Experimental and Computational Characterization of the Interaction between Gold Nanoparticles and Polyamidoamine Dendrimers.

Dendrimers provide a means to control the synthesis of gold nanoparticles and stabilize their suspensions. However, design of improved dendrimers for this application is hindered by a lack of understanding how the dendrimers and synthesis conditions determine nanoparticle morphology and suspension stability. In the present work, we evaluate the effect of polyamidoamine (PAMAM) dendrimers terminated with different functional groups (-OH or -NH3+) and different synthesis conditions on the morphology of the resulting gold nanoparticles and their stability in solution. We leverage molecular dynamics (MD) simulations to identify the atomic interactions that underlie adsorption of PAMAM dendrimers to gold surface and how the thermodynamics of this adsorption depends on the terminal functional groups of the dendrimers. We find that gold nanoparticles formed with hydroxyl-terminated PAMAM (PAMAM-OH) rapidly aggregate, whereas those formed with PAMAM-NH3+ are stable in solution for months of storage. Synthesis under ultrasound sonication is shown to be more rapid than that under agitation, with sonication producing smaller nanoparticles. Free-energy calculations in MD simulations show that all dendrimers have a high affinity for the gold surface, although PAMAM-OH and its oxidized aldehyde form (PAMAM-CHO) have a greater affinity for the nanoparticle surface than PAMAM-NH3+. Although adsorption of PAMAM-OH and PAMAM-CHO has both favorable entropy and enthalpy, adsorption of PAMAM-NH3+ is driven by a strong enthalpic component subject to an unfavorable entropic component.

Identification and functional expression of a glutamate- and avermectin-gated chloride channel from Caligus rogercresseyi, a southern Hemisphere sea louse affecting farmed fish.

Parasitic sea lice represent a major sanitary threat to marine salmonid aquaculture, an industry accounting for 7% of world fish production. Caligus rogercresseyi is the principal sea louse species infesting farmed salmon and trout in the southern hemisphere. Most effective control of Caligus has been obtained with macrocyclic lactones (MLs) ivermectin and emamectin. These drugs target glutamate-gated chloride channels (GluCl) and act as irreversible non-competitive agonists causing neuronal inhibition, paralysis and death of the parasite. Here we report the cloning of a full-length CrGluClα receptor from Caligus rogercresseyi. Expression in Xenopus oocytes and electrophysiological assays show that CrGluClα is activated by glutamate and mediates chloride currents blocked by the ligand-gated anion channel inhibitor picrotoxin. Both ivermectin and emamectin activate CrGluClα in the absence of glutamate. The effects are irreversible and occur with an EC(50) value of around 200 nM, being cooperative (n(H) = 2) for ivermectin but not for emamectin. Using the three-dimensional structure of a GluClα from Caenorabditis elegans, the only available for any eukaryotic ligand-gated anion channel, we have constructed a homology model for CrGluClα. Docking and molecular dynamics calculations reveal the way in which ivermectin and emamectin interact with CrGluClα. Both drugs intercalate between transmembrane domains M1 and M3 of neighbouring subunits of a pentameric structure. The structure displays three H-bonds involved in this interaction, but despite similarity in structure only of two these are conserved from the C. elegans crystal binding site. Our data strongly suggest that CrGluClα is an important target for avermectins used in the treatment of sea louse infestation in farmed salmonids and open the way for ascertaining a possible mechanism of increasing resistance to MLs in aquaculture industry. Molecular modeling could help in the design of new, more efficient drugs whilst functional expression of the receptor allows a first stage of testing of their efficacy.

Mechanistic Studies on the Self-Assembly of PLGA Patchy Particles and Their Potential Applications in Biomedical Imaging.

Currently, several challenges prevent poly(lactic-co-glycolic acid) (PLGA) particles from reaching clinical settings. Among these is a lack of understanding of the molecular mechanisms involved in the formation of these particles. We have been studying in depth the formation of patchy polymeric particles. These particles are made of PLGA and lipid-polymer functional groups. They have unique patch-core-shell structural features: hollow or solid hydrophobic cores and a patchy surface. Previously, we identified the shear stress as the most important parameter in a patchy particle's formation. Here, we investigated in detail the role of shear stress in the patchy particle's internal and external structure using an integrative experimental and computational approach. By cross-sectioning the multipatch particles, we found lipid-based structures embedded in the entire PLGA matrix, which represents a unique finding in the PLGA field. By developing novel computational fluid dynamics and molecular dynamics simulations, we found that the shear stress determines the internal structure of the patchy particles. Equally important, we discovered that these particles emit a photoacoustic (PA) signal in the optical clinical imaging window. Our results show that particles with multiple patches emit a higher PA signal than single-patch particles. This phenomenon most likely is due to the fact that multipatchy particles absorb more heat than single-patchy particles as shown by differential scanning calorimetry analysis. Furthermore, we demonstrated the use of patchy polymeric particles as photoacoustic molecular probes both in vitro and in vivo studies. The fundamental studies described here will help us to design more effective PLGA carriers for a number of medical applications as well as to accelerate their medical translation.

PAMAM G4 dendrimers as inhibitors of the iron storage properties of human L-chain ferritin.

Cationic dendrimers, such as PAMAM, are known to be positively charged at neutral pH allowing their unspecific interaction with proteins and other cellular components. Especially, ferritin, which has an important role in iron homeostasis, presents a negative electrostatic potential at the 3-fold channel. This channel is important in the functionality of ferritin because it allows the iron entry into its inner cavity. In this way, the interaction between the protonated terminal amines of the dendrimer and the negatively charged 3-fold channels of ferritin is expected. Experimental measurements demonstrated that PAMAM G4 inhibits the iron storage properties of L-chain human ferritin (L-Ftn). Molecular dynamics simulations have been used to analyze the specific interaction between PAMAM G4 and L-Ftn. Results show that PAMAM G4 effectively interacts with the 3-fold channels of L-Ftn, suggesting that this interaction is responsible for the inhibition of the iron storage properties of L-Ftn.

Synthesis and characterization of an insoluble polymer based on polyamidoamine: applications for the decontamination of metals in aqueous systems.

We present a novel, insoluble, low-generation polyamidoamine (PAMAM)-based polymer. The monomer and polymer were characterized by fourier transform infrared spectroscopy, electrospray ionization mass spectrometry and thermogravimetric measurement, revealing that G0 acryloyl-terminated PAMAM were synthesized and polymerized using ammonium persulfate as an initiator, producing a high-density PAMAM derivative (PAMAM-HD). PAMAM-HD was tested for its ability to remove Na(I), K(I), Ca(II), Mg(II), Cu(II), Mn(II), Cd(II), Pb(II) and Zn(II) ions from acidic, neutral and basic aqueous solutions. PAMAM-HD efficiently removed metals ions from all three solutions. The greatest absorption efficiency at neutral pH was observed against Cu(II), Cd(II) and Pb(II), and the experimental data were supported by the calculated Kd values. Our data could have a significant impact on water purification by providing an inexpensive and efficient polymer for the removal of metal ions.

Nanoinformatics: developing new computing applications for nanomedicine.

Nanoinformatics has recently emerged to address the need of computing applications at the nano level. In this regard, the authors have participated in various initiatives to identify its concepts, foundations and challenges. While nanomaterials open up the possibility for developing new devices in many industrial and scientific areas, they also offer breakthrough perspectives for the prevention, diagnosis and treatment of diseases. In this paper, we analyze the different aspects of nanoinformatics and suggest five research topics to help catalyze new research and development in the area, particularly focused on nanomedicine. We also encompass the use of informatics to further the biological and clinical applications of basic research in nanoscience and nanotechnology, and the related concept of an extended "nanotype" to coalesce information related to nanoparticles. We suggest how nanoinformatics could accelerate developments in nanomedicine, similarly to what happened with the Human Genome and other -omics projects, on issues like exchanging modeling and simulation methods and tools, linking toxicity information to clinical and personal databases or developing new approaches for scientific ontologies, among many others.

Experimental and theoretical structural/spectroscopical correlation of enterobactin and catecholamide.

Here we report the IR spectra of FeEnterobactin in catecholate conformations ([CatFeEB]3-) obtained by DFT calculations using PBE/QZVP and their correlation it with its experimental counterpart [SalH3FeEB]0. Fragments of FeEnterobactin and Enterobactin (H6EB) are elucidated from their MALDI-TOF mass spectrometry, and the dependence of the frontier orbitals (HOMO and LUMO) with the catecholamide dihedral angles of H6EB is reported. The frequency distribution of catecholamide dihedral angle of H6EB was carried-out using molecular dynamics (MD). The data presented enriches the understanding of [CatFeEB]3 - and H6EB frequency distribution and reactivity.

IR and NMR spectroscopic correlation of enterobactin by DFT.

Emerging and re-emerging epidemic diseases pose an ongoing threat to global health. Currently, Enterobactin and Enterobactin derivatives have gained interest, owing to their potential application in the pharmaceutical field. As it is known [J. Am. Chem. Soc (1979) 101, 20, 6097-6104], Enterobactin (H6EB) is an efficient iron carrier synthesized and secreted by many microbial species. In order to facilitate the elucidation of enterobactin and its analogues, here we propose the creation of a H6EB standard set using Density Functional Theory Infrared (IR) and NMR spectra. We used two exchange-correlation (xc) functionals (PBE including long-range corrections LC-PBE and mPW1), 2 basis sets (QZVP and 6-31G(d)) and 2 grids (fine and ultrafine) for most of the H6EB structures dependent of dihedral angles. The results show a significant difference between the OH and NH bands, while the CO amide and O(CO) IR bands are often found on top of each other. The NMR DFT calculations show a strong dependence on the xc functional, basis set, and grid used for the H6EB structure. Calculated 1H and 13C NMR spectra enable the effect of the solvent to be understood in the context of the experimental measurements. The good agreement between the experimental and the calculated spectra using LC-PBE/QZVP and ultrafine grid suggest the possibility of the systems reported here to be considered as a standard set. The dependence of electrostatic potential and frontier orbitals with the catecholamide dihedral angles of H6EB is described. The matrix-assisted laser desorption/ionization time of the flight mass spectrometry (MALDI-TOF MS) of H6EB is also reported of manner to enrich the knowledge about its reactivity.

Mutation Arg336 to Lys in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase originates an enzyme with increased oxaloacetate decarboxylase activity.

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes one of the first reactions in the biosynthesis of carbohydrates. Apart from the physiologically important reaction, the enzyme also presents low oxaloacetate decarboxylase and pyruvate kinase-like activities. Data from the crystalline structure of homologous Escherichia coli PEP carboxykinase suggest that Arg(333) may be involved in stabilization of enolpyruvate, a postulated reaction intermediate. In this work, the equivalent Arg(336) from the S. cerevisiae enzyme was changed to Lys or Gln. Kinetic analyses of the varied enzymes showed that a positive charge at position 336 is critical for catalysis of the main reaction, and further suggested different rate limiting steps for the main reaction and the secondary activities. The Arg336Lys altered enzyme showed increased oxaloacetate decarboxylase activity and developed the ability to catalyze pyruvate enolization. These last results support the proposal that enolpyruvate is an intermediate in the PEP carboxykinase reaction and suggest that in the Arg336Lys PEP carboxykinase a proton donor group has appeared.

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1.

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO(2). Mutations of PEP carboxykinase have been constructed where the residues His(225) and Asp(263), two residues of the enzyme's putative Mn(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16,800-fold reductions in V(max) relative to the wild-type enzyme, respectively, with minor alterations in K(m) for Mn(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N(epsilon-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.

Gating of two-pore domain K+ channels by extracellular pH.

Potassium channels have a conserved selectivity filter that is important in determining which ions are conducted and at what rate. Although K+ channels of different conductance characteristics are known, they differ more widely in the way their opening and closing, the gating, is governed. TASK and TALK subfamily proteins are two-pore region KCNK K+ channels gated open by extracellular pH. We discuss the mechanism for this gating in terms of electrostatic effects on the pore changing the occupancy and open probability of the channels in a way reminiscent of C-type inactivation gating at the selectivity filter. Essential to this proposed mechanism is the replacement of two highly conserved aspartate residues at the pore mouth by asparagine or histidine residues in the TALK and TASK channels.

Molecular modeling of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase and the ATP analogs pyridoxal 5'-diphosphoadenosine and pyridoxal 5'-triphosphoadenosine. Specific labeling of lysine 290.

Molecular mechanics calculations have been employed to obtain models of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate (PEP) kinase and the ATP analogs pyridoxal 5'-diphosphoadenosine (PLP-AMP) and pyridoxal 5'-triphosphoadenosine (PLP-ADP), using the crystalline coordinates of the ATP-pyruvate-Mn(2+)-Mg(2+) complex of Escherichia coli PEP carboxykinase [Tari et al. (1997), Nature Struct. Biol. 4, 990-994]. In these models, the preferred conformation of the pyridoxyl moiety of PLP-ADP and PLP-AMP was established through rotational barrier and simulated annealing procedures. Distances from the carbonyl-C of each analog to epsilon-N of active-site lysyl residues were calculated for the most stable enzyme-analog complex conformation, and it was found that the closest epsilon-N is that from Lys(290), thus predicting Schiff base formation between the corresponding carbonyl and amino groups. This prediction was experimentally verified through chemical modification of S. cerevisiae PEP carboxykinase with PLP-ADP and PLP-AMP. The results here described demonstrate the use of molecular modeling procedures when planning chemical modification of enzyme-active sites.