Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR

Constantly expressed genes are used as internal controls in relative quantification studies. Suitable internal controls for such studies have not yet been defined for Pseudomonas aeruginosa. In this study, the genes ampC, fabD, proC, pbp-2, rpoD and rpoS of P. aeruginosa were compared in terms of expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistance phenotypes were studied. Stability of expression among the housekeeping genes was assessed on the basis of correlation coefficients, with the best-correlated pair accepted as being the most stable one. Eventually, proC and rpoD formed the most stable pair (r = 0.958; P < 0.001). Next, in four ciprofloxacin-selected nfxC-like mutants, levels of oprD, oprM and oprN mRNA were compared with those of their wild-type counterparts. The comparison was made after correcting the raw values by the geometric mean of the internal control genes proC and rpoD. The level of oprN mRNA was significantly up-regulated, while the oprD gene was down-regulated (although this difference was statistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of nfxC-type mutants; this experiment therefore ends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies

N-acetylcysteine attenuates bacterial translocation after partial hepatectomy in rats.

Background. Translocating enteric bacteria have been suggested as playing a major role in the development of infections after partial hepatectomy. We investigated the effect of N-acetylcysteine (NAC) on bacterial translocation. (BT) and intestinal mucosa as the first line of defense against BT

Preparation of bow tie-type methacrylated poly(caprolactone-co-lactic acid) scaffolds: Effect of collagen modification on cell growth

A branched methacrylated poly(caprolactone-co-lactic acid) and methacrylated poly(tetramethylene ether glycol) (PTMG-IEM) resins were synthesized. 1H-NMR spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy, and gel permeation chromatography confirmed the chemical structures of copolymers. The photoinitiated polymerization of formulation composed of various amounts of methacrylated poly(caprolactone-co-lactic acid), PTMG-IEM, poly(ethylene glycol) diacrylate, water, and photoinitiator were performed. The curing reactions were followed by photo-DSC (Differential scanning calorimetry). Gel fraction was calculated from the insoluble part and found as =93%. Swelling and contact angles were measured, and all increased with the increasing amount of PTMG-IEM in network formulations. In vitro degradation studies were performed at 37 degrees C in phosphate-buffered saline (pH 7.4). Collagen-modified polymers were also prepared and introduced as a bioactive moiety to modify the polymer to enhance cell affinity. To compare the cell adhesion affinity to the polymer with and without collagen, cell growth experiments were performed. The results showed that collagen improves the cell adhesion onto the polymer surface. With the increasing amount of collagen, cell viability increases 86% (ECV304, p?<?0.05) and 83% (3?T3, p?<?0.05). Copyright (C) 2011 John Wiley & Sons, Ltd

Preparation of biocompatible, UV-cured fumarated poly(ether-ester)-based tissue-engineering hydrogels

The aim of this study was to develop biodegradable, photo-polymerizable in situ gel-forming systems prepared from a fumaric acid monoethyl ester (FAME) modified poly(lactide-co-glycolide) (PLGA) copolymer. By reacting lactide and glycolide in the presence of stannous octoate as a catalyst and 2-ethyl, 2-hydroxymethyl 1,3-propanediol as an initiator, hydroxyl terminated branched PLGA was synthesized. Afterwards, at room temperature hydroxyl terminated branched PLGA was reacted with fumaric acid monoethyl ester (FAME). N,N'-dicyclohexylcarbodiimide and triethylamine were used as a coupling agent and catalyst, respectively. The gel percentage, equilibrium mass swelling, degradation profile and polymerization kinetics of the hydrogels were investigated. All of the results were influenced by the amount of FAME modified PLGA co-polymer. Biocompatibility of the hydrogels was examined by using MTT cytotoxicity assay. According to the results, hydrogels are biocompatible and cell viability percentage depends on the amount of PLGA co-polymer. While the amount was 15% in hydrogel composition, cell viability was 100%, but after increasing the PLGA co-polymer amount to 30% the viability reduced to 78%. (C) Koninklijke Brill NV, Leiden, 201

Genetic and enzymatic properties of metallo-beta-lactamase VIM-5 from a clinical isolate of Enterobacter cloacae

A VIM-5-producing Enterobacter cloacae isolate (EDV/1) was identified in a collection of clinical strains stored before 2002. The gene, blav(VIM-5), was located on a 2,712-bp BamHI-HindIII fragment of a 23-kbp (approximately) nonconjugative plasmid (pEDV5) in a class 1 integron as a single gene cassette

Effect of carbapenems on the transcriptional expression of the oprD, oprM and oprN genes in Pseudomonas aeruginosa

The effects of imipenem and meropenem on the transcriptional expression of resistance-related genes oprD, oprM and oprN in Pseudomonas aeruginosa were studied by quantitative real-time PCR. Four strains were examined: the type strain PT5 (PAO1), its derivatives M7 and PT149, and a clinical isolate, PaKT3. The derivative M7 is a nalB mutant, overexpressing the MexAB-OprM pump, and the derivative PT149 is a nfxC-type mutant, overexpressing the MexEF-OprN pump while it is down-regulated for the OprD protein. After 18 h incubation in broth, the cultures were divided into three portions. Two were supplemented with antibiotics and the other was left antibiotic-free as the control. After a further 45 min incubation, total RNA was isolated from the strains by guanidine denaturation and acid-phenol/chloroform extraction. DNA-free total RNAs were immediately reverse-transcribed by MMuLV reverse transcriptase. Concentrations of mRNAs obtained by quantitative PCR were expressed relative to uninduced portions of the strains. The results showed that oprD was relatively stable against carbapenem antibiotics. oprM was induced significantly by imipenem in only one strain and oprN was induced by imipenem in most of the strains. The responses at the mRNA level found here were unexpected and suggested a chaotic, unpredictable regulatory mechanism