Isabel Sousa

Molecular basis of enrofloxacin translocation through OmpF, an outer membrane channel of Escherichia coli--when binding does not imply translocation.

The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.

Synthesis, characterization and antibacterial studies of a copper(II) levofloxacin ternary complex.

Solution behavior of levofloxacin (lvx) complexes with copper(II) in the presence and absence of phen was studied in aqueous solution, by potentiometry. The results obtained show that under physiological conditions (micromolar concentration range and pH 7.4) only copper(II):lvx:phen ternary complexes are stable. Hence, a novel copper(II) ternary complex of fluoroquinolone levofloxacin with nitrogen donor heterocyclic ligand phen was synthesized and characterized by means of UV-Visible and IR spectroscopy, elemental analysis and X-Ray crystallography. In the synthesized complex (1), [Cu(lvx)(phen)(H(2)O)](NO(3)).2H(2)O, levofloxacin acts as a bidentate ligand coordinating to the metal, in its anionic form, through the carbonyl and carboxyl oxygens and phen coordinates through two N-atoms forming the equatorial plane of a distorted square-pyramidal geometry. The fifth ligand of the penta-coordinated Cu(II) centre is occupied axially by an oxygen atom from a water molecule. Minimum inhibitory concentration (MIC) determinations of the complex and comparison with free levofloxacin in various E. coli strains indicated that the Cu-complex is as efficient an antimicrobial as the free antibiotic (both in the case of the dissolved synthesized complex and the complex formed following stoichiometric mixture of the individual components in solution). Moreover, results strongly suggest that the cell intake route of both species is different supporting, therefore, the complexs suitability as a candidate for further biological testing in fluoroquinolone-resistant microorganisms.

Characterization of membrane protein reconstitution in LUVs of different lipid composition by fluorescence anisotropy.

A major requirement to perform structural studies with membrane proteins is not only to define efficient reconstitution protocols, that assure a high incorporation degree in preformed liposomes, but also a protein directionality and topology that mimics its in vivo conditions. For this kind of studies, protein reconstitution in membranes systems via a detergent-mediated pathway is usually successfully adopted, since detergents are generally used in the initial isolation and purification of membrane proteins. In this study we report the reconstitution of OmpF in preformed DMPC and E. coli liposomes using two different techniques for detergent removal: (1) exclusion chromatography and (2) incubation with detergent-adsorbing beads. The incorporation degree was determined by bicinchoninic acid assay and fluorescence anisotropy was used to determine OmpF effect on the structural order of membrane lipids. These results show that protein insertion in membranes depends both on the technique used to remove detergent and on the lipids used to prepare the liposomes. Furthermore, it is possible to state that although the insertion is directly related to the size distributions of proteoliposomes, it could be efficiently recognized by steady-state fluorescence anisotropy. This technique, more popular among cell biologists, can be a very practical and straightforward alternative to DLS to confirm membrane protein insertion.

Study of the release of a microencapsulated acid dye in polyamide dyeing using mixed cationic liposomes

The main objective of this work was to increase the retarding effect of the acid dye Telon® Blue RR (C.I. Acid Blue 62; DyStar, Frankfurt, Germany) release on polyamide fibres dyeing by encapsulation of the dye in liposomes as an alternative to synthetic auxiliaries, in order to reduce effluent pollution. The retarding effect achieved with the use of mixed cationic liposomes of dioctadecyldimethylammonium bromide (DODAB)/soybean lecithin (containing a 10% molar fraction of DODAB) was better in comparison with either pure soybean lecithin liposomes or synthetic auxiliaries. The retarding effect of liposomes on the dye release was analysed through changes in the absorption and fluorescence spectra of the acid dye at different conditions. The effect of temperature (in the range of 25 °C - 70 °C) on the spectroscopic behaviour of the dye in the absence and in presence of polyamide was also studied, in order to simulate the dyeing conditions. Exhaustion curves obtained in dyeing experiments showed that, below 45 °C, the retarding effect of the mixed liposomes (lecithin/DODAB (9:1)) was similar to that of the auxiliaries, but better than the one of pure lecithin liposomes. At higher temperatures (above 45 °C), the system lecithin/DODAB presents a better performance, achieving a higher final exhaustion level when compared with the commercial leveling agent without losing the smoothing effect of lecithin.

A novel approach for immobilization of polyhexamethylene biguanide within silica capsules

Antibacterial agents have been in the spotlight over the past few years either due to their extensive applications or for the increasing concern with the spreading of biocide resistance. The encapsulation of large spectrum antibacterial agents, that are human safe, has been proposed as an alternative to overcome some of these problems but also to impart a controlled release of the encapsulated material. Polyhexamethylene biguanide (PHMB) is a bactericide polymer widely used in various applications, from industrial to household and everyday applications, with little to no documented bacterial resistance. In this work, the encapsulation/immobilization of antibacterial agent PHMB into silica capsules produced in a one-step emulsification process is reported for the first time. The synthesized capsules were characterized by Fourier transform infrared spectroscopy (FTIR), UV-Visible spectrophotometry, electron microscopies (SEM, STEM) and thermogravimetric analysis (TGA), confirming the presence of PHMB in the silica capsules. Furthermore, the incorporation of PHMB into the silica capsules led to significant changes in the textural properties, as revealed by nitrogen adsorption–desorption experiments. The biological performance of PHMB-loaded silica capsules was evaluated against a recombinant bioluminescent strain of Escherichia coli. Although after 60 min encapsulated PHMB has afforded the same bactericidal activity as an equivalent amount of free PHMB, the former demonstrated a delayed action consistent with a limited release of active compound from capsules, which may be advantageous in applications where a controlled release is required.

Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF

The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp214, Trp61), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp61 and iodide Trp214. Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp214 and moxifloxacin near Trp61. These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.

Opinions on a car smoking ban: a needed guide for public health decision-makers

This study was supported through FEDER from the Operational Programme Factors of Competitiveness COMPETE and through national funding from the Foundation for Science and Technology FCT (Portuguese Ministry of Education and Science) (FCOMP-01-0124-FEDER-009117).

Liposomal therapies in oncology: does one size fit all?

Liposomal therapies opened the chapter of nanomedicine, in 1995, with the approval of liposomal doxorubicin (Doxil®) for the treatment of numerous types of cancer. For the first time, liposomes permitted the employment of potent chemotherapeutic agents with improved pharmacokinetic and pharmacodynamic profiles, with less undesired side effects. Liposomal therapies allow the drug encapsulation and more selective delivery in the tumor bed, particularly due to the enhanced permeation and retention effect. These unique characteristics explain why liposomal therapies are being increasingly considered as alternatives in cancer therapy and represent an important research field with the recent approval for clinical use and emergent formulations in clinical trials. Even so, the response rate of liposomal therapies varies between 5 and 71%, and they are still indistinguishably given to every patient. As already well-demonstrated for conventional chemotherapies and targeted therapies, there is also the need for predictive biomarkers that allow a better use of liposomal drugs, with higher patient quality of life. Our aim in this review was to address the approved liposomal therapies and to summarize the information concerning possible predictive markers of response in their various clinical applications, to personalize each patient treatment and maximize its efficacy.

Ultra-high pressure modified cellulosic fibres with antimicrobial properties

In this work bleached E. globulus kraft pulp was doped with polyhexamethylene biguanide (PHMB) from an aqueous solution or from a suspension of silica capsules (PHMB@silica) by impregnation under atmospheric or ultra-high pressure (UHP) conditions (500MPa). The antimicrobial properties of pulps were evaluated towards gram-negative E. coli and gram-positive L. innocua bacteria. PHMB loads below 500mg per kg of pulp revealed negligible bacteriostatic properties, whereas PHMB loads of ca 3000-4000mg per kg demonstrated bactericidal properties of pulp without significant deterioration of its mechanical strength. The UHP impregnation allowed significant improvement of PHMB uptake. Thus, under equal conditions, PHMB uptake was ca 25% greater under UHP than under atmospheric pressure impregnation, whereas the leachable amounts of PHMB in both pulps were comparable. The sorption of PHMB@silica on pulp in suspension under UHP conditions was ca 17% greater than under atmospheric pressure with almost 70% increase of leachable PHMB.