy Indices and Synthesis, Assay, and Theoretical Study of New Asymmetric 1,2-Rasagiline Carbamates

In a multi-target complex network, the links (L-ij) represent the interactions between the drug (d(i)) and the target (t(j)), characterized by different experimental measures (K-i, K-m, IC50, etc.) obtained in pharmacological assays under diverse boundary conditions (c(j)). In this work, we handle Shannon entropy measures for developing a model encompassing a multi-target network of neuroprotective/neurotoxic compounds reported in the CHEMBL database. The model predicts correctly >8300 experimental outcomes with Accuracy, Specificity, and Sensitivity above 80%-90% on training and external validation series. Indeed, the model can calculate different outcomes for >30 experimental measures in >400 different experimental protocolsin relation with >150 molecular and cellular targets on 11 different organisms (including human). Hereafter, we reported by the first time the synthesis, characterization, and experimental assays of a new series of chiral 1,2-rasagiline carbamate derivatives not reported in previous works. The experimental tests included: (1) assay in absence of neurotoxic agents; (2) in the presence of glutamate; and (3) in the presence of H2O2. Lastly, we used the new Assessing Links with Moving Averages (ALMA)-entropy model to predict possible outcomes for the new compounds in a high number of pharmacological tests not carried out experimentally.

High prevalence of HIV-1 and hepatitis C virus coinfection among injection drug users in the southeastern region of Yunnan, China

The southeastern region of Yunnan province is a key site for drug trafficking and HIV-1 infection spread from the west of Yunnan and Laos to southeastern China. To investigate the prevalence of HIV-1 infection and hepatitis C virus (HCV) coinfection among injection drug users (IDUs) in southeastern Yunnan, three cohorts of 285 addicts, including 242 IDUs and 43 oral drug users, living in the cities of Gejiu and Kaiyuan and the county of Yanshan were studied. HIV-1 and HCV infections were detected by enzyme-linked immunosorbent assay and/or polymerase chain reaction. Data on the age, sex, risk behavior, drug use history, employment, ethnic background, and marriage status were obtained by interview. The overall prevalence of HIV-1 infection was 71.9%. The rate of HCV coinfection among 138 HIV-1-infected IDUs was 99.3%. Most HIV-infected IDUs were 20 to 35 years old (86.7%) and were ethnic Han (75.9%), suggesting that the epidemic in Yunnan is no longer confined to non-Han ethnic minorities, HIV prevalence in female IDUs (81.2%) was significantly higher than in male IDUs (68.2%) (p <.05). The prevalence of HIV infection reached 68.4% after 1 year of injection drug use. Needle/syringe sharing is the major high risk factor for the spread of HIV-1 and HCV infections. Large-scale educational campaigns are urgently needed to reduce the spread of HIV and HCV infection in these regions.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide Ligation assay on DNA chips

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

In Situ Observation of Tensile Deformation Processes of Soft Colloidal Crystalline Latex Fibers

The deformation mechanism or styrene/n-butyl acrylate copolymer latex films with fiber symmetric crystalline structure subjected to uniaxial stretching was studied using synchrotron small-angle X-ray scattering technique. The fibers were drawn at angles or 0, 35, and 55 degrees with respect to the Fiber axis. In all cases, the microscopic deformation within the crystallites was Found to deviate from affine deformation behavior with respect to the macroscopic deformation ratio. Moreover, the extent of this deviation is different in the three cases. This peculiar behavior can be attributed to the relative orientation of the (111) plane of the crystals, the plane of densest packing, with respect to the stretching direction in each case. When the stretching direction coincides with the crystallographic (111) plane, which is the case for stretching directions of 0 and 55 degrees with respect to the fiber axis, the microscopic deformation deviates less from affine behavior than when the stretching direction is arbitrarily oriented with respect to the crystallographic (111) plan.

Anticancer Drug-DNA Interactions Measured Using a Photoinduced Electron-Transfer Mechanism Based on Luminescent Quantum Dots

A sensing system based on the photoinduced electron transfer of quantum dots (QDs) was designed to measure the interaction of anticancer drug and DNA, taking mitoxantrone (MTX) as a model drug. MTX adsorbed on the surface of QDs can quench the photoluminescence (PL) of QDs through the photoinduced electron-transfer process; and then the addition of DNA will bring the restoration of QDs PL intensity, as DNA can bind with MTX and remove it from QDs. Sensitive detection of MTX with the detection limit of 10 nmol L-1 and a linear detection range from 10 nmol L-1 to 4.5 mu mol L-1 was achieved. The dependence of PL intensity on DNA amount was successfully utilized to investigate the interactions between MTX and DNA. Both the binding constants and the sizes of binding site of MTX-DNA interactions were calculated based on the equations deduced for the PL recovery process. The binding constant obtained in our experiment was generally consistent with previous reports. The sensitive and speedy detection of MTX as well as the avoidance of modification or immobilization process made this system suitable and promising in the drug-DNA interaction studies.

The structural transition of DNA-Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol-water solution

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when e is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)(3)(3+) in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B-A conformation transition takes place at more than 60% ethanol solution.

Determination of five pharmacologically active compounds extracted from Rhodiola for natural product drug discovery with HPLC-APCI-MS

A rapid and sensitive liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS) assay for the determination of five pharmacologically active compounds (PAC) extracted from the traditional Chinese medicine, Rhodiola , namely salidroside, tyrosol, rhodionin, gallic acid, and ethyl gallate has been developed. In this method, PAC could be baseline separated and detected with DAD at 275 nm. The validation of the method, including sensitivity, linearity, repeatability, and recovery, was examined. The linear calibration curves were acquired with correlation coefficient >0.999 and the limits of detection LOD (at a signal-to-noise ratio=3:1) were between 0.058 and 1.500 mu mol/L. It was found, that the amounts of PAC varied with different species of Rhodiola . The established method is rapid and reproducible for the separation of five natural pharmacologically active compounds from extracts of Rhodiola with satisfactory results.

Two genes on A/J chromosome 18 are associated with susceptibility to Staphylococcus aureus infection by combined microarray and QTL analyses.

Although it has recently been shown that A/J mice are highly susceptible to Staphylococcus aureus sepsis as compared to C57BL/6J, the specific genes responsible for this differential phenotype are unknown. Using chromosome substitution strains (CSS), we found that loci on chromosomes 8, 11, and 18 influence susceptibility to S. aureus sepsis in A/J mice. We then used two candidate gene selection strategies to identify genes on these three chromosomes associated with S. aureus susceptibility, and targeted genes identified by both gene selection strategies. First, we used whole genome transcription profiling to identify 191 (56 on chr. 8, 100 on chr. 11, and 35 on chr. 18) genes on our three chromosomes of interest that are differentially expressed between S. aureus-infected A/J and C57BL/6J. Second, we identified two significant quantitative trait loci (QTL) for survival post-infection on chr. 18 using N(2) backcross mice (F(1) [C18A]xC57BL/6J). Ten genes on chr. 18 (March3, Cep120, Chmp1b, Dcp2, Dtwd2, Isoc1, Lman1, Spire1, Tnfaip8, and Seh1l) mapped to the two significant QTL regions and were also identified by the expression array selection strategy. Using real-time PCR, 6 of these 10 genes (Chmp1b, Dtwd2, Isoc1, Lman1, Tnfaip8, and Seh1l) showed significantly different expression levels between S. aureus-infected A/J and C57BL/6J. For two (Tnfaip8 and Seh1l) of these 6 genes, siRNA-mediated knockdown of gene expression in S. aureus-challenged RAW264.7 macrophages induced significant changes in the cytokine response (IL-1 beta and GM-CSF) compared to negative controls. These cytokine response changes were consistent with those seen in S. aureus-challenged peritoneal macrophages from CSS 18 mice (which contain A/J chromosome 18 but are otherwise C57BL/6J), but not C57BL/6J mice. These findings suggest that two genes, Tnfaip8 and Seh1l, may contribute to susceptibility to S. aureus in A/J mice, and represent promising candidates for human genetic susceptibility studies.

Characterisation of freeze-dried wafers and solvent evaporated films as potential drug delivery systems to mucosal surfaces

Freeze-dried (lyophilised) wafers and solvent cast films from sodium alginate (ALG) and sodium carboxymethylcellulose (CMC) have been developed as potential drug delivery systems for mucosal surfaces including wounds. The wafers (ALG, CMC) and films (CMC) were prepared by freeze-drying and drying in air (solvent evaporation) respectively, aqueous gels of the polymers containing paracetamol as a model drug. Microscopic architecture was examined using scanning electron microscopy, hydration characteristics with confocal laser scanning microscopy and dynamic vapour sorption. Texture analysis was employed to investigate mechanical characteristics of the wafers during compression. Differential scanning calorimetry was used to investigate polymorphic changes of paracetamol occurring during formulation of the wafers and films. The porous freeze-dried wafers exhibited higher drug loading and water absorption capacity than the corresponding solvent evaporated films. Moisture absorption, ease of hydration and mechanical behaviour were affected by the polymer and drug concentration. Two polymorphs of paracetamol were observed in the wafers and films, due to partial conversion of the original monoclinic to the orthorhombic polymorph during the formulation process. The results showed the potential of employing the freeze-dried wafers and solvent evaporated films in diverse mucosal applications due to their ease of hydration and based on different physical mechanical properties exhibited by both type of formulations.

Simultaneous monitoring of drug and solvent diffusion across a model membrane using ATR-FTIR spectroscopy

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used to simultaneously follow the diffusion of model drugs and solvent across polydimethylsiloxane (silicone) membrane. Three model drugs, cyanophenol (CNP), methyl nicotinate (MN) and butyl paraben (BP) were selected to cover a range of lipophilicities. Isostearyl isostearate (ISIS) was chosen as the solvent because its large molecular weight should facilitate observation of whether the drug molecules are able to diffuse through the membrane independently of the solvent. The diffusion of the three drugs and the solvent was successfully described by a Fickian model. The effects of parameters such as the absorption wavelength used to follow diffusion on the calculated diffusion coefficient were investigated. Absorption wavelength which affects the depth of penetration of the infrared radiation into the membrane did not significantly affect the calculated diffusion coefficient over the wavelength range tested. Each of the model drugs was observed to diffuse independently of the solvent across the membrane. The diffusion of a CNP-ISIS hydrogen bonded complex across the membrane was also monitored. The relative diffusion rates of the solute and solvent across the membrane can largely be accounted for by the molecular size of the permeant.

Microscopic models for dielectric relaxation in disordered systems

It is shown how the Debye rotational diffusion model of dielectric relaxation of polar molecules (which may be described in microscopic fashion as the diffusion limit of a discrete time random walk on the surface of the unit sphere) may be extended to yield the empirical Havriliak-Negami (HN) equation of anomalous dielectric relaxation from a microscopic model based on a kinetic equation just as in the Debye model. This kinetic equation is obtained by means of a generalization of the noninertial Fokker-Planck equation of conventional Brownian motion (generally known as the Smoluchowski equation) to fractional kinetics governed by the HN relaxation mechanism. For the simple case of noninteracting dipoles it may be solved by Fourier transform techniques to yield the Green function and the complex dielectric susceptibility corresponding to the HN anomalous relaxation mechanism.