Poly(Sitosterol)-Based Hydrophobic Blocks in Amphiphilic Block Copolymers for the Assembly of Hybrid Vesicles.

Amphiphilic block copolymer and lipids can be assembled into hybrid vesicles (HVs), which are an alternative to liposomes and polymersomes. Block copolymers that have either poly(sitostryl methacrylate) or statistical copolymers of sitosteryl methacrylate and butyl methacrylate as the hydrophobic part and a poly(carboxyethyl acrylate) hydrophilic segment are synthesized and characterized. These block copolymers assemble into small HVs with soybean L-α-phosphatidylcholine (soyPC), confirmed by electron microscopy and small-angle X-ray scattering. The membrane's hybrid nature is illustrated by fluorescence resonance energy transfer between labeled building blocks. The membrane packing, derived from spectra when using Laurdan as an environmentally sensitive fluorescent probe, is comparable between small HVs and the corresponding liposomes with molecular sitosterol, although the former show indications of transmembrane asymmetry. Giant HVs with homogenous distribution of the block copolymers and soyPC in their membranes are assembled using the electroformation method. The lateral diffusion of both building blocks is slowed down in giant HVs with higher block copolymer content, but their permeability toward (6)-carboxy-X-rhodamine is higher compared to giant vesicles made of soyPC and molecular sitosterol. This fundamental effort contributes to the rapidly expanding understanding of the integration of natural membrane constituents with designed synthetic compounds to form hybrid membranes.

On-Site Measurement of Fat and Protein Contents in Milk Using Mobile NMR Technology.

Robust and easy-to-use NMR sensor technology is proposed for accurate, on-site determination of fat and protein contents in milk. The two parameters are determined using fast consecutive 1H and 35Cl low-field NMR experiments on milk samples upon the 1:1 addition of a low-cost contrast solution. Reliable and accurate measurements are obtained without tedious calibrations and the need for extensive database information and may readily be conducted by non-experts in production site environments. This enables on-site application at farms or dairies, or use in laboratories harvesting significant reductions in costs and time per analysis as compared to wet-chemistry analysis. The performance is demonstrated for calibration samples, various supermarket milk products, and raw milk samples, of which some were analyzed directly in the milking room. To illustrate the wide application range, the supermarket milk products included both conventionally/organically produced, lactose-free milk, cow's, sheep's and goat's milk, homogenized and unhomogenized milk, and a broad nutrient range (0.1-9% fat, 1-6% protein). Excellent agreement between NMR measurements and reference values, without corrections or changes in calibration for various products and during extensive periods of experiment conduction (4 months) demonstrates the robustness of the procedure and instrumentation. For the raw milk samples, correlations between NMR and IR, NMR and wet-chemistry, as well as IR and wet-chemistry results, show that NMR, in terms of accuracy, compares favorably with the other methods.

Cryogen-free dissolution dynamic nuclear polarization polarizer operating at 3.35 T, 6.70 T, and 10.1 T.

PURPOSE: A novel dissolution dynamic nuclear polarization (dDNP) polarizer platform is presented. The polarizer meets a number of key requirements for in vitro, preclinical, and clinical applications. METHOD: It uses no liquid cryogens, operates in continuous mode, accommodates a wide range of sample sizes up to and including those required for human studies, and is fully automated. RESULTS: It offers a wide operational window both in terms of magnetic field, up to 10.1 T, and temperature, from room temperature down to 1.3 K. The polarizer delivers a 13 C liquid state polarization for [1-13 C]pyruvate of 70%. The build-up time constant in the solid state is approximately 1200 s (20 minutes), allowing a sample throughput of at least one sample per hour including sample loading and dissolution. CONCLUSION: We confirm the previously reported strong field dependence in the range 3.35 to 6.7 T, but see no further increase in polarization when increasing the magnetic field strength to 10.1 T for [1-13 C]pyruvate and trityl. Using a custom dry magnet, cold head and recondensing, closed-cycle cooling system, combined with a modular DNP probe, and automation and fluid handling systems, we have designed a unique dDNP system with unrivalled flexibility and performance.

A unified heteronuclear decoupling picture in solid-state NMR under low radio-frequency amplitude and fast magic-angle-spinning frequency regime.

Heteronuclear spin decoupling is a highly important component of solid-state NMR experiments to remove undesired coupling interactions between unlike spins for spectral resolution. Recently, experiments using a unification strategy of standard decoupling schemes were presented for high radio-frequency (RF) amplitudes and slow-intermediate magic-angle-spinning (MAS) frequencies, in the pursuit of deeper understanding of spin decoupling under phase-modulated RF irradiation [A. Equbal et al., J. Chem. Phys. 142, 184201 (2015)]. The approach, unified two-pulse heteronuclear decoupling (UTPD), incorporates the simultaneous time- and phase-modulation strategies, commonly used in solid-state NMR. Here, the UTPD based decoupling scheme is extended to the experimentally increasingly important regime of low RF amplitudes and fast MAS frequencies. The unified decoupling approach becomes increasingly effective in identifying the deleterious dipole-dipole and, in particular, J recoupling conditions which become critical for the low-amplitude RF regime. This is because J coupling is isotropic and therefore not averaged out by sample spinning unlike the anisotropic dipole-dipole coupling. Numerical simulations and analytic theory are used to understand the effects of various nuclear spin interactions on the decoupling performance of UTPD, in particular, the crucial difference between the low-phase and high-phase UTPD conditions with respect to J coupling. In the UTPD scheme, when the cycle-frequency of the pulse-sequence is comparable to the RF nutation frequency, the existence of a non-zero effective rotation in the basic two-pulse scheme becomes an essential feature for the efficient and robust averaging out of the scalar J coupling. This broad viewpoint is expected to bring different optimum low-power decoupling pulse schemes under a common footing.

Fast Wide-Line Solid-State NMR on a Low-Cost Benchtop Spectrometer.

Solid-state NMR may provide access to a wealth of information on molecular structure and dynamics. However, for many applications, the acquisition is challenged by broad resonances implying large spectral linewidths and low sensitivity. Conventionally, this is tackled by using costly and laboratory-fixed spectrometers based on large high-field superconducting magnets. In this Communication, we demonstrate that a range of challenging wide-line solid-state NMR spectra can be acquired on a robust, maintenance-free, low-cost benchtop/mobile NMR spectrometer with a sensitivity comparable to common high-field instruments. The performance and versatility for recording sensitive wide-line spectra is demonstrated through acquisition of 31 P NMR of paramagnetic FePO4 and full quadupolar lineshapes of Al2 O3 (27 Al) and KNO3 (14 N). Also, we introduce interleaved acquisition of frequency-stepped slices providing a dramatic reduction of the required experiment time.

Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation.

Protease inhibition by metastable serine protease inhibitors (serpins) is mediated by one of the largest functional intradomain conformational changes known in biology. In this extensive structural rearrangement, protease-serpin complex formation triggers cleavage of the serpin reactive center loop (RCL), its subsequent insertion into central ß-sheet A, and covalent trapping of the target protease. In this study, we present the first detailed accelerated molecular dynamics simulation of the insertion of the fully cleaved RCL in α-1-antitrypsin (α1AT), the archetypal member of the family of human serpins. Our results reveal internal water pathways that allow the initial incorporation of side chains of RCL residues into the protein interior. We observed structural plasticity of the helix F (hF) element that blocks the RCL path in the native state, which is in excellent agreement with previous experimental reports. Furthermore, the simulation suggested a novel role of hF and the connected turn (thFs3A) as chaperones that support the insertion process by reducing the conformational space available to the RCL. Transient electrostatic interactions of RCL residues potentially fine-tune the serpin inhibitory activity. On the basis of our simulation, we generated the α1AT mutants K168E, E346K, and K168E/E346K and analyzed their inhibitory activity along with their intrinsic stability and heat-induced polymerization. Remarkably, the E346K mutation exhibited enhanced inhibitory activity along with an increased rate of premature structural collapse (polymerization), suggesting a significant role of E346 in the gatekeeping of the strain in the metastable native state.

Local SAR, global SAR, and power-constrained large-flip-angle pulses with optimal control and virtual observation points.

PURPOSE: To present a constrained optimal-control (OC) framework for designing large-flip-angle parallel-transmit (pTx) pulses satisfying hardware peak-power as well as regulatory local and global specific-absorption-rate (SAR) limits. The application is 2D and 3D spatial-selective 90° and 180° pulses. THEORY AND METHODS: The OC gradient-ascent-pulse-engineering method with exact gradients and the limited-memory Broyden-Fletcher-Goldfarb-Shanno method is proposed. Local SAR is constrained by the virtual-observation-points method. Two numerical models facilitated the optimizations, a torso at 3 T and a head at 7 T, both in eight-channel pTx coils and acceleration-factors up to 4. RESULTS: The proposed approach yielded excellent flip-angle distributions. Enforcing the local-SAR constraint, as opposed to peak power alone, reduced the local SAR 7 and 5-fold with the 2D torso excitation and inversion pulse, respectively. The root-mean-square errors of the magnetization profiles increased less than 5% with the acceleration factor of 4. CONCLUSION: A local and global SAR, and peak-power constrained OC large-flip-angle pTx pulse design was presented, and numerically validated for 2D and 3D spatial-selective 90° and 180° pulses at 3 T and 7 T. Magn Reson Med 77:374-384, 2017. © 2015 Wiley Periodicals, Inc.

Significance of symmetry in the nuclear spin Hamiltonian for efficient heteronuclear dipolar decoupling in solid-state NMR: A Floquet description of supercycled rCW schemes.

Symmetry plays an important role in the retention or annihilation of a desired interaction Hamiltonian in NMR experiments. Here, we explore the role of symmetry in the radio-frequency interaction frame Hamiltonian of the refocused-continuous-wave (rCW) pulse scheme that leads to efficient 1H heteronuclear decoupling in solid-state NMR. It is demonstrated that anti-periodic symmetry of single-spin operators (Ix, Iy, Iz) in the interaction frame can lead to complete annihilation of the 1H-1H homonuclear dipolar coupling effects that induce line broadening in solid-state NMR experiments. This symmetry also plays a critical role in cancelling or minimizing the effect of 1H chemical-shift anisotropy in the effective Hamiltonian. An analytical description based on Floquet theory is presented here along with experimental evidences to understand the decoupling efficiency of supercycled (concatenated) rCW scheme.

Parameter independent low-power heteronuclear decoupling for fast magic-angle spinning solid-state NMR.

Major advances have recently been made in the field of heteronuclear dipolar decoupling in solid-state nuclear magnetic resonance (NMR). These developments have improved the resolution and sensitivity of the NMR spectrum of spins coupled to protons. One such new scheme, denoted as rCWApA, has proven to be robust with practically no need for parameter optimization [A. Equbal et al. Chem. Phys. Lett., 635, 339 (2015)]. Most of the experiments with rCWApA have been carried out in the regimes of slow to moderate magic-angle spinning while simultaneously applying high decoupling radio-frequency amplitudes. Here, we explore the performance of the rCWApA sequence and its predecessor rCWA in the regime of low-power radio-frequency irradiation and fast magic-angle spinning. The robustness of the refocused continuous-wave (rCW) schemes to experimental parameters such as pulse lengths and offset irradiation is demonstrated. Numerical simulations and analytical theory have been used to understand the effects of various nuclear spin interactions on the decoupling performance of the low-power rCW decoupling scheme relative to other decoupling methods. This has lead to the design of an "optimum low-power decoupling sequence" that can be used without parameter optimization. This result is particularly important in the context of samples with low signal to noise.

A general theoretical description of the influence of isotropic chemical shift in dipolar recoupling experiments for solid-state NMR.

We present a general theoretical description that allows us to describe the influence of isotropic chemical shift in homonuclear and heteronuclear dipolar recoupling experiments in magic-angle-spinning solid-state NMR. Through a transformation of the Hamiltonian into an interaction frame with the combined radio-frequency irradiation and the isotropic chemical shift, we determine an effective Hamiltonian to first order with respect to the relevant internal nuclear spin interactions. This unravels the essential resonance conditions for efficient dipolar recoupling. Furthermore, we propose how to handle situations where the resonance conditions are not exactly fulfilled. To verify the general theoretical description, we compare numerical simulations using a time-sliced time-dependent Hamiltonian with simulations using the calculated effective Hamiltonian for propagation. The comparisons are exemplified for the homonuclear dipolar recoupling experiments C721 and POST-C721.

Theranostic poly(lactic-co-glycolic acid) nanoparticle for magnetic resonance/infrared fluorescence bimodal imaging and efficient siRNA delivery to macrophages and its evaluation in a kidney injury model.

In this work, a theranostic nanoparticle was developed for multimodal imaging and siRNA delivery. The core of the nanoparticles (NP) was formed by encapsulation of superparamagnetic iron oxides and indocyanine green in a PLGA matrix to serve as a multimodal probe for near-infrared (NIFR) and magnetic resonance (MR) imaging. The surface of the particle was coated with polyethylenimine (PEI) for siRNA delivery. Macrophages efficiently took up the nanoparticles and emitted strong NIFR and MR contrast. When transfected with siRNA targeting the pro-inflammatory enzyme cyclooxygenase-2 (COX-2), significant down-regulation of COX-2 was achieved in activated macrophages. Furthermore, after injection into a unilateral ureteral obstruction (UUO)-induced kidney injury model, NIFR and MRI imaging revealed accumulation of nanoparticles in the injury kidney. In addition, in vivo silencing of COX-2 was achieved by NP/PEI/siCOX-2, which further attenuated kidney injury. Our theranostic platform represents a promising approach for simultaneous diagnosis and treatment of inflammatory diseases.

Mild heat treatments induce long-term changes in metabolites associated with energy metabolism in Drosophila melanogaster.

Heat-induced hormesis, the beneficial effect of mild heat-induced stress, increases the average lifespan of many organisms. Yet little is known about the mechanisms underlying this effect. We used nuclear magnetic resonance spectroscopy to investigate the long-term effects of repeated mild heat treatments on the metabolome of male Drosophila melanogaster. 10 days after the heat treatment, metabolic aging appears to be slowed down, and a treatment response with 40 % higher levels of alanine and lactate and lower levels of aspartate and glutamate were measured. All treatment effects had disappeared 16 days later. Metabolic reprogramming has been associated with the life extending effects of dietary restriction. The metabolite changes induced by the hormetic treatment suggest that the positive effects might not be limited to the repair pathways induced, but that there also is a change in energy metabolism. A possible direct link between changes in energy metabolism and heat induced increase in Hsp70 expression is discussed.

Highly efficient 19F heteronuclear decoupling in solid-state NMR spectroscopy using supercycled refocused-CW irradiation.

We present heteronuclear 19F refocused CW (rCW) decoupling pulse sequences for solid-state magic-angle-spinning NMR applications. The decoupling sequences have been designed specifically to ensure suppression of the pertinent 13C-19F dipolar coupling interactions while simultaneously suppressing strong anisotropic chemical shift as well as homonuclear 19F-19F dipolar coupling effects as typically present in perfluorated compounds. In an extensive numerical and experimental analysis using a rigid, organic solid as a model compound, it becomes evident that the supercycled rCW schemes markedly improve the decoupling efficiency, leading to substantial enhancements in resolution and sensitivity when compared to previous state-of-the-art methods. Furthermore, considerable gains in robustness toward rf mismatch as well as offset in the radio-frequency carrier frequency are observed, all of which clearly render the new rCW schemes the methods of choice for 19F decoupling in rigid, fluorinated compounds - which is further supported by a Floquet-based theoretical analysis.

Handling the influence of chemical shift in amplitude-modulated heteronuclear dipolar recoupling solid-state NMR.

We present a theoretical analysis of the influence of chemical shifts on amplitude-modulated heteronuclear dipolar recoupling experiments in solid-state NMR spectroscopy. The method is demonstrated using the Rotor Echo Short Pulse IRrAdiaTION mediated Cross-Polarization ((RESPIRATION)CP) experiment as an example. By going into the pulse sequence rf interaction frame and employing a quintuple-mode operator-based Floquet approach, we describe how chemical shift offset and anisotropic chemical shift affect the efficiency of heteronuclear polarization transfer. In this description, it becomes transparent that the main attribute leading to non-ideal performance is a fictitious field along the rf field axis, which is generated from second-order cross terms arising mainly between chemical shift tensors and themselves. This insight is useful for the development of improved recoupling experiments. We discuss the validity of this approach and present quaternion calculations to determine the effective resonance conditions in a combined rf field and chemical shift offset interaction frame transformation. Based on this, we derive a broad-banded version of the (RESPIRATION)CP experiment. The new sequence is experimentally verified using SNNFGAILSS amyloid fibrils where simultaneous (15)N → (13)CO and (15)N → (13)Cα coherence transfer is demonstrated on high-field NMR instrumentation, requiring great offset stability.

Improved transfer efficiencies in radio-frequency-driven recoupling solid-state NMR by adiabatic sweep through the dipolar recoupling condition.

The homonuclear radio-frequency driven recoupling (RFDR) experiment is commonly used in solid-state NMR spectroscopy to gain insight into the structure of biological samples due to its ease of implementation, stability towards fluctuations/missetting of radio-frequency (rf) field strength, and in general low rf requirements. A theoretical operator-based Floquet description is presented to appreciate the effect of having a temporal displacement of the π-pulses in the RFDR experiment. From this description, we demonstrate improved transfer efficiency for the RFDR experiment by generating an adiabatic passage through the zero-quantum recoupling condition. We have compared the performances of RFDR and the improved sequence to mediate efficient (13)CO to (13)Cα polarization transfer for uniformly (13)C,(15)N-labeled glycine and for the fibril forming peptide SNNFGAILSS (one-letter amino acid codes) uniformly (13)C,(15)N-labeled at the FGAIL residues. Using numerically optimized sweeps, we get experimental gains of approximately 20% for glycine where numerical simulations predict an improvement of 25% relative to the standard implementation. For the fibril forming peptide, using the same sweep parameters as found for glycine, we have gains in the order of 10%-20% depending on the spectral regions of interest.

Theranostic tumor targeted nanoparticles combining drug delivery with dual near infrared and 19F magnetic resonance imaging modalities.

Combining imaging and drug delivery of "theranostic" nanoparticles has enabled concurrent diagnosis and therapy of diseases. Here, we describe a novel theranostic system that combines two imaging tracers, perfluorooctyl bromide (PFOB) for 19F magnetic resonance imaging (MRI) and indocyanine green (ICG) for near infrared (NIR) imaging, with the chemotherapeutic agent doxorubicin (Dox) into poly (lactic-co-glycolic acid)- poly (ethylene-glycol)-folate (PLGA-PEG-folate) nanoparticles. Cell culture studies using flow cytometry, confocal laser scanning microscope imaging, and 19F MRI showed enhanced uptake of nanoparticles via folate receptors expressed on human nasopharyngeal epidermal carcinoma (KB) cells. In vivo, higher MRI and fluorescence signals were obtained from tumors with 19F MRI and NIR, respectively, using folate-receptor-targeted nanoparticles compared with non-targeted equivalents. An in vitro cytotoxicity assay showed that folate-targeted nanoparticles were able to kill cancer cells more efficiently than non-folate conjugated particles. Our results suggest a potential use of PLGA-PEG-folate PFOB/ICG/Dox nanoparticles as a targeted chemotherapy agent traceable by either 19F MRI or NIR imaging.

Natural abundant (17) O NMR in a 1.5-T Halbach magnet.

We present mobile, low-field (17) O NMR as a means for monitoring oxygen in liquids. Whereas oxygen is one of the most important elements, oxygen NMR is limited by a poor sensitivity related to low natural abundance and gyro-magnetic ratio of the NMR active (17) O isotope. Here, we demonstrate (17) O NMR detection at a Larmor frequency of 8.74 MHz in a 1.5-T Halbach neodymium magnet with a home-built digital NMR instrument suitable for large-scale production and in-line monitoring applications. The proposed (17) O NMR sensor may be applied for direct, noninvasive measurements of water content in, for example, oil, manure, or food in automated quality or process control. Copyright © 2015 John Wiley & Sons, Ltd.

Early Events in the Amyloid Formation of the A546T Mutant of Transforming Growth Factor ß-Induced Protein in Corneal Dystrophies Compared to the Nonfibrillating R555W and R555Q Mutants.

The human transforming growth factor ß-induced protein (TGFBIp) is involved in several types of corneal dystrophies where protein aggregation and amyloid fibril formation severely impair vision. Most disease-causing mutations are located in the last of four homologous fasciclin-1 (FAS1) domains of the protein, and it has been shown that when isolated, the fourth FAS1 domain (FAS1-4) mimics the behavior of full-length TGFBIp. In this study, we use molecular dynamics simulations and principal component analysis to study the wild-type FAS1-4 domain along with three disease-causing mutations (R555W, R555Q, and A546T) to decipher any internal difference in dynamical properties of the domains that may explain their varied stabilities and aggregation properties. In addition, we use a protein-protein docking method in combination with chemical cross-linking experiments and mass spectrometry of the cross-linked species to obtain information about interaction faces between identical FAS1-4 domains. The results show that the pathogenic mutations A546T and R555W affect the packing in the hydrophobic core of FAS1-4 in different directions. We further show that the FAS1-4 monomers associate using their ß-rich regions, consistent with peptides observed to be part of the amyloid fibril core in lattice corneal dystrophy patients.

Optimized co-solute paramagnetic relaxation enhancement for the rapid NMR analysis of a highly fibrillogenic peptide.

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T 1 relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T 1 values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in 'proton-less' NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α1-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.

NPK NMR Sensor: Online Monitoring of Nitrogen, Phosphorus, and Potassium in Animal Slurry.

Knowledge of the actual content of nitrogen, phosphorus, and potassium (NPK) in animal slurry is highly important to optimize crop production and avoid environmental pollution when slurry is spread on agricultural fields. Here, we present a mobile, low-field nuclear magnetic resonance (NMR) sensor suitable for online monitoring of the NPK content in animal slurry as an alternative to crude estimates or tedious nonspecific, off-site laboratory analysis. The sensor is based on (14)N, (17)O, (31)P, and (39)K NMR in a digital NMR instrument equipped with a 1.5 T Halbach magnet for direct detection of ammonium N, total P, and K and indirect evaluation of the organic N content, covering all practical components of NPK in animal slurry. In correlation studies, the obtained NMR measurements show good agreement with reference measurements from commercial laboratories.