Magnetic resonance imaging analysis predicts nanoparticle concentration delivered to the brain parenchyma.

Ultrasound in combination with the introduction of microbubbles into the vasculature effectively opens the blood brain barrier (BBB) to allow the passage of therapeutic agents. Increased permeability of the BBB is typically demonstrated with small-molecule agents (e.g., 1-nm gadolinium salts). Permeability to small-molecule agents, however, cannot reliably predict the transfer of remarkably larger molecules (e.g., monoclonal antibodies) required by numerous therapies. To overcome this issue, we developed a magnetic resonance imaging analysis based on the ΔR2* physical parameter that can be measured intraoperatively for efficient real-time treatment management. We demonstrate successful correlations between ΔR2* values and parenchymal concentrations of 3 differently sized (18 nm-44 nm) populations of liposomes in a rat model. Reaching an appropriate ΔR2* value during treatment can reflect the effective delivery of large therapeutic agents. This prediction power enables the achievement of desirable parenchymal drug concentrations, which is paramount to obtaining effective therapeutic outcomes.

Electron-nuclear interactions as probes of domain motion in proteins.

Long range interactions between nuclear spins and paramagnetic ions can serve as a sensitive monitor of internal motion of various parts of proteins, including functional loops and separate domains. In the case of interdomain motion, the interactions between the ion and NMR-observable nuclei are modulated in direction and magnitude mainly by a combination of overall and interdomain motions. The effects on observable parameters such as paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS) can, in principle, be used to characterize motion. These parameters are frequently used for the purpose of structural refinements. However, their use to probe actual domain motions is less common and is lacking a proper theoretical treatment from a motional perspective. In this work, a suitable spin Hamiltonian is incorporated in a two body diffusion model to produce the time correlation function for the nuclear spin-paramagnetic ion interactions. Simulated observables for nuclei in different positions with respect to the paramagnetic ion are produced. Based on these simulations, it demonstrated that both the PRE and the PCS can be very sensitive probes of domain motion. Results for different nuclei within the protein sense different aspects of the motions. Some are more sensitive to the amplitude of the internal motion, others are more sensitive to overall diffusion rates, allowing separation of these contributions. Experimentally, the interaction strength can also be tuned by substitution of different paramagnetic ions or by varying magnetic field strength (in the case of lanthanides) to allow the use of more detailed diffusion models without reducing the reliability of data fitting.

Single-scan 2D Hadamard NMR spectroscopy.

Scan and deliver: By combining imaging-based spectral/spatial 2D radiofrequency manipulations (see scheme, left) with Hadamard-weighting principles, 2D NMR spectra can be retrieved within a single scan (right). This approach can give homo- or heteronuclear correlations with an enhanced sensitivity over conventional ultrafast 2D NMR spectroscopy.

Single Scan 2D NMR Spectroscopy on a 25 T Bitter Magnet.

2D NMR relies on monitoring systematic changes in the phases incurred by spin coherences as a function of an encoding time t(1), whose value changes over the course of independent experiments. The intrinsic multiscan nature of such protocols implies that resistive and/or hybrid magnets, capable of delivering the highest magnetic field strengths but possessing poor temporal stabilities, become unsuitable for 2D NMR acquisitions. It is here shown with a series of homo- and hetero-nuclear examples that such limitations can be bypassed using recently proposed 2D "ultrafast" acquisition schemes, which correlate interactions along all spectral dimensions within a single scan.

Spatially encoded pulse sequences for the acquisition of high resolution NMR spectra in inhomogeneous fields.

We have recently proposed a protocol for retrieving nuclear magnetic resonance (NMR) spectra based on a spatially-dependent encoding of the MR interactions. It has also been shown that the spatial selectivity with which spins are manipulated during such encoding opens up new avenues towards the removal of magnetic field inhomogeneities; not by demanding extreme Bo field uniformities, but rather by compensating for the dephasing effects introduced by the field distribution at a radiofrequency excitation and/or refocusing level. The present study discusses in further detail a number of strategies deriving from this principle, geared at acquiring both uni- as well as multi-dimensional spectroscopic data at high resolution conditions. Different variants are presented, tailored according to the relative sensitivity and chemical nature of the spin system being explored. In particular a simple multi-scan experiment is discussed capable of affording substantial improvements in the spectral resolution, at nearly no sensitivity or scaling penalties. This new compensation scheme is therefore well-suited for the collection of high-resolution data in low-field systems possessing limited signal-to-noise ratios, where magnetic field heterogeneities might present a serious obstacle. Potential areas of applications of these techniques include high-field in vivo NMR studies in regions near tissue/air interfaces, clinical low field MR spectroscopy on relatively large off-center volumes difficult to shim, and ex situ NMR. The principles of the different compensation methods are reviewed and experimentally demonstrated for one-dimensional inhomogeneities; further improvements and extensions are briefly discussed.

Symmetric spatial encoding in ultrafast 2D NMR spectroscopy.

Single-scan multidimensional spectroscopy utilizes spatial dimensions for encoding the indirect-domain internal spin interactions. Various strategies have been hitherto demonstrated for fulfilling the encoding needs underlying this methodology; in analogy with their time-domain counterparts all of them have in common the fact that they proceed monotonically-starting at one end of the sample and concluding at the other. The present manuscript discusses another possibility that arises for the case of amplitude-modulated ultrafast nD NMR, whereby the spatial encoding progresses from both ends of the sample simultaneously towards the center. Such symmetric encoding is compatible with continuous or discrete excitations as well as with homonuclear or heteronuclear correlations, and exhibits a number of advantages vis-à-vis the unidirectional encodings that have been used so far: it originates echoes that are free from large first-order phase distortions, and yields nD peaks possessing a purely-absorptive character. It has the added advantage that for a given indirect-domain spectral resolution it can complete its task in half the time required by a conventional monotonic spatial encoding, leading to potentially important gains in sensitivity. The main features underlying this new spatially symmetric encoding protocol are derived, and its advantages are demonstrated with a series of amplitude-modulated homo- and hetero-nuclear 2D ultrafast NMR examples.

A continuous phase-modulated approach to spatial encoding in ultrafast 2D NMR spectroscopy.

Ultrafast 2D NMR replaces the time-domain parametrization usually employed to monitor the indirect-domain spin evolution, with an equivalent encoding along a spatial geometry. When coupled to a gradient-assisted decoding during the acquisition, this enables the collection of complete 2D spectra within a single transient. We have presented elsewhere two strategies for carrying out the spatial encoding underlying ultrafast NMR: a discrete excitation protocol capable of imparting a phase-modulated encoding of the interactions, and a continuous protocol yielding amplitude-modulated signals. The former is general but has associated with it a number of practical complications; the latter is easier to implement but unsuitable for certain 2D NMR acquisitions. The present communication discusses a new protocol that incorporates attractive attributes from both alternatives, imparting a continuous spatial encoding of the interactions yet yielding a phase modulation of the signal. This in turn enables a number of basic experiments that have shown particularly useful in the context of in vivo 2D NMR, including 2D J-resolved and 2D H,H-COSY spectroscopies. It also provides a route to achieving sensitivity-enhanced acquisitions for other homonuclear correlation experiments, such as ultrafast 2D TOCSY. The main features underlying this new spatial encoding protocol are derived, and its potential demonstrated with a series of phase-modulated homonuclear single-scan 2D NMR examples.

Ultrafast 2D NMR spectroscopy using a continuous spatial encoding of the spin interactions.

A new protocol for acquiring multidimensional NMR spectra within a single scan is introduced and illustrated. The approach relies on applying a pair of frequency-chirped excitation and storage pulses in combination with echoing magnetic field gradients, in order to impart the kind of linear spatial encoding of the NMR interactions that is required by ultrafast 2D NMR spectroscopy. It is found that when dealing with 2D NMR experiments involving a t1 amplitude-modulation of the spin evolution, such continuous encoding scheme presents a number of advantages over alternatives employing discrete excitation pulses. From an experimental standpoint this is mainly reflected by the use of a single pair of bipolar gradients during the course of the indirect-domain encoding, as opposed to the numerous (and more intense) gradient echoes required so far. In terms of the spectral outcome, main advantages of the continuous spatial encoding scheme are the avoidance of "ghost peaks" and of "enveloping effects" associated to the discrete excitation mode. The principles underlying this new spatial encoding protocol are derived, and its applicability is demonstrated with homo- and heteronuclear 2D ultrafast NMR applications on small molecule and on protein samples.

Sub-second 2D NMR spectroscopy at sub-millimolar concentrations.

A recently proposed protocol enables the acquisition of two-dimensional nuclear magnetic resonance (2D NMR) spectra within a single scan. A promising application opened up by this new data acquisition mode concerns its combination with active nuclear polarization methods, whereby spectroscopy is carried out on analytes whose spin magnetizations have been significantly enhanced over their Boltzmann thermal values. The present paper explores the potential of such combination, with the acquisition of peptide and protein 2D NMR 1H correlation spectra recorded after the samples had been subject to laser-driven chemically induced dynamic nuclear polarization (CIDNP). It is demonstrated that the speed and sensitivity enhancement afforded by these combined processes enables the acquisition of quality 2D NMR data sets within a fraction of a second, at analyte concentrations that are under 1 mM.

Spatial encoding and the acquisition of high-resolution NMR spectra in inhomogeneous magnetic fields.

A scheme enabling the acquisition of high-resolution nuclear magnetic resonance (NMR) spectra within inhomogeneous magnetic fields is introduced and exemplified. The scheme is based on the spatial encoding protocol recently introduced for collecting multidimensional NMR data within a single scan, which retrieves spectral information via interference phenomena between spin packets located at distinct positions within the sample. This in turn enables compensating for field inhomogeneities over the sample as a whole by shifting the phases of the radio-frequency pulses involved in the spatial encoding, rather than by demanding an extreme uniformity in the employed magnetic field. The upper tolerable field inhomogeneity limit thus becomes orders of magnitude higher than that in conventional time-domain acquisitions. No particular spatial dependencies are demanded by the new scheme, which can yield its high-resolution results on a single-scan basis.

Hydrophobic complementarity in protein-protein docking.

Formation of hydrophobic contacts across a newly formed interface is energetically favorable. Based on this observation we developed a geometric-hydrophobic docking algorithm that estimates quantitatively the hydrophobic complementarity at protein-protein interfaces. Each molecule to be docked is represented as a grid of complex numbers, storing information regarding the shape of the molecule in the real part and information regarding the hydropathy of the surface in the imaginary part. The grid representations are correlated using fast Fourier transformations. The algorithm is used to compare the extent of hydrophobic complementarity in oligomers (represented by D2 tetramers) and in hetero-dimers of soluble proteins (complexes). We also test the implication of hydrophobic complementarity in distinguishing correct from false docking solutions. We find that hydrophobic complementarity at the interface exists in oligomers and in complexes, and in both groups the extent of such complementarity depends on the size of the interface. Thus, the non-polar portions of large interfaces are more often juxtaposed than non-polar portions of small interfaces. Next we find that hydrophobic complementarity helps to point out correct docking solutions. In oligomers it significantly improves the ranks of nearly correct reassembled and modeled tetramers. Combining geometric, electrostatic and hydrophobic complementarity for complexes gives excellent results, ranking a nearly correct solution < 10 for 5 of 23 tested systems, < 100 for 8 systems and < 1000 for 19 systems.

Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation.

We have recently proposed and demonstrated an approach that enables the acquisition of multidimensional nuclear magnetic resonance (NMR) spectra within a single scan. A promising application opened up by this new accelerated form of data acquisition concerns the possibility of monitoring in real time the chemical nature of analytes subject to a continuous flow. The present paper illustrates such potential, with the real-time acquisition of a series of 2D 1H NMR spectra arising from a mixture of compounds subject to a continuous liquid chromatography (LC) separation. This real-time 2D NMR identification of chemicals eluted minutes apart under usual LC-NMR conditions differs from the way in which LC-2D NMR has hitherto been carried out, which relies on stopped-flow modes of operations whereby fractions are first collected and then subject to individual, aliquot-by-aliquot analyses. The real-time LC-2D NMR experiment hereby introduced can be implemented in a straightforward manner using modern commercial LC-NMR hardware, thus opening up immediate possibilities in high-throughput characterizations of complex molecules.

Line shape considerations in ultrafast 2D NMR.

We have recently proposed and demonstrated an approach that enables the acquisition of 2D nuclear magnetic resonance (NMR) spectra within a single scan. The approach is based on spatially encoding the spins' evolution along the indirect domain with the aid of a magnetic field gradient, and subsequently decoding this information numerous times over the course of the signal acquisition while spins are subject to a train of gradient echoes. The present paper discusses further considerations pertaining the 2D line shapes arising from this new way of collecting NMR data. Specific issues that are hereby addressed include (i) the effects introduced by fast relaxation onto the spatial encoding process, particularly the line widths and line shapes that will then arise in the frequency domain; (ii) approaches capable of correcting for the mixed-phase kernels resulting in these fast-relaxation cases, corresponding in essence to spatially encoded analogs of the TPPI and hypercomplex time-domain acquisition procedures; (iii) the enveloping characteristics imposed by the use of discrete excitation pulses on the attainable spectral widths along the indirect domain; and (iv) an analysis of the signal-to-noise characteristics of the methodology, with experimental corroborations of theoretical predictions and an illustration of the method's capabilities to analyze protein solutions in the mM-range concentration.

Arrayed acquisition of 2D exchange NMR spectra within a single scan experiment.

We have recently demonstrated that magnetic field gradients in combination with frequency selective pulses, can be employed to collect a complete multi-dimensional NMR spectrum within a single scan. Following similar guidelines, field gradients could also be exploited to parallelize other types of NMR experiments where the final results arise from the collection and analysis of a series of time-incremented spectra. The present Communication exemplifies this concept by showing how a combination of gradients can be employed to monitor within a single continuous acquisition, a slow dynamic process which is in turn followed by systematic increments in the duration of a magnetization transfer time. Further, since 2D exchange NMR spectra can nowadays be themselves collected within one scan, the acquisition of a complete set of mixing-incremented 2D exchange patterns could be achieved within a single experiment entailing a total time of approximately 1 s.

Prediction of the unknown: inspiring experience with the CAPRI experiment.

We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our submitted models included acceptable, medium accuracy predictions of the structures of the complexes, and for a fifth target we identified the location of the binding site of one of the molecules. We used a weighted-geometric docking algorithm in which contacts involving specified parts of the surfaces of either one or both molecules were up-weighted or down-weighted. The weights were based on available structural and biochemical data or on sequence analyses. The weighted-geometric docking proved very useful for five targets, improving the complementarity scores and the ranks of the nearly correct solutions, as well as their statistical significance. In addition, the weighted-geometric docking promoted formation of clusters of similar solutions, which include more accurate predictions.