Identification of potential drug targets in Salmonella Typhimurium using metabolic modelling and experimental validation

almonella enterica serovar Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of Salmonella Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in energy demand, while growing in glucose minimal medium. By grouping reactions with similar flux responses, a sub-network of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions, that when removed from the genome-scale model interfered with energy and biomass generation. 11 such sets were found to be essential for the production of biomass precursors. Experimental investigation of 7 of these showed that knock-outs of the associated genes resulted in attenuated growth for 4 pairs of reactions, while 3 single reactions were shown to be essential for growth.

Assessment of the CMD Mini-Explorer, a new low-frequency multi-coil electromagnetic device, for archaeological investigations

In this article we assess the abilities of a new electromagnetic (EM) system, the CMD Mini-Explorer, for prospecting of archaeological features in Ireland and the UK. The Mini-Explorer is an EM probe which is primarily aimed at the environmental/geological prospecting market for the detection of pipes and geology. It has long been evident from the use of other EM devices that such an instrument might be suitable for shallow soil studies and applicable for archaeological prospecting. Of particular interest for the archaeological surveyor is the fact that the Mini-Explorer simultaneously obtains both quadrature (‘conductivity’) and in-phase (relative to ‘magnetic susceptibility’) data from three depth levels. As the maximum depth range is probably about 1.5 m, a comprehensive analysis of the subsoil within that range is possible. As with all EM devices the measurements require no contact with the ground, thereby negating the problem of high contact resistance that often besets earth resistance data during dry spells. The use of the CMD Mini-Explorer at a number of sites has demonstrated that it has the potential to detect a range of archaeological features and produces high-quality data that are comparable in quality to those obtained from standard earth resistance and magnetometer techniques. In theory the ability to measure two phenomena at three depths suggests that this type of instrument could reduce the number of poor outcomes that are the result of single measurement surveys. The high success rate reported here in the identification of buried archaeology using a multi-depth device that responds to the two most commonly mapped geophysical phenomena has implications for evaluation style surveys. Copyright © 2013 John Wiley & Sons, Ltd.

Fungicide-resistance management tactics: impacts on Zymoseptoria tritici populations

Azoles and Succinate Dehydrogenase Inhibitors (SDHIs) are the main fungicides available for septoria tritici blotch control, causal agent Zymoseptoria tritici. Decline in azole sensitivity, in combination with European legislation, poses a threat to wheat production in Ireland. Azole fungicides select CYP51 mutations differentially; it was hypothesised that using combinations of azoles could be an effective anti-resistance tool. Naturally inoculated field experiments were carried out in order to understand the impacts of using combinations of azoles, epoxiconazole and metconazole, on azole sensitivity. Approximately 3700 isolates were isolated and their sensitivity to both azoles analysed. Findings showed that limiting the number of applications, by alternating each fungicide, slowed selection for reduced azole sensitivity. Limiting azole use by reducing doses did not reduce selection for decreased azole sensitivity. Although not complete, cross-resistance was observed between the two azoles, which will lead to general reduction in azole sensitivity. A sub-selection of isolates from each treatment at each location were analysed for changes in the CYP51 gene. Sequence analysis identified 49 combinations of mutations in the CYP51 gene, and three different inserts in the CYP51 promoter. Intragenic recombination also featured in these populations. Baseline studies of five new SDHIs were carried out on 209 naturally infected, non-SDHI-treated isolates. With the exception of fluopyram, cross-resistance was apparent between the SDHIs. Analysis of 2300 isolates found that when compared to the solo products, mixing the SDHI isopyrazam and the azole epoxiconazole increased epoxiconazole sensitivity, but had no apparent effect on isopyrazam sensitivity. SDHI resistance-conferring mutations were absent in the baseline and experimental isolates. As long as azoles are used, Z. tritici populations will continue to evolve towards resistance. Combining different modes-of-action, SDHIs and multi-sites, with azoles will relieve some of that selective pressure. To get the best out of available fungicides, they should be used in combination with host resistance and good crop management practices.

Optimization of amaranth flour films plasticized with glycerol and sorbitol by multi-response analysis

The optimal formulation for the preparation of amaranth flour films plasticized with glycerol and sorbitol was obtained by a multi-response analysis. The optimization aimed to achieve films with higher resistance to break, moderate elongation and lower solubility in water. The influence of plasticizer concentration (Cg, glycerol or Cs, sorbitol) and process temperature (Tp) on the mechanical properties and solubility of the amaranth flour films was initially studied by response surface methodology (RSM). The optimized conditions obtained were Cg 20.02 g glycerol/100 g flour and Tp 75 degrees C, and Cs 29.6 g sorbitol/100 g flour and Tp 75 degrees C. Characterization of the films prepared with these formulations revealed that the optimization methodology employed in this work was satisfactory. Sorbitol was the most suitable plasticizer. It furnished amaranth flour films that were more resistant to break and less permeable to oxygen, due to its greater miscibility with the biopolymers present in the flour and its lower affinity for water. (C) 2011 Elsevier Ltd. All rights reserved.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, < 0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 mu g/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 mu g/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 mu g/mL). The quantitation of cytokines revealed a predominance of IFN-gamma and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

From Rational Design of Drug Crystals to Understanding of Nucleic Acid Structures: Lamivudine Duplex

A DNA-like duplex of nucleosides is probable to exist even without the 5`-phosphate groups needed to assemble the chain backbone. However, double-stranded helical structures of nucleosides are unknown. Here, we report a duplex of nucleoside analogs that is spontaneously assembled due to stacking of the neutral and protonated molecules of lamivudine, a nucleoside reverse transcriptase inhibitor (NTRI) widely used in anti-HIV drug combinatory medication. The left-handed lamivudine duplex has features similar to those of i-motif DNA, as the face-to-face base stacking and the helix rise per base pair. Furthermore, the protonation pattern on alternate bases expected for it DNA-like duplex stabilized by pairing of neutral and protonated cytosine fragments was observed for the first time in the lamivudine double-stranded helix. This structure demonstrates that hydrogen bonds can substitute for covalent phosphodiester linkage in the stabilization of the duplex backbone. This interesting example of spontaneous molecular self-organization indicates that the 5`-phosphate group could not be a requirement for duplex assembly.

Mechanical and tribological properties of PVD-coated cemented carbide as evaluated by a new multi-pass scratch testing method

A new test method based on multipass scratch testing has been developed for evaluating the mechanical and tribological properties of thin, hard coatings. The proposed test method uses a pin-on-disc tribometer and during testing a Rockwell C diamond stylus is used as the “pin” and loaded against the rotating coated sample. The influence of normal load on the number of cycles to coating damage is investigated and the resulting coating damage mechanisms are evaluated by posttest scanning electron microscopy. The present study presents the test method by evaluating the performance of Ti0.86Si0.14N, Ti0.34Al0.66N, and (Al0.7Cr0.3)2O3 coatings deposited by cathodic arc evaporation on cemented carbide inserts. The results show that the test method is quick, simple, and reproducible and can preferably be used to obtain relevant data concerning the fatigue, wear, chipping, and spalling characteristics of different coating-substrate composites. The test method can be used as a virtually nondestructive test and, for example, be used to evaluate the fatigue and wear resistance as well as the cohesive and adhesive interfacial strength of coated cemented carbide inserts prior to cutting tests.

Nanotechnology-based Drug Delivery Systems for Dermatomycosis Treatment

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Rheological, mechanical and mucoadhesive properties of thermoresponsive, bioadhesive binary mixtures composed of poloxamer 407 and carbopol 974P designed as platforms for implantable drug delivery systems for use in the oral cavity

This study described the formulation and characterisation of the viscoelastic, mechanical and mucoadhesive properties of thermoresponsive, binary polymeric systems composed of poloxamer (P407) and poly(acrylic acid, C974P) that were designed for use as a drug delivery platform within the oral cavity. Monopolymeric and binary polymeric formulations were prepared containing 10, 15 and 20% (w/w) poloxamer (407) and 0.10-0.25% (w/w) poly(acrylic acid, 934P). The flow theological and viscoelastic properties of the formulations were determined using controlled stress and oscillatory rheometry, respectively, the latter as a function of temperature. The mechanical and mucoadhesive properties (namely the force required to break the bond between the formulation and a pre-hydrated mucin disc) were determined using compression and tensile analysis, respectively. Binary systems composed of 10% (w/w) P407 and C934P were elastoviscous, were easily deformed under stress and did not exhibit mucoadhesion. Formulations containing 15 or 20% (w/w) Pluronic P407 and C934P exhibited a sol-gel temperature T(sol/gel), were viscoelastic and offered high elasticity and resistance to deformation at 37 degrees C. Conversely these formulations were elastoviscous and easily deformed at temperatures below the sol-gel transition temperature. The sol-gel transition temperatures of systems containing 15% (w/w) P407 were unaffected by the presence of C934P; however, increasing the concentration of C934P decreased the T(sol/gel) in formulations containing 20%(w/w) P407. Rheological synergy between P407 and C934P at 37 degrees C was observed and was accredited to secondary interactions between these polymers, in addition to hydrophobic interactions between P407 micelles. Importantly, formulations composed of 20% (w/w) P407 and C934P exhibited pronounced mucoadhesive properties. The ease of administration (below the T(sol/gel)) in conjunction with the viscoelastic (notably high elasticity) and mucoadhesive properties (at body temperature) render the formulations composed of 20% (w/w) P407 and C934P as potentially useful platforms for mucoadhesive, controlled topical drug delivery within the oral cavity. (c) 2009 Published by Elsevier B.V.

Darunavir: A Critical Review of Its Properties, Use and Drug Interactions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

AC Biosusceptometry as a Tool for Monitoring the Gastrointestinal Transit of Multiparticulate Drug Delivery System

Drug delivery systems based on natural polysaccharides, such as chitosan (CS) and pectin (PC), rather than on synthetic polymers, have been widely studied. Some reasons for that are low toxicity and costs and high biodegradability of the formers. A multiparticulate system based on CS and PC was developed in our laboratories, including the addition of an enteric polymer, cellulose acetate phtalate (CAP). Such improvement promoted stronger gastric and enteric resistances, as assessed in vitro, making the systems more selective to enzymatic degradation in the colon. Although in vitro dissolution tests can simulate some properties concerning the gastrointestinal transit (GT), collaborating to characterize the systems behavior in the biological fluids, frequently they do not result in satisfactory in vitro/in vivo correlations. The objective of this work was to follow in vivo the GT of the particles developed by means of AC biosusceptometry (ACB), a non-invasive and of low cost methodology. The particles containing ferrite in powder form were prepared by complex coacervation using an ideal 3:1:1 mass ratio for PC:CS:CAP. The magnetic particles were administered to healthy volunteers by oral route. The GT was monitored by using multi-sensor ACB system and the signal acquisition was performed every IS min until the colonic region was reached. By means of ACB technique, it was possible to acquiring images generated by the magnetic particles within the whole gastrointestinal tract including the colonic region. Variable particles transit times were observed among the volunteers, but without interference on the mapping of the particles until the colonic region. The particles were able to produce magnetic field strong enough to generate signals adequate for mapping the particles. The results suggest that integral particles reached the colon, after they resisted against gastric and enteric media. Studies associating transit time and in vivo drug release are in development in order to confirm the efficiency of the systems.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Oxacillin Resistance of Staphylococcus aureus Isolated from the University Hospital of Botucatu Medical School in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prevalence and antifungal resistance profile of Candida spp. oral isolates from patients with type 1 and 2 diabetes mellitus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Multi-spectroscopic monitoring of cisplatin-derived species delivery from ureasil polyether hybrid matrix

Driven by the challenges involved in the development of new advanced materials with unusual drug delivery profiles capable of improving the therapeutic and toxicological properties of existing cancer chemotherapy, the one-pot sol-gel synthesis of flexible, transparent and insoluble urea-cross-linked polyether-siloxane hybrids has been recently developed. In this one-pot synthesis, the strong interaction between the antitumor cisplatin (CisPt) molecules and the ureasil-poly(propylene oxide) (PPO) hybrid matrix gives rise to the incorporation and release of an unknown CisPt-derived species, hindering the quantitative determination of the drug release pattern from the conventional UV-Vis absorption technique. In this article, we report the use of an original synchrotron radiation calibration method based on the combination of XAS and UV-Vis for the quantitative determination of the amount of Pt-based molecules released in water. Thanks to the combination of UV-Vis, XAS and Raman techniques, we demonstrated that both the CisPt molecules and the CisPt-derived species are loaded into an ureasil-PPO/ureasil-poly(ethylene oxide) (PEO) hybrid blend matrix. The experimentally determined molar extinction coefficient of the CisPt-derived species loaded into ureasil-PPO hybrid matrix enabled the simultaneous time-resolved monitoring of each Pt species released from this hybrid blend matrix.