Jorge Martins

Molecular Mechanism of Lateral Diffusion of py10-PC and Free Pyrene in Fluid DMPC Bilayers

From the study of the kinetics of the fluorescence self-quenching of pyrene in multilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the fluid phase we obtain the molecular diffusion parameters, diffusion coefficients, and activation energies for lateral diffusion for three probes: 1-palmitoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (py(10)-PC), pyrene, and 1-pyrenebutanoic acid (py-but). The experiments are done in a range of temperatures for which the reversibility of excimer formation is negligible and the probe/phospholipid ratios used are low, avoiding non-ideal mixing of the probe. The time-resolved fluorescence decays are, in all cases, accurately and consistently described by the two-dimensional (2D) formalism for bimolecular diffusion-controlled reactions. From the parameters obtained in this way we conclude that the primary step of the diffusion of the pyrene-labeled phospholipid involves the simultaneous jump of phospholipid and fluorophore moieties, and also that although in the case of py(10)-PC the pyrene from molecules pertaining to different layers may interact during the lifetime of the exited state, this is not the case for free pyrene.

Development of a highly sensitive bacteria detection assay using fluorescent pH-responsive polymeric micelles

The detection and control of bacteria is extremely important in the safety of food products and health systems. The conventional microbiological methods based on culture enrichment techniques and plating procedures are highly sensitive and selective for bacterial detection but are expensive, cumbersome and time-consuming. Here we report the development of a simple and sensitive bioassay to detect Escherichia coli (E. coli) bacteria by using self assembled pH-responsive polymeric micelles that have been bioconjugated to anti-E. coli (capturing agent). Poly(ethylene glycol-b-trimethylsilyl methacrylate), containing silicon moieties that can be cleaved under mildly acidic conditions, was synthesized and self-assembled into micelles, that were loaded with a fluorescent dye (1-methylpyrene). The polymer silicon protecting groups are used as a tool to remotely activate the dye release by means of pH. The high sensitivity of the newly developed bioassay, which is capable of detecting 15 bacteria per milliliter of solution, is due to an amplification effect generated by the optical signal of millions of fluorophores released from a single micelle upon attachment to a bacterium. Fluorescence probing involves the measurements of changes in the emission spectra, through the disappearance of the excimer band, which only occurs when the dye molecules are trapped within the polymeric micelles.

Sensing hydration and behavior of pyrene in POPC and POPC/cholesterol bilayers: a molecular dynamics study.

Molecular dynamics (MD) simulations of bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with varying amounts of cholesterol (0, 5, 20, and 40mol%) were carried out in the absence and presence of inserted pyrene molecules. Both fluorophore and bilayer parameters were computed, for characterization of probe location and dynamics, as well as its effects on the host bilayer. In agreement with previous studies in fluid disordered bilayers, pyrene prefers to be located in the hydrophobic acyl chain region of POPC bilayers, close to the glycerol group of lipid molecules and causes ordering of the lipid acyl chains. However, incorporation of pyrene in binary POPC/cholesterol bilayers decreases the acyl chain order parameter (especially near the end of the chains), opposing the ordering effect of cholesterol. These effects are modest and mainly felt locally. Significantly, as the bilayer is enriched with cholesterol, the relative position of pyrene and the POPC carbonyl and phosphocholine groups is invariant, and the local water density around the probe decreases. This work clarifies and supports the cautious use of pyrene Ham effect to effectively measure equivalent polarity in lipid bilayers. Within the time scale of the MD simulations, which is of the magnitude of the fluorescence lifetime of pyrene, the thermally averaged polarity of lipid bilayers is nearly out of influence of spurious uncertainty in the transverse location of pyrene in the bilayers. This renders the values of equivalent polarity measurements through the pyrene Ham effect more reliable and reproducible than previously expected.

Reappraisal of four different approaches for finding the mean reaction time in the multi-trap variant of the Adam–Delbrück problem

Adam and Delbrück argued that the dimensionality of the diffusion space determines the average lifetime of a diffusing particle confined to a region with a central trap. Doubts have often been aired as to whether their calculation is relevant to real biological systems, where the number of traps is usually much larger than unity; or whether the rate enhancement is merely a manifestation of an increase in the concentration of the traps; or whether the diverse multi-trap versions of their expression for the mean lifetime in two dimensions are trustworthy. These issues are addressed, and the long-standing problem of finding the low-density limit of trapping time in two dimensions solved, by examining previous treatments of the problem, and by carrying out simulations of two-dimensional systems in which the particles undergo a Pearsonian random walk, and the traps are distributed randomly or on a square lattice. The mean lifetimes are found to be different in the two situations, and it is concluded that the neglect of this aspect lies at the root of the conflict between some of the existing expressions for the mean lifetime. Relations expressing the mean lifetime as a function of the concentration of the traps are presented together with a discussion of their applicability.

Self-assembled polymeric nanoparticles as new, smart contrast agents for cancer early detection using magnetic resonance imaging

Early cancer detection is a major factor in the reduction of mortality and cancer management cost. Here we developed a smart and targeted micelle-based contrast agent for magnetic resonance imaging (MRI), able to turn on its imaging capability in the presence of acidic cancer tissues. This smart contrast agent consists of pH-sensitive polymeric micelles formed by self-assembly of a diblock copolymer (poly(ethyleneglycol-b-trimethylsilyl methacrylate)), loaded with a gadolinium hydrophobic complex (tBuBipyGd) and exploits the acidic pH in cancer tissues. In vitro MRI experiments showed that tBuBipyGd-loaded micelles were pH-sensitive, as they turned on their imaging capability only in an acidic microenvironment. The micelle-targeting ability toward cancer cells was enhanced by conjugation with an antibody against the MUC1 protein. The ability of our antibody-decorated micelles to be switched on in acidic microenvironments and to target cancer cells expressing specific antigens, together with its high Gd(III) content and its small size (35–40 nm) reveals their potential use for early cancer detection by MRI.

Partitioning of 1-pyrenesulfonate into zwitterionic and mixed zwitterionic/anionic fluid phospholipid bilayers.

Molecular partitioning into biomembranes is of fundamental importance in diverse biochemical processes and reactions. The majority of aqueous/membrane partition data using model membrane systems, is obtained with pure phosphatidylcholine bilayers, being lipid mixtures less used, while studies involving bilayers containing zwitterionic/anionic mixtures of phospholipids are even more scarce. In this study, the solvatochromic effects of 1-pyrenesulfonate observed at 375 nm in aqueous liposome suspensions, and monitored by second derivative absorption spectrophotometry, enabled the determination of its partition constants into defined phospholipid bilayers. We compare, under cautiously settled experimental conditions, the partition of the anionic amphiphile PSA into fluid zwitterionic bilayers of POPC (Kp=6.7 x 10(3), at 25 degrees C), and into fluid mixed zwitterionic/anionic bilayers containing small proportions of anionic phospholipids. At the same temperature, we found increasing K(p) values in parallel with the proportion of POPS mixed with POPC (Kp=3.4 x 10(4) and Kp=7.3 x 10(4), with 5 and 10 mol% of POPS, respectively). Our interpretation is based on the interfacial properties of fluid and flexible mixed zwitterionic/anionic phospholipid bilayers.

Induction of defence responses by cinnamomins against Phytophthora cinnamomi in Quercus suber and Quercus ilex subs. rotundifolia

The strong association between Phytophthora cinnamomi and the mortality and decline of Quercus suber and Q. ilex subsp. rotundifolia has been known for two decades. The ability of elicitins secreted by this pathogen to trigger defence responses in these Quercus against itself was evaluated in this work. Biomass quantification by quantitative real-time PCR revealed a significant decrease in pathogen colonization of Q. suber roots after 24xa0h pre-treatment with α- and β-cinnamomin. In Q. suber and Q. ilex roots pre-treated with α-cinnamomin, hyphae were unable to reach and colonize the vascular cylinder and showed cytoplasmic disorganization in all the roots observed as contrasted with non-pre-treated roots. The pathogen was restricted to the intercellular spaces of the cortical parenchyma and the concomitant accumulation of electron dense materials was observed in contact with the hyphae. Furthermore, ROS (reactive oxygen species) production and the enzymatic activities of superoxide dismutase, catalase and peroxidase were compared in infected and non-infected Quercus roots in time course trials. There was a significant increase in the production of hydrogen peroxide (H2O2) and superoxide anion (O2•-) and an enhanced activity of the enzymes in infected roots was observed at each time point. When comparing with elicitin non-treated roots, the α-cinnamomin-treated roots in interaction with P. cinnamomi showed a decrease in ROS accumulation and an increase of the enzyme activities. The overall results were consistent with an induction by the cinnamomins which initiated defence responses against the pathogen invasion of roots. Finally, elicitins were immunolocalized in the contact zone of P. cinnamomi hyphae with epidermal host cells, plasmalemma outer cytoplasm and around the intracellular hyphae in the vacuoles of invaded epidermal cells.

Can pyrene be localized inside lipid bilayers by simultaneously measuring Py values, and fulfilling the excimer formation conditions?

In a recent article in Chemistry and Physics of Lipids, Mazor t al. (2012) present a wavelength selective fluorescence approach o monitor pyrene localization within lipid bilayers and biologial membranes. There are obvious connections between this work nd our experimental findings in closely related systems: quanitative analysis of pyrene excimer formation in homogeneous hosphatidylcholine bilayers (Melo and Martins, 2006; Manuel nd Martins, 2011) and measuring of model membrane equivalent olarity in pure and mixed phospholipid/cholesterol lipid bilayers Arrais and Martins, 2007, 2011). Fishov and co-workers (Mazor t al., 2012) state that they are presenting the first observation of a ed edge-like excitation shift (REES) for pyrene incorporated within ilayers. For instance, the claimed excitation-dependent changes n the vibronic emission spectra of pyrene go along with alleged ariations in the excimer/monomer ratio (E/M), and these outomes are interpreted together as proof of different environments nside bilayers in terms of fluidity and polarity. These occurrences re observed exclusively in model and biological membranes, but ot in liquid homogeneous solvents of varying polarity and viscosty. They correlate the dependence on the excitation wavelength ith a selective UV absorption of pyrene molecules located close o the bilayer interface, or deep in the hydrophobic core of lipid embranes. In our opinion, some of the findings and statements f Mazor et al. (2012) are inconsistent due to improper experiental procedures and unsuitable theoretical assumptions, which ay lead to misinterpretations of results. From the experimental tandpoint, the average location of pyrene within lipid bilayers is rmly established. It was demonstrated that pyrene is inserted in he hydrophobic acyl chain region, close to the glycerol moiety nd to the first methylenic groups of the ordered portion of the ilayer palisade structure (from C2 to C8 of the acyl methylenic hains of phospholipids). This was achieved directly by NMR (Podo nd Blasie, 1977; Hoff et al., 2005) and molecular dynamics simlations (Hoff et al., 2005; Čurdova et al., 2007), and indirectly, hrough a self-consistent kinetic analysis of pyrene excimer foration in 1,2-dimiristoyl-3-glycerophosphatidylcholine (DMPC) iposomes (Martins and Melo, 2001) and by measurements of lipid ilayer equivalent polarity in diverse model membrane systems Arrais and Martins, 2007, 2011).