Isotope effects observed in diluted D2O/H2O mixtures identify HOD-induced low-density structures in D2O but not H2O.

Normal and heavy water are solvents most commonly used to study the isotope effect. The isotope effect of a solvent significantly influences the behavior of a single molecule in a solution, especially when there are interactions between the solvent and the solute. The influence of the isotope effect becomes more significant in D2O/H2O since the hydrogen bond in H2O is slightly weaker than its counterpart (deuterium bond) in D2O. Herein, we characterize the isotope effect in a mixture of normal and heavy water on the solvation of a HOD molecule. We show that the HOD molecule affects the proximal solvent molecules, and these disturbances are much more significant in heavy water than in normal water. Moreover, in D2O, we observe the formation of low-density structures indicative of an ordering of the solvent around the HOD molecule. The qualitative differences between HOD interaction with D2O and H2O were consistently confirmed with Raman spectroscopy and NMR diffusometry.

NKX2-5 Variant in Two Siblings with Thyroid Hemiagenesis.

Thyroid hemiagenesis (THA) is an inborn absence of one thyroid lobe of largely unknown etiopathogenesis. The aim of the study was to reveal genetic factors responsible for thyroid maldevelopment in two siblings with THA. None of the family members presented with congenital heart defect. The samples were subjected to whole-exome sequencing (WES) (Illumina, TruSeq Exome Enrichment Kit, San Diego, CA 92121, USA). An ultra-rare variant c.839C>T (p.Pro280Leu) in NKX2-5 gene (NM_004387.4) was identified in both affected children and an unaffected father. In the mother, the variant was not present. This variant is reported in population databases with 0.0000655 MAF (GnomAD v3, dbSNP rs761596254). The affected amino acid position is moderately conserved (positive scores in PhyloP: 1.364 and phastCons: 0.398). Functional prediction algorithms showed deleterious impact (dbNSFP v4.1, FATHMM, SIFT) or benign (CADD, PolyPhen-2, Mutation Assessor). According to ACMG criteria, variant is classified as having uncertain clinical significance. For the first time, NKX2-5 gene variants were found in two siblings with THA, providing evidence for its potential contribution to the pathogenesis of this type of thyroid dysgenesis. The presence of the variant in an unaffected parent, carrier of p.Pro280Leu variant, suggests potential contribution of yet unidentified additional factors determining the final penetrance and expression.

Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV-vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.

The Natural Oligoribonucleotides Functionalized by D-Mannitol Affected Interactions of Hemagglutinin with Glycan Receptor Indicating Anti-Influenza Activity.

Hemagglutinin (HA), the class I influenza A virus protein is responsible for the attachment of virus particles to the cell by binding to glycan receptors, subsequent virion internalization, and cell entry. Consequently, the importance of HA makes it a primary target for the development of anti-influenza drugs. The natural oligoribonucleotides (ORNs) as well as their derivatives functionalized with D-mannitol (ORNs-D-M) possess anti-influenza properties in vitro and in vivo due to interaction with HA receptor sites. This activity suppresses the viral infection in host cells. In the present work, the complexes of ORNs and ORNs-D-M with HA protein were studied by agglutination assay, fluorescence spectroscopy, as well as molecular docking simulations. Acquired experimental data exhibited a decrease in HA titer by 32 times after incubation with the ORNs-D-M for 0.5-24 h. Quenching fluorescence intensity of the HA suggests that titration by ORNs and ORNs-D-M probably leads to changes in the HA structure. Detailed structural data were obtained with the molecular docking simulations performed for ORNs and ORNs-D-M ligands containing three and six oligoribonucleotides. The results reveal that a majority of the ORNs and ORNs-D-M bind in a non-specific way to the receptor-binding domain of the HA protein. The ligand's affinity to the hemagglutinin was estimated at the micromolar level. Presented experimental data confirmed that both natural ORNs and functionalized ORNs-D-M inhibit the interactions between HA and glycan receptors and demonstrate anti-influenza activity.

Diffusion, Interactions, and Disparate Kinetic Trapping of Water-Hydrocarbon Mixtures in Nanoporous Solids.

Mixed fluids confined in porous solid hosts present challenges for the accurate characterization of individual-component behavior. NMR diffusometry with chemical resolution is used to identify unexpected loading- and composition-dependent anomalous diffusion in water/cyclohexane mixtures confined to solid nanoporous glass (NPG) hosts. Diffusion NMR results indicate that data obtained on pure-component liquids in confinement cannot be extrapolated to their nonideal liquid mixtures confined in the same solid host. Loading-dependent data must be obtained on each component in the confined mixture in order to determine which of the liquid components exhibits chemical affinity for the host and, conversely, which of the components exhibits anomalous diffusivity. Most notably, NMR diffusometry revealed that cyclohexane diffusivity varied by 2 orders of magnitude in a water-rich mixture depending on the total fluid loading in the NPG host, ranging from anomalously high diffusivities that significantly exceeded that for pure cyclohexane in NPG at low fluid loadings to kinetically trapped sequestration at high fluid loadings. NMR diffusometry indicates that nonideal solution behavior in fluids confined within nanoporous hosts may have practical implications for enhanced oil recovery methods. Specifically, kinetic trapping of hydrocarbons in water-flooding regimes can result from complex liquid-vapor equilibrium that is significantly perturbed from that which exists in bulk or microporous confinement.

The Palladium(II) Complex of Aß4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids.

The Aß4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aß4-16 and Aß4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aß4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aß4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aß4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aß4-16, Pd(II)-Aß4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aß complexes.

DMPC Phospholipid Bilayer as a Potential Interface for Human Cystatin C Oligomerization: Analysis of Protein-Liposome Interactions Using NMR Spectroscopy.

Studies revolving around mechanisms responsible for the development of amyloid-based diseases lay the foundations for the recognition of molecular targets of future to-be-developed treatments. However, the vast number of peptides and proteins known to be responsible for fibril formation, combined with their complexity and complexity of their interactions with various cellular components, renders this task extremely difficult and time-consuming. One of these proteins, human cystatin C (hCC), is a well-known and studied cysteine-protease inhibitor. While being a monomer in physiological conditions, under the necessary stimulus-usually a mutation, it tends to form fibrils, which later participate in the disease development. This process can potentially be regulated (in several ways) by many cellular components and it is being hypothesized that the cell membrane might play a key role in the oligomerization pathway. Studies involving cell membranes pose several difficulties; therefore, an alternative in the form of membrane mimetics is a very attractive solution. Here, we would like to present the first study on hCC oligomerization under the influence of phospholipid liposomes, acting as a membrane mimetic. The protein-mimetic interactions are studied utilizing circular dichroism, nuclear magnetic resonance, and size exclusion chromatography.

AT101-Loaded Cubosomes as an Alternative for Improved Glioblastoma Therapy.

INTRODUCTION: AT101, the R-(-)-enantiomer of the cottonseed-derived polyphenol gossypol, is a promising drug in glioblastoma multiforme (GBM) therapy due to its ability to trigger autophagic cell death but also to facilitate apoptosis in tumor cells. It does have some limitations such as poor solubility in water-based media and consequent low bioavailability, which affect its response rate during treatment. To overcome this drawback and to improve the anti-cancer potential of AT101, the use of cubosome-based formulation for AT101 drug delivery has been proposed. This is the first report on the use of cubosomes as AT101 drug carriers in GBM cells. MATERIALS AND METHODS: Cubosomes loaded with AT101 were prepared from glyceryl monooleate (GMO) and the surfactant Pluronic F-127 using the top-down approach. The drug was introduced into the lipid prior to dispersion. Prepared formulations were then subjected to complex physicochemical and biological characterization. RESULTS: Formulations of AT101-loaded cubosomes were highly stable colloids with a high drug entrapment efficiency (97.7%) and a continuous, sustained drug release approaching 35% over 72 h. Using selective and sensitive NMR diffusometry, the drug was shown to be efficiently bound to the lipid-based cubosomes. In vitro imaging studies showed the high efficiency of cubosomal nanoparticles uptake into GBM cells, as well as their marked ability to penetrate into tumor spheroids. Treatment of GBM cells with the AT101-loaded cubosomes, but not with the free drug, induced cytoskeletal rearrangement and shortening of actin fibers. The prepared nanoparticles revealed stronger in vitro cytotoxic effects against GBM cells (A172 and LN229 cell lines), than against normal brain cells (SVGA and HMC3 cell lines). CONCLUSION: The results indicate that GMO-AT101 cubosome formulations are a promising basic tool for alternative approaches to GBM treatment.

Effect of PAMAM Dendrimers on Interactions and Transport of LiTFSI and NaTFSI in Propylene Carbonate-Based Electrolytes.

Poly(amidoamine) (PAMAM)-based electrolytes are prepared by dissolving the PAMAM half-generations G1.5 or G2.5 in propylene carbonate (PC), either with lithium bis(trifluoromethylsulfonyl)imide (LiTFSI) or sodium bis(trifluoromethylsulfonyl)imide (NaTFSI) salts. The solutions, designed for ion battery applications, are studied in terms of ions transport properties. Raman Spectroscopy reveals information about the interactions between cations and PAMAM dendrimers as well as full dissociation of the salts in all solutions. Pulsed-field gradient Nuclear Magnetic Resonance (PFG NMR), measured as a function of both temperature and PAMAM concentration, are obtained for the cation, anion, solvent, and dendrimer molecules using lithium (7Li), sodium (23Na), fluorine (19F), and hydrogen (1H) NMR, respectively. It was found that lithium diffusion is slow compared to the larger TFSI anion and decreases with PAMAM concentration due to interactions between cation and dendrimer. Comparison of conductivities calculated from diffusion coefficients using the Nernst-Einstein equation, with conductivity measurements obtained from Impedance Spectroscopy (IS), shows slightly higher IS conductivities, caused among others by PAMAM conductivity.

Structural Analysis and Dynamic Processes of the Transmembrane Segment Inside Different Micellar Environments-Implications for the TM4 Fragment of the Bilitranslocase Protein.

The transmembrane (TM) proteins are gateways for molecular transport across the cell membrane that are often selected as potential targets for drug design. The bilitranslocase (BTL) protein facilitates the uptake of various anions, such as bilirubin, from the blood into the liver cells. As previously established, there are four hydrophobic transmembrane segments (TM1-TM4), which constitute the structure of the transmembrane channel of the BTL protein. In our previous studies, the 3D high-resolution structure of the TM2 and TM3 transmembrane fragments of the BTL in sodium dodecyl sulfate (SDS) micellar media were solved using Nuclear Magnetic Resonance (NMR) spectroscopy and molecular dynamics simulations (MD). The high-resolution 3D structure of the fourth transmembrane region (TM4) of the BTL was evaluated using NMR spectroscopy in two different micellar media, anionic SDS and zwitterionic DPC (dodecylphosphocholine). The presented experimental data revealed the existence of an α -helical conformation in the central part of the TM4 in both micellar media. In the case of SDS surfactant, the α -helical conformation is observed for the Pro258-Asn269 region. The use of the zwitterionic DPC micelle leads to the formation of an amphipathic α -helix, which is characterized by the extension of the central α -helix in the TM4 fragment to Phe257-Thr271. The complex character of the dynamic processes in the TM4 peptide within both surfactants was analyzed based on the relaxation data acquired on 15 N and 31 P isotopes. Contrary to previously published and present observations in the SDS micelle, the zwitterionic DPC environment leads to intensive low-frequency molecular dynamic processes in the TM4 fragment.

The domain swapping of human cystatin C induced by synchrotron radiation.

Domain swapping is observed for many proteins with flexible conformations. This phenomenon is often associated with the development of conformational diseases. Importantly, domain swapping has been observed for human cystatin C (HCC), a protein capable of forming amyloid deposits in brain arteries. In this study, the ability of short exposure to high-intensity X-ray radiation to induce domain swapping in solutions of several HCC variants (wild-type HCC and V57G, V57D, V57N, V57P, and L68V mutants) was determined. The study was conducted using time-resolved small-angle X-ray scattering (TR-SAXS) synchrotron radiation. The protein samples were also analysed using small-angle neutron scattering and NMR diffusometry. Exposing HCC to synchrotron radiation (over 50 ms) led to a gradual increase in the dimeric fraction, and for exposures longer than 150 ms, the oligomer fraction was dominant. In contrast, the non-irradiated protein solutions, apart from the V57P variant, were predominantly monomeric (e.g., V57G) or in monomer/dimer equilibrium. This work might represent the first observation of domain swapping induced by high-intensity X-rays.

Nano-mediated delivery of double-stranded RNA for gene therapy of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fueled a search for new therapeutic targets and treatment modalities. Here we report new approach towards RNA interference therapy of glioblastoma multiforme based on the magnetic nanoparticles delivery of the double-stranded RNA (dsRNA) with homological sequences to mRNA of tenascin-C (TN-C), named ATN-RNA. The obtained nanocomposite consisted of polyethyleneimine (PEI) coated magnetic nanoparticles conjugated to the dsRNA show high efficiency in ATN-RNA delivery, resulting not only in significant TN-C expression level suppressesion, but also impairing the tumor cells migration. Moreover, synthesized nanomaterials show high contrast properties in magnetic resonance imaging (MRI) and low cytotoxicity combining with lack of induction of interferon response. We believe that the present work is a successful combination of effective, functional, non-immunostimulatory dsRNA delivery system based on magnetic nanoparticles with high potential for further application in GBM therapy.

Dendrimer based theranostic nanostructures for combined chemo- and photothermal therapy of liver cancer cells in vitro.

Here we report the synthesis of multifunctional nanocarriers based on PAMAM dendrimers generation (G) 4.0, 5.0 and 6.0 fixed to polydopamine (PDA) coated magnetite nanoparticles (Fe3O4). Synthesized nanoplatforms were characterized by transmission electron microscopy (TEM), the electrokinetic (zeta) potential, Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and magnetic resonance imaging (MRI). Further, we show as a proof of concept that nanocarriers functionalized with G 5.0 could be successfully applied in combined chemo- and photothermal therapy (CT-PTT) of the liver cancer cells. The cooperative effect of the modalities mentioned above led to higher mortality of cancer cells when compared to their individual performance. Moreover, the performed in vitro studies revealed that the application of dual therapy triggered the desired cell death mechanism-apoptosis. Furthermore, performed tests using Magnetic Resonance Imaging (MRI) showed that our materials have competitive contrast properties. Overall, the functionality of dendrimers has been extended by merging them with magnetic nanoparticles resulting in multifunctional hybrid nanostructures that are promising smart drug delivery system for cancer therapy.

Cyclodextrin-Based Magnetic Nanoparticles for Cancer Therapy.

Polydopamine (PDA)-coated magnetic nanoparticles functionalized with mono-6-thio-ß-cyclodextrin (SH-ßCD) were obtained and characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), Nuclear and Magnetic Resonance Imaging (NMR and MRI), and doxorubicin (DOXO)-loading experiments. The liver cancer cellular internalization of DOXO-loaded nanoparticles was investigated by confocal imaging microscopy. Synthesized nanomaterials bearing a chemotherapeutic drug and a layer of polydopamine capable of absorbing near-infrared light show high performance in the combined chemo- and photothermal therapy (CT-PTT) of liver cancer due to the synergistic effect of both modalities as demonstrated in vitro. Moreover, our material exhibits improved T2 contrast properties, which have been verified using Carr-Purcell-Meiboom-Gill pulse sequence and MRI Spin-Echo imaging of the nanoparticles dispersed in the agarose gel phantoms. Therefore, the presented results cast new light on the preparation of polydopamine-based magnetic theranostic nanomaterials, as well as on the proper methodology for investigation of magnetic nanoparticles in high field MRI experiments. The prepared material is a robust theranostic nanoasystem with great potential in nanomedicine.

Clear distinction between CAC and CMC revealed by high-resolution NMR diffusometry for a series of bis-imidazolium gemini surfactants in aqueous solutions.

The aggregation behavior in the transition region was studied for a series of dicationic surfactants 3,3'-[α,ω-(dioxaalkane)]bis(1-dodecylimidazolium)dichlorides with varied spacer length from two to twelve carbon atoms. We employed Nuclear Magnetic Resonance diffusometry and Bayesian DOSY analysis to obtain the aggregate size distribution in the transition region. The critical concentrations CC were independently obtained from surface tension, electric conductivity, UV-Vis and NMR methods. The micelle aggregation numbers were estimated from the self-diffusion coefficients and were independently confirmed using steady-state fluorescence quenching. The morphology of the aggregates was characterized by small-angle scattering of synchrotron radiation and molecular dynamics simulations. The obtained CC values are identified as critical aggregation concentrations CAC. A broad transition region was observed, and stable micelles were obtained at much higher concentrations than CAC. The accurate CMC values could not be identified for the systems in the study. We indicated that the distribution of aggregate size becomes small and the system becomes homogeneous at much larger concentrations than CAC (typically 15-20 mM). The existence of a slow exchange between two environments, an aggregate and aqueous environment, was confirmed by 1H NMR and 2D HSQC NMR spectroscopy.

Molecular dynamics of 1-ethyl-3-methylimidazolium triflate ionic liquid studied by 1H and 19F nuclear magnetic resonances.

The molecular dynamics of an ionic liquid (IL) composed of a 1-ethyl-3-methylimidazolium cation and a triflate (trifluoromethanesulfonate) anion, abbreviated as [Emim][TfO], were studied by NMR spectroscopy. By measuring the temperature-dependent high-field 1H and 19F spin-lattice relaxation (SLR) rates, the frequency-dependent 1H and 19F SLR dispersion curves using fast-field-cycling relaxometry, and the temperature-dependent 1H and 19F diffusion constants, and by utilizing the fact that the primary NMR-active nucleus on the Emim cation is 1H, whereas on the TfO anion it is 19F, the cationic and anionic dynamics were studied separately. A single theoretical relaxation model successfully reproduced all the experimental data of both types of resonant nuclei by fitting all the data simultaneously with the same set of fit parameters. Upon cooling, [Emim][TfO] exhibited a supercooled liquid phase between TSL = 256 K and the crystallization temperature TCr ≈ 227-222 K, as confirmed by differential scanning calorimetry (DSC) experiments. Theoretical analysis revealed that within the liquid and the supercooled liquid states of [Emim][TfO], the 1H and 19F relaxation rates are affected by both the rotational and translational diffusional processes with no discontinuous change at TSL. While the rotational diffusion is well described as an Arrhenius thermally activated process, the translational diffusion undergoes strong freezing dynamics that are well described by the Vogel-Fulcher model assuming a freezing temperature of T0 = 157 K. The existence of the supercooled liquid region in the [Emim][TfO] IL should be taken into account when using this IL for a specific application.

Spectroscopic, structural and in vitro cytotoxicity evaluation of luminescent, lanthanide doped core@shell nanomaterials GdVO4:Eu(3+)5%@SiO2@NH2.

The luminescent GdVO4:Eu(3+)5%@SiO2@NH2 core@shell nanomaterials were obtained via co-precipitation method, followed by hydrolysis and co-condensation of silane derivatives: tetraethyl orthosilicate and 3-aminopropyltriethoxysilane. Their effect on human erythrocytes sedimentation and on proliferation of human lung microvascular endothelial cells was examined and discussed. The luminescent nanoparticles were synthesized in the presence of polyacrylic acid or glycerin in order to minimalize the agglomeration and excessive growth of nanostructures. Surface coating with amine functionalized silica shell improved their biocompatibility, facilitated further organic conjugation and protected the internal core. Magnetic measurements revealed an enhanced T1-relaxivity for the synthesized GdVO4:Eu(3+)5% nanostructures. Structure, morphology and average grain size of the obtained nanomaterials were determined by X-ray diffraction, transmission electron microscopy and dynamic light scattering analysis. The qualitative elemental composition of the nanomaterials was established using energy-dispersive X-ray spectroscopy. The spectroscopic characteristic of red emitting core@shell nanophosphors was completed by measuring luminescence spectra and decays. The emission spectra revealed characteristic bands of Eu(3+) ions related to the transitions (5)D0-(7)F0,1,2,3,4 and (5)D1-(7)F1. The luminescence lifetimes consisted of two components, associated with the presence of Eu(3+) ions located at the surface of the crystallites and in the bulk.

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data.

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.

Long-range ordering in the lyotropic lamellar phase studied by high-resolution magnetic resonance diffusion-weighted imaging.

Diffusion-weighted magnetic resonance imaging (DW MRI) was applied to the lyotropic lamellar phase of the dodecylammonium chloride/water system (DDACl/H(2)O). In the course of employing a well-known medical imaging method, namely, diffusion tensor imaging (DTI), the system morphology was assessed accurately in the most straightforward way by two-dimensional visualization of eigenvectors associated with planar distribution of effective diffusion tensors throughout the whole slice with 40 microm in-plane resolution. Long-range order was observed in the studied lamellar phase, and morphology was best described by a combination of three- and one-dimensional diffusion.

Effective and accurate single-shot NMR diffusion experiments based on magnetization grating.

Current single-shot diffusion methods based on magnetization gratings suffer from low sensitivity due to small rf tip angles and, consequently, from inefficient use of the total equilibrium magnetization. Here, we propose and illustrate the use of a slightly modified form of the magnetization encoding scheme OUFIS for single-shot diffusion experiments. In a detailed theoretical and experimental analysis, we compare the performance of the proposed method to other encoding schemes such as the one-phase or two-phase DANTE and conclude that the OUFIS-based experiment is a superior one. The primary reason is that this scheme allows one to use a larger total pulse area. Hence, one can encode a far larger portion of the initial magnetization into a frequency grating before the onset of various nonlinear effects. In the experimental illustration, we present a single-shot measurement of multicomponent diffusion.