Interaction of MRI Contrast Agent [Gd(DOTA)]- with Lipid Membranes: A Molecular Dynamics Study.

Contrast agents are important imaging probes in clinical MRI, allowing the identification of anatomic changes that otherwise would not be possible. Intensive research on the development of new contrast agents is being made to image specific pathological markers or sense local biochemical changes. The most widely used MRI contrast agents are based on gadolinium(III) complexes. Due to their very high charge density, they have low permeability through tight biological barriers such as the blood-brain barrier, hampering their application in the diagnosis of neurological disorders. In this study, we explore the interaction between the widely used contrast agent [Gd(DOTA)]- (Dotarem) and POPC lipid bilayers by means of molecular dynamics simulations. This metal complex is a standard reference where several chemical modifications have been introduced to improve key properties such as bioavailability and targeting. The simulations unveil detailed insights into the agent's interaction with the lipid bilayer, offering perspectives beyond experimental methods. Various properties, including the impact on global and local bilayer properties, were analyzed. As expected, the results indicate a low partition coefficient (KP) and high permeation barrier for this reference compound. Nevertheless, favorable interactions are established with the membrane leading to moderately long residence times. While coordination of one inner-sphere water molecule is maintained for the membrane-associated chelate, the physical-chemical attributes of [Gd(DOTA)]- as a MRI contrast agent are affected. Namely, increases in the rotational correlation times and in the residence time of the inner-sphere water are observed, with the former expected to significantly increase the water proton relaxivity. This work establishes a reference framework for the use of simulations to guide the rational design of new contrast agents with improved relaxivity and bioavailability and for the development of liposome-based formulations for use as imaging probes or theranostic agents.

Fluorescent Probes cis- and trans-Parinaric Acids in Fluid and Gel Lipid Bilayers: A Molecular Dynamics Study.

Fluorescence probes are indispensable tools in biochemical and biophysical membrane studies. Most of them possess extrinsic fluorophores, which often constitute a source of uncertainty and potential perturbation to the host system. In this regard, the few available intrinsically fluorescent membrane probes acquire increased importance. Among them, cis- and trans-parinaric acids (c-PnA and t-PnA, respectively) stand out as probes of membrane order and dynamics. These two compounds are long-chained fatty acids, differing solely in the configurations of two double bonds of their conjugated tetraene fluorophore. In this work, we employed all-atom and coarse-grained molecular dynamics simulations to study the behavior of c-PnA and t-PnA in lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), representative of the liquid disordered and solid ordered lipid phases, respectively. All-atom simulations indicate that the two probes show similar location and orientation in the simulated systems, with the carboxylate facing the water/lipid interface and the tail spanning the membrane leaflet. The two probes establish interactions with the solvent and lipids to a similar degree in POPC. However, the almost linear t-PnA molecules have tighter lipid packing around them, especially in DPPC, where they also interact more with positively charged lipid choline groups. Probably for these reasons, while both probes show similar partition (assessed from computed free energy profiles across bilayers) to POPC, t-PnA clearly partitions more extensively than c-PnA to the gel phase. t-PnA also displays more hindered fluorophore rotation, especially in DPPC. Our results agree very well with experimental fluorescence data from the literature and allow deeper understanding of the behavior of these two reporters of membrane organization.

Modeling Gd3+ Complexes for Molecular Dynamics Simulations: Toward a Rational Optimization of MRI Contrast Agents.

The correct parametrization of lanthanide complexes is of the utmost importance for their characterization using computational tools such as molecular dynamics simulations. This allows the optimization of their properties for a wide range of applications, including medical imaging. Here we present a systematic study to establish the best strategies for the correct parametrization of lanthanide complexes using [Gd(DOTA)]- as a reference, which is used as a contrast agent in MRI. We chose the bonded model to parametrize the lanthanide complexes, which is especially important when considering the study of the complex as a whole (e.g., for the study of the dynamics of its interaction with proteins or membranes). We followed two strategies: a so-called heuristic approach employing strategies already published by other authors and another based on the more recent MCPB.py tool. Adjustment of the Lennard-Jones parameters of the metal was required. The final topologies obtained with both strategies were able to reproduce the experimental ion to oxygen distance, vibrational frequencies, and other structural properties. We report a new strategy to adjust the Lennard-Jones parameters of the metal ion in order to capture dynamic properties such as the residence time of the capping water (τm). For the first time, the correct assessment of the τm value for Gd-based complexes was possible by recording the dissociative events over up to 10 µs all-atom simulations. The MCPB.py tool allowed the accurate parametrization of [Gd(DOTA)]- in a simpler procedure, and in this case, the dynamics of the water molecules in the outer hydration sphere was also characterized. This sphere was divided into the first hydration layer, an intermediate region, and an outer hydration layer, with a residence time of 18, 10 and 19 ps, respectively, independent of the nonbonded parameters chosen for Gd3+. The Lennard-Jones parameters of Gd3+ obtained here for [Gd(DOTA)]- may be used with similarly structured gadolinium MRI contrast agents. This allows the use of molecular dynamics simulations to characterize and optimize the contrast agent properties. The characterization of their interaction with membranes and proteins will permit the design of new targeted contrast agents with improved pharmacokinetics.

Interactions between Rhodamine Dyes and Model Membrane Systems-Insights from Molecular Dynamics Simulations.

BACKGROUND: rhodamines are dyes widely used as fluorescent tags in cell imaging, probing of mitochondrial membrane potential, and as P-glycoprotein model substrates. In all these applications, detailed understanding of the interaction between rhodamines and biomembranes is fundamental. METHODS: we combined atomistic molecular dynamics (MD) simulations and fluorescence spectroscopy to characterize the interaction between rhodamines 123 and B (Rh123 and RhB, respectively) and POPC bilayers. RESULTS: while the xanthene moiety orients roughly parallel to the membrane plane in unrestrained MD simulations, variations on the relative position of the benzoic ring (below the xanthene for Rh123, above it for RhB) were observed, and related to the structure of the two dyes and their interactions with water and lipids. Subtle distinctions were found among different ionization forms of the probes. Experimentally, RhB displayed a lipid/water partition coefficient more than two orders of magnitude higher than Rh123, in agreement with free energy profiles obtained from umbrella sampling MD. CONCLUSIONS: this work provided detailed insights on the similarities and differences in the behavior of bilayer-inserted Rh123 and RhB, related to the structure of the probes. The much higher affinity of RhB for the membranes increases the local concentration and explains its higher apparent affinity for P-glycoprotein reconstituted in model membranes.

Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aß and Amylin*.

Invited for the cover of this issue are Jean-François Morfin and Éva Tóth at the CNRS in Orléans, and their collaborators from University of Debrecen, University of Coimbra and Université de Toulouse. The image depicts that when an amphiphilic compound is intravenously injected, monomer, pre-micellar and micellar forms can co-exist in the blood and have different affinities for amyloid peptides. Read the full text of the article at 10.1002/chem.202004000.

Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aß and Amylin*.

Metal chelates targeted to amyloid peptides are widely explored as diagnostic tools or therapeutic agents. The attachment of a metal complex to amyloid recognition units typically leads to a decrease in peptide affinity. We show here that by separating a macrocyclic GdL chelate and a PiB targeting unit with a long hydrophobic C10 linker, it is possible to attain nanomolar affinities for both Aß1-40 (Kd =4.4 nm) and amylin (Kd =4.5 nm), implicated, respectively in Alzheimer's disease and diabetes. The Scatchard analysis of surface plasmon resonance data obtained for a series of amphiphilic, PiB derivative GdL complexes indicate that their Aß1-40 or amylin binding affinity varies with their concentration, thus micellar aggregation state. The GdL chelates also affect peptide aggregation kinetics, as probed by thioflavin-T fluorescence assays. A 2D NMR study allowed identifying that the hydrophilic region of Aß1-40 is involved in the interaction between the monomer peptide and the Gd3+ complex. Finally, ex vivo biodistribution experiments were conducted in healthy mice by using 111 In labeled analogues. Their pancreatic uptake, ∼3 %ID g-1 , is promising to envisage amylin imaging in diabetic animals.

Photophysical studies on lanthanide(III) chelates conjugated to Pittsburgh compound B as luminescent probes targeted to Aß amyloid aggregates.

The photophysical properties of Eu3+ and Tb3+ complexes of DOTAGA and DO3A-monoamide conjugates of the Pittsburgh compound B (PiB) chromophore, prepared using linkers of different lengths and flexibilities, and which form stable negatively charged (LnL1), and uncharged (LnL2) complexes, respectively, were studied as potential probes for optical detection of amyloid aggregates. The phenylbenzothiazole (PiB) moiety absorbs light at wavelengths longer than 330 nm with a high molar absorption coefficient in both probes, and acts as an antenna in these systems. The presence of the luminescent Ln3+ ion quenches the excited states of PiB through an energy transfer process from the triplet state of PiB to the metal centre, and structured emission is seen from Eu3+ and Tb3+. The luminescence study indicates the presence of a 5D4 → T1 back transfer process in the Tb3+ complexes. It also provides insights on structural properties of the Eu3+ complexes, such as the high symmetry environment of the Eu3+ ion in a single macrocyclic conformation and the presence of one water molecule in its inner coordination sphere. The overall quantum yield of luminescence of EuL1 is higher than for EuL2. However, their low values reflect the low overall sensitization efficiency of the energy transfer process, which is a consequence of the large distances between the metal center and the antenna, especially in the EuL2 complex. DFT calculations confirmed that the most stable conformation of the Eu3+ complexes involves a combination of a square antiprismatic (SAP) geometry of the chelate and an extended conformation of the linker. The large calculated average distances between the metal center and the antenna point to the predominance of the Förster energy transfer mechanism, especially for EuL2. This study provides insights into the behavior of amyloid-targeted Ln3+ complexes as optical probes, and contributes towards their rational design.

Accuracy of High-resolution Small-volume Cone-Beam Computed Tomography in the Diagnosis of Vertical Root Fracture: An In Vivo Analysis.

INTRODUCTION: The purpose of this in vivo study was to evaluate the accuracy of small-volume cone-beam computed tomographic (CBCT) imaging compared with conventional periapical radiography (CPR) in the diagnosis of vertical root fractures (VRFs) using exploratory surgery as the reference standard. METHODS: Eighty-two dental records of 85 teeth with suspected VRFs that underwent CPR, CBCT imaging, and exploratory surgery were included. Two observers assessed CPR and CBCT images independently for the presence or absence of root fractures, and findings from the exploratory surgery were considered the reference standard. Diagnostic sensitivity, specificity, accuracy, and the receiver operating characteristic curve values were obtained. The effect of single- and multirooted teeth on diagnostic accuracy as well as the association between clinical symptoms and the presence of VRFs were also assessed. RESULTS: VRFs were surgically detected in 64 of the 85 teeth (75.3%), of which 62.5% were multirooted and 76.6% had intracanal posts. CBCT imaging was more sensitive and accurate (65.6% and 64%) than CPR (27.3% and 40.5%). Both CPR and CBCT diagnostic accuracies were higher in single- than multirooted teeth. Pain on percussion, a localized periodontal pocket, and tooth mobility were associated with the presence of VRFs (P < .05; odds ratio = 4.15, 13.5 and 4.1, respectively). CONCLUSIONS: The accuracy of CBCT imaging for the diagnosis of VRFs was poor, although it was higher than with CPR. Multirooted teeth in the presence of intracanal posts may limit its diagnostic value.

Associating a negatively charged GdDOTA-derivative to the Pittsburgh compound B for targeting Aß amyloid aggregates.

We have conjugated the tetraazacyclododecane-tetraacetate (DOTA) chelator to Pittsburgh compound B (PiB) forming negatively charged lanthanide complexes, Ln(L4), with targeting capabilities towards aggregated amyloid peptides. The amphiphilic Gd(L4) chelate undergoes micellar aggregation in aqueous solution, with a critical micellar concentration of 0.68 mM, lower than those for the neutral complexes of similar structure. A variable temperature (17)O NMR and NMRD study allowed the assessment of the water exchange rate, k ex (298) = 9.7 × 10(6) s(-1), about the double of GdDOTA, and for the description of the rotational dynamics for both the monomeric and the micellar forms of Gd(L4). With respect to the analogous neutral complexes, the negative charge induces a significant rigidity of the micelles formed, which is reflected by slower and more restricted local motion of the Gd(3+) centers as evidenced by higher relaxivities at 20-60 MHz. Surface Plasmon Resonance results indicate that the charge does not affect significantly the binding strength to Aß1-40 [K d = 194 ± 11 µM for La(L4)], but it does enhance the affinity constant to human serum albumin [K a = 6530 ± 68 M(-1) for Gd(L4)], as compared to neutral counterparts. Protein-based NMR points to interaction of Gd(L4) with Aß1-40 in the monomer state as well, in contrast to neutral complexes interacting only with the aggregated form. Circular dichroism spectroscopy monitored time- and temperature-dependent changes of the Aß1-40 secondary structure, indicating that Gd(L4) stabilizes the random coil relative to the α-helix and ß-sheet. TEM images confirm that the Gd(L4) complex reduces the formation of aggregated fibrils.

Personalized Modeling for Prediction with Decision-Path Models.

Deriving predictive models in medicine typically relies on a population approach where a single model is developed from a dataset of individuals. In this paper we describe and evaluate a personalized approach in which we construct a new type of decision tree model called decision-path model that takes advantage of the particular features of a given person of interest. We introduce three personalized methods that derive personalized decision-path models. We compared the performance of these methods to that of Classification And Regression Tree (CART) that is a population decision tree to predict seven different outcomes in five medical datasets. Two of the three personalized methods performed statistically significantly better on area under the ROC curve (AUC) and Brier skill score compared to CART. The personalized approach of learning decision path models is a new approach for predictive modeling that can perform better than a population approach.

Tomografia computadorizada de feixe cônico na odontologia veterinária: descrição e padronização da técnica / Tapered beam computed tomography in Veterinary Dentistry: description and technical standardization

Onze cães e quatro gatos, portadores de alterações buco-dentárias e atendidos no Centro Veterinário do Gama, em Brasília, DF, foram submetidos à tomografia computadorizada de feixe cônico. Os exames foram realizados em um tomógrafo i-CAT, utilizando para aquisição das imagens, altura de seis centímetros, tempo de 40 segundos, 0,2 voxel, 120 kilovolts e 46,72 miliampéres por segundo. O melhor posicionamento dos animais para realização do exame foi definido neste estudo. Esse é um fator fundamental para a realização do exame, que necessitou um protocolo anestésico simples e seguro, em função do tempo mínimo necessário à obtenção das imagens. Várias alterações e enfermidades foram identificadas, com extrema acurácia, credenciando a tomografia computadorizada de feixe cônico como um exame seguro, acessível e exeqüível e que pode ser incorporado à rotina odontológica das clínicas de pequenos animais.

Eleven dogs and four cats with buccodental alterations, treated in the Centro Veterinário do Gama, in Brasilia, DF, Brazil, were submitted to cone beam computed tomography. The exams were carried out in a i-CAT tomograph, using for image acquisition six centimeters height, 40 seconds time, 0.2 voxel, 120 kilovolts and 46.72 milliamperes per second. The ideal positioning of the animal for the exam was also determined in this study and it proved to be fundamental for successful examination, which required a simple and safe anesthetic protocol due to the relatively short period of time necessary to obtain the images. Several alterations and diseases were identified with accurate imaging, demonstrating that cone beam computed tomography is a safe, accessible and feasible imaging method which could be included in the small animal dentistry routine diagnosis.