Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

3D Extrusion Printing of Biphasic Anthropomorphic Brain Phantoms Mimicking MR Relaxation Times Based on Alginate-Agarose-Carrageenan Blends.

The availability of adapted phantoms mimicking different body parts is fundamental to establishing the stability and reliability of magnetic resonance imaging (MRI) methods. The primary purpose of such phantoms is the mimicking of physiologically relevant, contrast-creating relaxation times T1 and T2. For the head, frequently examined by MRI, an anthropomorphic design of brain phantoms would imply the discrimination of gray matter and white matter (WM) within defined, spatially distributed compartments. Multichannel extrusion printing allows the layer-by-layer fabrication of multiple pastelike materials in a spatially defined manner with a predefined shape. In this study, the advantages of this method are used to fabricate biphasic brain phantoms mimicking MR relaxation times and anthropomorphic geometry. The printable ink was based on purely naturally derived polymers: alginate as a calcium-cross-linkable gelling agent, agarose, ι-carrageenan, and GdCl3 in different concentrations (0-280 µmol kg-1) as the paramagnetic component. The suggested inks (e.g., 3Alg-1Agar-6Car) fulfilled the requirements of viscoelastic behavior and printability of large constructs (>150 mL). The microstructure and distribution of GdCl3 were assessed by scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX). In closely monitored steps of technological development and characterization, from monophasic and biphasic samples as printable inks and cross-linked gels, we describe the construction of large-scale phantom models whose relaxation times were characterized and checked for stability over time.

Comprehensive Quantitative and Calibration-Free Evaluation of Hyperpolarized Xenon-Host Interaction by Multiparametric NMR.

The probing of microscopic environments by hyperpolarized xenon NMR has spurred investigations in supramolecular chemistry as well as important biosensing and molecular imaging applications. While xenon exchange with host structures at micromolar concentrations and below can be readily detected, a quantitative analysis is limited, requiring complementary experimentation by different methodologies and thus lacking completeness and compromising the validity and comparability of numerical results. Here, a new NMR measurement and data analysis approach is introduced for the comprehensive characterization of the host-xenon binding dynamics. The application of chemical exchange saturation transfer of hyperpolarized 129Xe under parametric modulation of the saturation RF amplitude and xenon gas saturation of the solution enables a delineation of exchange mechanisms and, through modeling, a numerical estimation of the various reaction rate constants (and thus magnetization exchange rate constants), the xenon affinity, and the total host molecule concentration. Only the numerical xenon solubility is additionally required for input, a quantity that has a low impact on the measurement uncertainty and is derivable from metrological data collections. Signal calibration by a reference material may thus be avoided, qualifying the method as calibration-free. For demonstration a xenon exchange with the host cucurbit[6]uril at low concentration is investigated, with the numerical results being validated by standard quantitative NMR data obtained at high concentration. The readiness to evaluate xenon exchange for the one sample at hand and in a single experimental attempt by the proposed method may allow comprehensive quantitative studies in supramolecular chemistry, biomacromolecular structure and dynamics, and sensing.

A built-in coil system attached to the inside walls of a magnetically shielded room for generating an ultra-high magnetic field homogeneity.

The homogeneity of the magnetic field generated by a coil inside a magnetic shield is essential for many applications, such as ultra-low field nuclear magnetic resonance or spin precession experiments. In the course of upgrading the Berlin Magnetically Shielded Room (BMSR-2) with a new inserted Permalloy layer of side length 2.87 m, we designed a built-in coil consisting of four identical square windings attached to its inside walls. The spacings of the four windings were optimized using a recently developed semi-analytic model and finite element analysis. The result reveals a strong dependence of the field homogeneity on the asymmetric placement of the inner two windings and on the chosen material permeability value µs. However, our model calculations also show that these experimental variations can be counterbalanced by an adjustment of the inner winding positions in the millimeter range. Superconducting quantum interference device-based measurements yield for our implementation after fine adjustments of a single winding position a maximum field change of less than 10 pT for a total field of B0 = 2.3 µT within a 10 cm region along the coil axis, which is already better than the residual field of the upgraded BMSR-2.1 after degaussing. Measurements of free spin precession decay signals of polarized Xe129 nuclei show that the transverse relaxation time for the used cell is not limited by the inhomogeneity of the new built-in coil system.

Quantitative Assessment of Xenon Exchange Kinetics with Cucurbit[6]uril in Physiological Saline.

Cucurbit[6]uril and xenon form supramolecular complexes that are of great potential for biosensing by NMR. This host-guest system acts alike a signaler in sensors facilitating the ultrasensitive detection of biomarkers by saturation transfer of chemically exchanging, hyperpolarized 129 Xe. Here, the exchange process is evaluated by NMR exchange spectroscopy utilizing the preparation of anti-parallel longitudinal magnetization with respect to free and host-bound xenon and the variation of xenon concentration. Evidence for dissociative as well as degenerate exchange mechanisms is revealed by a linear regression analysis of the determined exchange rates resulting in rate coefficients of 1131±11 s-1 (2390±70 s-1 ) and 108500±4900 M-1 s-1 (174200±13900 M-1 s-1 ), respectively, and an affinity constant of 289±8 M-1 (278±14 M-1 ) in physiological saline at 298 K (310 K). The results elucidate the supramolecular exchange and underpin the high efficacy for biosensing of this host-guest system. The approach is generally applicable to enhanced host-xenon exchange dynamics, yet slow on the NMR timescale, for quantitative kinetics and biosensing analyses.

Transversal modes and Higgs bosons in electroweak vector-boson scattering at the LHC.

Processes where W and Z bosons scatter into pairs of electroweak bosons W, Z, and Higgs, are sensitive probes of new physics in the electroweak sector. We study simplified models that describe typical scenarios of new physics and parameterize the range of possible LHC results between the standard-model prediction and unitarity limits. Extending the study beyond purely longitudinal scattering, we investigate the role of transversally polarized gauge bosons. Unitarity becomes an essential factor, and limits on parameters matched to the naive perturbative low-energy effective theory turn out to be necessarily model-dependent. We discuss the implications of our approach for the interpretation of LHC data on vector-boson scattering and Higgs-pair production.

Design and comparison of exchange spectroscopy approaches to cryptophane-xenon host-guest kinetics.

Exchange spectroscopy is used in combination with a variation of xenon concentration to disentangle the kinetics of the reversible binding of xenon to cryptophane-A. The signal intensity of either free or crytophane-bound xenon decays in a manner characteristic of the underlying exchange reactions when the spins in the other pool are perturbed. Three experimental approaches, including the well-known Hyper-CEST method, are shown to effectively entail a simple linear dependence of the signal depletion rate, or of a related quantity, on free xenon concentration. This occurs when using spin pool saturation or inversion followed by free exchange. The identification and quantification of contributions to the binding kinetics is then straightforward: in the depletion rate plot, the intercept at the vanishing free xenon concentration represents the kinetic rate coefficient for xenon detachment from the host by dissociative processes while the slope is indicative of the kinetic rate coefficient for degenerate exchange reactions. Comparing quantified kinetic rates for hyperpolarized xenon in aqueous solution reveals the high accuracy of each approach but also shows differences in the precision of the numerical results and in the requirements for prior knowledge. Because of their broad range of applicability the proposed exchange spectroscopy experiments can be readily used to unravel the kinetics of complex formation of xenon with host molecules in the various situations appearing in practice.

Degeneracy in cryptophane-xenon complex formation in aqueous solution.

The reversible binding of xenon to cryptophane molecules is currently heavily explored for application as a reporter system in NMR. Herein, for aqueous solution, first evidence of degenerate exchange in this host-guest system is presented based on a novel approach using hyperpolarized (129)Xe.

Configuration and Performance of a Mobile (129)Xe Polarizer.

A stand-alone, self-contained and transportable system for the polarization of (129)Xe by spin exchange optical pumping with Rb is described. This mobile polarizer may be operated in batch or continuous flow modes with medium amounts of hyperpolarized (129)Xe for spectroscopic or small animal applications. A key element is an online nuclear magnetic resonance module which facilitates continuous monitoring of polarization generation in the pumping cell as well as the calculation of the absolute (129)Xe polarization. The performance of the polarizer with respect to the crucial parameters temperature, xenon and nitrogen partial pressures, and the total gas flow is discussed. In batch mode the highest (129)Xe polarization of P(Xe) = 40 % was achieved using 0.1 mbar xenon partial pressure. For a xenon flow of 6.5 and 26 mln/min, P(Xe) = 25 % and P(Xe) = 13 % were reached, respectively. The mobile polarizer may be a practical and efficient means to make the applicability of hyperpolarized (129)Xe more widespread.

A xenon-129 biosensor for monitoring MHC-peptide interactions.

Caged in: The formation of a complex between a peptide ligand and a major histocompatibility complex (MHC) class II protein is detected by a (129)Xe biosensor. Cryptophane molecules that trap Xe atoms are modified with a hemagglutinin (HA) peptide, which binds to the MHC protein. The interaction can be monitored by an NMR chemical shift change of cage-HA bound (129)Xe.

Dynamic NMR spectroscopy of hyperpolarized (129)Xe in human brain analyzed by an uptake model.

Hyperpolarized (129)Xe (HpXe) NMR not only holds promise for functional lung imaging, but for measurements of tissue perfusion as well. To investigate human brain perfusion, several time-series of (129)Xe MR spectra were recorded from one healthy volunteer after HpXe inhalation. The time-dependent amplitudes of the MR spectra were analyzed by using a compartment model for xenon uptake modified to account for the loss of (129)Xe polarization due to RF-excitation and for the breathhold technique used in the experiments. This analysis suggests that the resonances detected at 196.5 +/- 1 ppm and 193 +/- 1 ppm originate from HpXe dissolved in gray and white matter, respectively, and that T(1) relaxation times of HpXe are different in gray and white matter (T(1g) > T(1w)).

Platelet function test HemoSTATUS 2: tool or toy for an optimized management of hemostasis?

BACKGROUND: Bleeding after open heart surgery is a common but unintended problem, which is unequivocally related to platelet function. The target of our study was to correlate platelet activation levels and postoperative blood loss as well as the predictive power of measurements focusing on postoperative hemostasis. MATERIALS AND METHODS: The prospective trial comprised 100 patients (mean age: 64.3 years, 68% male) undergoing cardiac surgery. Platelet activation was measured by the new and modified HemoSTATUS test. Blood samples were drawn pre-, intra- and postoperatively. Standard hemostasis tests, including activated clotting time (ACT), partial thromboplastin time (PTT), hemoglobin, platelet count, antithrombin III (AT III) and fibrinogen, were measured according to the clinical routine. Blood loss and consumed blood products were documented up to the 24th hour after the operation. RESULTS: Platelet activation showed a typical change, with lowest levels after the end of extracorporeal circulation and a restitution to preoperative levels after 24 h. Mean blood loss was 461 ml. Statistical analysis showed neither a correlation to the platelet activation measurements nor to low pre-, intra- or postoperative levels. CONCLUSION: The HemoSTATUS platelet function test is not suitable for a reliable monitoring of platelet pathophysiology and patient outcome after extracorporeal circulation. Furthermore, no correlation of preoperative platelet activation and blood loss could be shown.