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The monitoring of location and degradation rates of injectable biomaterials is an area of particular interest in the design and implementation of therapeutic scaffolds and carriers for tissue repair and replacement. We describe here the fabrication and characterization of gadolinium (Gd)-labeled temperature-responsive hydrogels that can be detected noninvasively using T1-weight magnetic resonance. Two acrylamide-functionalized GdIIIDOTA-monoamide complexes with either a short n-butylene spacer (GdIII-C4-AA) or a long hydrophilic spacer (GdIII-PEG-AA) were synthesized and incorporated into the hydrogels. At temperatures above the lower critical solution temperature (LCST), 37 °C, these hydrogels have the capacity to enhance relaxivity (r1) due to the hydrophobic interactions of the polyamide chains around the gadolinium chelates. This effect is further accentuated by the presence of the polyethylene glycol groups of the Gd complex GdIII-PEG-AA.

Fluorine MRI ((19) F MRI) is receiving an increasing attention as a viable alternative to proton-based MRI ((1) H MRI) for dedicated application in molecular imaging. The (19) F nucleus has a high gyromagnetic ratio, a 100% natural abundance and is furthermore hardly present in human tissues allowing for hot spot MR imaging. The applicability of (19) F MRI as a molecular and cellular imaging technique has been exploited, ranging from cell tracking to detection and imaging of tumors in preclinical studies. In addition to applications, developing new contrast materials with improved relaxation properties has also been a core research topic in the field, since the inherently low longitudinal relaxation rates of perfluorocarbon compounds result in relatively low imaging efficiency. Borrowed from (1) H MRI, the incorporation of lanthanides, specifically Gd(III) complexes, as signal modulating ingredients in the nanoparticle formulation has emerged as a promising approach to improvement of the fluorine signal. Three different perfluorocarbon emulsions were investigated at five different magnetic field strengths. Perfluoro-15-crown-5-ether was used as the core material and Gd(III)DOTA-DSPE, Gd(III)DOTA-C6-DSPE and Gd(III)DTPA-BSA as the relaxation altering components. While Gd(III)DOTA-DSPE and Gd(III)DOTA-C6-DSPE were favorable constructs for (1) H NMR, Gd(III)DTPA-BSA showed the strongest increase in (19F) R1 . These results show the potential of the use of paramagnetic lipids to increase (19F) R1 at clinical field strengths (1.5-3 T). At higher field strengths (6.3-14 T), gadolinium does not lead to an increase in (19F) R1 compared with emulsions without gadolinium, but leads to an significant increase in (19F) R2 . Our data therefore suggest that the most favorable situation for fluorine measurements is at high magnetic fields without the inclusion of gadolinium constructs. Copyright © 2014 John Wiley & Sons, Ltd

Forced degradation studies are an important tool for a systematic assessment of decomposition pathways and identification of reactive sites in active pharmaceutical ingredients (APIs). Two methodologies have been combined in order to provide a deeper understanding of singlet oxygen-related degradation pathways of APIs under light irradiation. First, we report that a dark singlet oxygen test enables the investigation of drug reactivity toward singlet oxygen independently of photolytic irradiation processes. Second, the photosensitizing properties of the API producing the singlet oxygen was proven and quantified by spin trapping and electron paramagnetic resonance analysis. A combination of these techniques is an interesting addition to the forced degradation portfolio as it can be used for (1) revealing unexpected degradation pathways of APIs due to singlet oxygen, (2) clarifying photolytic drug-drug interactions in fixed-dose combinations, and (3) synthesizing larger quantities of hardly accessible oxidative drug degradants.

Synthesis of the modified decadeoxynucleotides 16–20 having the sequence d(G1pC2pG)3pT4x1T5x2T6x3T7pG8pC9pG10) and containing one (x2 = CH2, x1 = x3 = p, i.e., 16), two (x1 = x3 = CH2, x2 = p, i.e., 17, x2 = x3 = CH2, x1 = p, i.e., 18 and x1 = x2 = CH2, x3 = p. i.e., 19) or three (x1 = x2 = x3 = CH2, i.e., 20) -O-CH2-O- linkages could be accomplished using the respective mono-, di- or tri(-O-CH2-O) 3′-O-phosphoramidites 6, 10 and 14 as the incoming synthons. Hybridization of 16–20 with their native complementary strand afforded stable duplexes, which were studied by 1D- and 2D-1H-NMR and UV-hyperchromicity techniques.

Micron-sized particles with a narrow size distribution are prepared by ink-jet printing technology. Droplets of a polymer solution are printed with the nozzle submerged into an aqueous phase. The particles are formed upon removal of the solvent for the polymer. The particle size can be accurately predicted from the initial drop size and the polymer concentration. The method is also suitable for the preparation of monodisperse capsules with a well-defined core and shell using an additional non-solvent for the polymer. Hollow capsules are prepared by removal of the non-solvent from core of the capsules using freeze-drying. This leads to gas-filled capsules with a well-defined polymeric shell and a diameter in the range of 5 μm, which may be applied as an ultrasound contrast agent.

Contains fulltext : 57305.pdf (Publisher’s version ) (Closed access) Localized in vivo 1H magnetic resonance spectroscopy (MRS) was used to investigate metabolite levels in the brain of adult Zucker Diabetic Fatty (ZDF) rats, an animal model for type 2 diabetes mellitus. This study focussed on the hippocampus, assumed to be one of the main brain areas affected by this disease. Together with an almost 5-fold increase in blood glucose concentration measured by glucose oxidation, significant increases were found in the hippocampal concentrations of glucose (4.93 vs 1.66 mM p < 0.001), myo-inositol (6.52 vs 4.30 mM; p < 0.05), and total creatine (12.71 vs 10.50 mM; p < 0.05) in ZDF rats (n = 5) compared with littermates (n = 5). Although no obvious alterations were detected in the hippocampal levels of other metabolites, including NAA + NAAG and choline-containing compounds in the ZDF rats, the increase in Glc and Ins levels is in line with elevated brain tissue contents of these metabolites in patients with diabetes mellitus.

We have previously shown that the dense vascular network in mouse brain allows for growth of human melanoma xenografts (Mel57) by co-option of preexisting vessels. Overexpression of recombinant vascular endothelial growth factor-A (VEGF-A) by such xenografts induced functional and morphologic alterations of preexisting vessels. We now describe the effects of VEGF-A expression on visualization of these brain tumors in mice by magnetic resonance imaging (MRI), using gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA) and ultra small paramagnetic iron oxide particles (USPIO) as contrast agents. Brain lesions derived from (mock-transfected) Mel57 cells were undetectable in MRI after Gd-DTPA injection. However, the majority of such lesions became visible after injection of USPIO, due to the lower vascular density in the lesions as compared to the surrounding parenchyma. In contrast, VEGF-A-expressing lesions were visualized using Gd-DTPA-enhanced MRI by a rapid circumferential enhancement, due to leaky peritumoral vasculature. USPIO-enhanced MRI of these tumors corroborated the immunohistochemic finding that peritumorally located, highly irregular and dilated vessels were present, while intratumoral vessel density was low. Our study shows that VEGF-A is a key factor in imaging of brain neoplasms. Our data also demonstrate that, at least in brain, blood-pool agent-enhanced MRI may be a valuable diagnostic tool to detect malignancies that are not visible on Gd-DTPA-enhanced MRI. Furthermore, the involvement of VEGF-A in MRI visibility suggests that care must be taken with MRI-based evaluation of antiangiogenic therapy, as anti-VEGF treatment might revert a tumor to a co-opting phenotype, resulting in loss of contrast enhancement in MRI.

Molecular imaging is likely to have a significant impact onhealthcare through the early detection of disease on a cellularand molecular level. Among the clinical imaging modalities,magnetic resonance imaging (MRI) offers a unique combi-nation of advantages including the recording of anatomicaland contrast-enhanced images with a high spatial resolution,while avoiding the use of ionizing radiation. The use of MRIfor imaging sparse molecular epitopes present on diseasedcells is hampered by its low sensitivity, which can potentiallybe overcome with new contrast-amplifying nanocarriers.

We have investigated the thermodynamic properties of two homologous DNA four-way junctions, J4 and J4M, based on 46-mer linear DNA molecules. J4 and J4M have the same base sequence with the only difference that the latter contains an uncharged methylene–acetal linkage, -O3′-CH 2 -O5′, instead of the phosphodiester linkage, -O3′-PO 2 -O5′-, between the residues T18 and C19. The comparison of the thermal unfolding of the J4 junction and J4M junction serves to investigate the effect of the uncharged methylene–acetal linkage on the stability of the junction. Our analysis is based on CD, UV absorbance spectroscopy, DSC, and chemical footprinting. The aim is to characterize in detail the structure and stability of the junctions. As demonstrated before by NMR, in the presence of 5 mM MgCl 2 ± 50 mM NaCl, both J4 and J4M form a complete four-way junction. This is now evidenced by protection from OsO 4 cleavage (chemical footprinting). We can assume that full base pairing occurs throughout the arms even at the center of the junction. CD spectra suggest that the helices within the junctions adopt the regular B-DNA conformation. Almost identical melting temperatures and unfolding enthalpies are obtained for J4 and J4M both by UV and DSC. Furthermore, the Van't Hoff enthalpy (Δ H VH ) derived from UV melting equals the calorimetric enthalpy (Δ H cal ), which means that the melting process of the structures proceeds in a two-state manner. All results taken together support the conclusion that there are no major conformational and energetic differences between J4 and J4M. The inclusion of the uncharged methylene–acetal group into the junction has no effect on its stability.

The DNA Holliday junction is a central intermediate in genetic recombination. We have designed and synthesized a DNA oligomer, J1a, as a model compound for the Holliday junction suitable to be studied by NMR spectroscopy and future molecular modelling. The design was based on a 46-base oligomer, J4, previously studied by Pikkemaat, J. A., van den Elst, H., van Boom, J. H. & Altona, C. [Biochemistry 33, 14896-14907 (1994)], including the propensity to undergo a self-folding process to give a four-way junction in which three of the four arms are capped with a hairpin loop. J1a, however, is considerably shortened by eight bases and thus contains only 38 residues which significantly facilitates the proton resonance assignments. The base sequence at the branch point is identical to that in J4. 1H-NMR data clearly point to the presence of three hairpin loops in J1a and show that the double-helical arms adopt the B-DNA form. Quasicontinuous pairwise stacking between helical arms to give a single preferred stacked X-conformation is evident. The extent of folding into this stacked conformation is strongly dependent upon the magnesium concentration. Full Watson-Crick base pairing at the branch point is completely preserved. The A/D-stacking preference of the small junction is the same as that exhibited by J4.

The three-dimensional spatial structure of a methylene-acetal-linked thymine dimer presentin a 10 base-pair (bp) sense–antisense DNA duplex was studied with a geneticalgorithm designed to interpret NOE distance restraints. Trial solutions were represented bytorsion angles. This means that bond angles for the dimer trial structures are kept fixed duringthe genetic algorithm optimization. Bond angle values were extracted from a 10 bpsense–antisense duplex model that was subjected to energy minimization by means ofa modified AMBER force field. A set of 63 proton–proton distance restraints definingthe methylene-acetal-linked thymine dimer was available. The genetic algorithm minimizesthe difference between distances in the trial structures and distance restraints. A largeconformational search space could be covered in the genetic algorithm optimization byallowing a wide range of torsion angles. The genetic algorithm optimization in all cases ledto one family of structures. This family of the methylene-acetal-linked thymine dimer in theduplex differs from the family that was suggested from distance geometry calculations. It isdemonstrated that the bond angle geometry around the methylene-acetal linkage plays animportant role in the optimization.

Electrostatic interactions appear to be important in the conformation and dynamics of DNA four-way junctions. Particularly, the Coulomb repulsion between two approaching phosphate groups at the site of strand exchange is commonly supposed to have a negative influence on the thermodynamic stability of the fully stacked conformation. We synthesized a unimolecular DNA four-way junction containing an uncharged methylene-acetal linkage, -O3'-CH2-O5'-, instead of a regular phosphodiester linkage, -O3'-PO2-O5'-, between two specific residues at the branch-point to examine the effect of charge removal. Complete sequential 1H-NMR assignments of the non-exchangeable base protons and H1'/H2'/H2" sugar protons were obtained with the aid of NOESY and TOCSY experiments. On the basis of the NMR data it is concluded that the stacking arrangement at the branch-point of the modified oligonucleotide is similar to that of the previously studied parent four-way junction. Surprisingly, this is not the stacking arrangement in which the close phosphate-phosphate contact at the site of strand exchange would be absent. Some implications of this novel information regarding the role of the phosphate-phosphate repulsion in four-way junctions are discussed.

DNA three-way junctions (TWJs) containing unpaired residues at the branch point can adopt a conformation in which one helix is stacked upon another, forming a coaxial, quasicontinuous double helix. As in four-way junctions (FWJs), two conformers with different stacking arrangements between the arms are possible. However, in both types of structures a markedly strong preference for one conformer has been observed. To investigate the basis for this preference, in particular the influence of the stacking proclivity of the base-pairs at the centre of the junction, two linear oligomers (36 nucleotides), TWJ1 and TWJ2, differing only in one base-pair (G.C versus C.G, respectively) at the branch point, were designed and chemically synthesized. Each one is expected to fold into a stable three-way junction, containing two unpaired thymidine bases at the junction region and two arms capped with a hairpin loop. The data obtained from 1H and 31P-NMR spectroscopy confirm that both oligomers are present as stable three-way junctions. In both TWJs two of the helical arms stack preferentially upon each other. However, the stacking arrangement is similar in both molecules. From this it is deduced that purine-purine stacking across the junction cannot be considered as a major factor that determines the preferred stacking arrangement.

In recent years various examples of highly stable two-residue hairpin loops (miniloops) in DNA have been encountered. As the detailed structure and stability of miniloops appear to be determined not only by the nature and sequence of the two bases in the loop, but also by the closing base pair, it is desirable to carry out in-depth studies of especially designed small model DNA compounds. Therefore, a circular DNA dumbbell-like molecule is tailored to consist of a stem of three Watson–Crick base pairs, flanked on each side by a minihairpin loop. The resulting circular DNA decanter 5′-d〈pCGC- TT-GCG- TT〉 -3′ (I) is studied in solution by means of nmr spectroscope. At a temperature of 269 K the molecule occurs in a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4). L2L2 contains three Watson–Crick C-G base pairs and two two-residue loops (H2-family type) in opposite parts of the molecule. On raising the temperature from 269 to 314 K. The L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop with only one remaining solvent-accessible hydrogen bond between NHα of the cytosine C(1) and O6 of the guanine G(8), whereas the opposite 5′-CTTG-3′ loop remains stable. The disruption of the C(1)-G(8) base pair in the L2L4 form is correlated with the presence of a syn orientation for the C(1) base at the 5′-3′ loop-stem junction in the 5′-GTTC-3′ loop. The two conformers. L2L2 and L2L4, occur in slow equilibrium (2–20 s−1). Moderate line broadening of specific 1H, 13C, and 31P resonances of residues C(1), G(8), T(9), and T(10) at low temperatures, due to chemical exchange between L2L2 and L2L4, show that the interconversion from an anti to syn conformer in residue C(1) has a small local effect on the structure of the dumbbell. T1 relaxation measurements, chemical-shift considerations, and complete hand-shape calculations of the exchange process of the G(8) imino proton reveal a possibility for the existence of multiconformational slates in the anti–syn equilibrium. © 1995 John Wiley & Sons, Inc.

The conformational behavior of DNA minihairpin loops is sensitive to the directionality of the base pair that closes the loop. Especially tailored circular dumbbells, consisting of a stem of three Watson–Crick base pairs capped on each side with a minihairpin loop, serve as excellent model compounds by means of which deeper insight is gained into the relative stability and melting properties of hairpin loops that differ only in directionality of the closing pair: C-G vs G-C. For this reason the thermodynamic properties of the circular DNA decamers 5′-d〈pCGC-TT-GCG-TT〉-3′(I) and reference compounds 5′-d〈pGGC-TT-GCC-TT≤-3′(II) and 5′-d(GCG-TC-CGC)-3′(III) are studied by means of nmr spectroscopy. MoleculesIandIIadopt dumbbell structures closed on both sides by a two-membered hairpin hop. At low temperatureIconsists of a mixture of two slowly exchanging forms, denotedL2L2andL2L4. The low-temperatureL2L2form is the fully intact minihairpin structure with three Watson–Crick C-G base pairs. The high-temperature form,L2L4,contains a partially disrupted closing G-C base pair in the 5′-GTTC-3′ loop, with the cytosine base placed in a syn orientation. The opposite 5′-CTTG-3′ loop remains stable. A study of the noncircular hairpin structureIIIshows similar conformational behavior for the 5′-GTTC-3′ loop as found inIa syn orientation for C(6) and two slowly exchanging imino proton signals for G(3). The melting point Tm of IIwas estimated to lie above 365 K. The Tm value of the duplex stem and the 5′-CTTG-3′ loop of theL2L4form ofIis 352 ± 2 K. The ΔH° is calculated as −89 ± 10 kJ/mol. The Tm value determined for the individual residues of the 5′-GTTC-3′ loop lies 4°–11° lower. The enthalpy ΔH° of melting the thymine residues in the 5′-GTTC-3′ loop is calculated to be -61± 7 kJ/mol. Thermodynamic data of the equilibrium between the slowly exchanging two- and four-membered loop conformers of I reveal an upper limit for ΔH° of +30 kJ/mol in going from a two-memberedto a four-membered loop, in agreement with the enthalpy difference of +28 k.j/mol between the two loops at the Tm midpoint. For hairpin III the upper limit for ΔH° going from a two-membered to a four-membered loop amounts to ±21 kJ/mol. The mutual exchange rate between the L2 and L4 form in III is estimated as 13.6 s−1. Our results clearly suggest that small four-way DNA junctions(model for immobilized Holliday junctions) can be designed that consist of a single DNA strandthat features -CTTG-caps on three of the four arms of the junction. © 1995 John Wiley & Sons, Inc.

Fluorine MRI ((19) F MRI) is receiving an increasing attention as a viable alternative to proton-based MRI ((1) H MRI) for dedicated application in molecular imaging. The (19) F nucleus has a high gyromagnetic ratio, a 100% natural abundance and is furthermore hardly present in human tissues allowing for hot spot MR imaging. The applicability of (19) F MRI as a molecular and cellular imaging technique has been exploited, ranging from cell tracking to detection and imaging of tumors in preclinical studies. In addition to applications, developing new contrast materials with improved relaxation properties has also been a core research topic in the field, since the inherently low longitudinal relaxation rates of perfluorocarbon compounds result in relatively low imaging efficiency. Borrowed from (1) H MRI, the incorporation of lanthanides, specifically Gd(III) complexes, as signal modulating ingredients in the nanoparticle formulation has emerged as a promising approach to improvement of the fluorine signal. Three different perfluorocarbon emulsions were investigated at five different magnetic field strengths. Perfluoro-15-crown-5-ether was used as the core material and Gd(III)DOTA-DSPE, Gd(III)DOTA-C6-DSPE and Gd(III)DTPA-BSA as the relaxation altering components. While Gd(III)DOTA-DSPE and Gd(III)DOTA-C6-DSPE were favorable constructs for (1) H NMR, Gd(III)DTPA-BSA showed the strongest increase in (19F) R(1). These results show the potential of the use of paramagnetic lipids to increase (19F) R(1) at clinical field strengths (1.5-3 T). At higher field strengths (6.3-14 T), gadolinium does not lead to an increase in (19F) R(1) compared with emulsions without gadolinium, but leads to an significant increase in (19F) R(2). Our data therefore suggest that the most favorable situation for fluorine measurements is at high magnetic fields without the inclusion of gadolinium constructs.

Objectives: To validate near-infrared (NIR)-based optical spectroscopy measurements of hepatic fat content using a minimally invasive needle-like probe with integrated optical fibers, enabling real-time feedback during percutaneous interventions. The results were compared with magnetic resonance spectroscopy (MRS) as validation and with histopathology, being the clinical gold standard. Additionally, ex vivo magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography were performed for comparison. Materials and Methods: Ten mice were used for the study, of which half received a regular chow diet and the other half received a high-fat diet to induce obesity and hepatosteatosis. The mice were imaged with a clinical 3-Tesla MR to select a region of interest within the right and left lobes of the liver, where MRS measurements were acquired in vivo. Subsequently, optical spectra were measured ex vivo at the surface of the liver at 6 different positions immediately after resection. Additionally, hepatic fat was determined by magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography. Histopathologic analyses were performed and used as the reference standard. Pearson correlation and linear regression analyses were performed to assess the correlation of the various techniques with NIR. A 1-way analysis of variance including post hoc Tukey multiple comparison tests was used to study the difference in fat estimation between the various techniques. Results: For both the mice groups, the estimated fat fractions by the various techniques were significantly similar (P = 0.072 and 0.627 for chow diet and high-fat diet group, respectively). The Pearson correlation value between NIR and the other techniques for fat determination showed the same strong linear correlation (P above 0.990; P < 0.001), whereas for histopathologic analyses, which is a rather qualitative measure, the Pearson correlation value was slightly lower (P = 0.925, P < 0.001) . Linear regression coefficient computed to compare NIR with the other techniques resulted in values close to unity with MRS having the narrowest confidence interval (r = 0.935, 95% confidence interval: 0.860-1.009), demonstrating highly correlating results between NIR and MRS. Conclusions: NIR spectroscopy measurements from a needle-like probe with integrated optical fibers for sensing at the tip of the needle can quickly and accurately determine hepatic fat content during an interventional procedure and might therefore be a promising novel diagnosing tool in the clinic. © 2012 by Lippincott Williams & Wilkins.

Synthesis of the modified decadeoxynucleotides 16–20 having the sequence d(G1pC2pG)3pT4x1T5x2T6x3T7pG8pC9pG10) and containing one (x2 = CH2, x1 = x3 = p, i.e., 16), two (x1 = x3 = CH2, x2 = p, i.e., 17, x2 = x3 = CH2, x1 = p, i.e., 18 and x1 = x2 = CH2, x3 = p. i.e., 19) or three (x1 = x2 = x3 = CH2, i.e., 20) -O-CH2-O- linkages could be accomplished using the respective mono-, di- or tri(-O-CH2-O) 3′-O-phosphoramidites 6, 10 and 14 as the incoming synthons. Hybridization of 16–20 with their native complementary strand afforded stable duplexes, which were studied by 1D- and 2D-1H-NMR and UV-hyperchromicity techniques.

The circular DNA decamer 5′-d 3′ is studied in solution by means of NMR spectroscopy. At low temperature the molecule adopts a dumbbell structure with three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature another conformer appears, in which the closing C-G base pair in the 5′-GTTC-3′ loop is disrupted, whereas the opposite 5′-CTTG-3′ loop remains stable. The two conformers are in slow equilibrium over a limited temperature range.

The solid state and solution structure of a series of lanthanide complexes of the decadentate ligand triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetamide, (ttham), its two decadentate derivatives di-tert-butyl N,N,N''',N'''-tetra(carbamoylmethyl)-triethylenetetramine-N',N''-diacetate (Bu(2)ttha-tm) and N,N,N''',N'''-tetra(carbamoylmethyl)-triethylenetetramine-N',N''-diacetic acid (H(2)ttha-tm), and its two nonadentate derivatives N-benzyl-triethylenetetramine-N,N',N'',N''',N'''-pentaacetamide (1bttpam) and N'-benzyl-triethylenetetramine-N,N,N'',N''',N'''-pentaacetamide (4bttpam) have been investigated by infrared and Raman spectroscopy, X-ray crystallography, cyclovoltammetry, and NMR spectroscopy. In these mononuclear lanthanide complexes, the first coordination sphere is generally saturated by four amine nitrogens of the triethylenetetramine ligand backbone and five or six carbonyl oxygen atoms of the pendent amide or acetate donor groups. In the [Ln(ttham)](3+) complex series, a switch from a decadentate to a nonadentate coordination occurs between [Er(ttham)](3+) and [Tm(ttham)](3+). This switch in coordination mode, which is caused by decreasing metal ion radii going from lanthanum to lutetium (lanthanide contraction), has no significant effect on the T(1)-relaxivity of these complexes. It is shown that the T(1)-relaxivity is dominated by second coordination sphere interactions, with an ascendant contribution of the classical dipolar relaxation mechanism for the earlier (Ce-Sm) and very late (Tm, Yb) lanthanides, and a prevailing Curie relaxation mechanism for most of the remaining paramagnetic lanthanide ions. In chemical exchange-dependent saturation transfer (CEST) (1)H NMR experiments, most of the above complexes exhibit multiple strong CEST peaks of the paramagnetically shifted amide protons spread over a100 ppm chemical shift range. The effective CEST effect of the studied thulium complexes strongly depends on temperature and pH. The pH at which the CEST effect maximizes (generally between pH 7 and 8) is determined by the overall charge of the complex. Depending on the used saturation frequency offset, the temperature-dependence varies between the extremes of strongly linearly dependent and fully independent in the case of [Tm(ttham)](3+). In combination with the strong pH-dependence of the CEST effect at the latter frequency offset, this complex is highly suitable for simultaneous temperature and pH mapping using magnetic resonance imaging.

The ligand triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetamide (ttham) was synthesized with the aim of forming lanthanide complexes suitable as contrast agents for magnetic resonance imaging applications utilizing the chemical exchange-dependent saturation transfer (CEST) effect. It was designed to exclude water molecules from the first coordination sphere and provide a high number of CEST active amide protons per lanthanide ion. The ligand was characterized by its protonation behavior and its complexation properties with ytterbium ions in aqueous solution. The basicity of the ttham backbone amine protons decreases in the order N(central(1))N(terminal(1))N(terminal(2))N(central(2)), as deduced from NMR titration experiments and from a comparison of its protonation constants with those of two ttham derivatives, in which either a terminal (N-benzyl-triethylenetetramine-N,N',N'',N''',N'''-pentaacetamide, 1bttpam) or a central acetamide group (N'-benzyl-triethylenetetramine-N,N,N'',N''',N'''-pentaacetamide, 4bttpam) is substituted with a benzyl group. This protonation sequence results from the combined influence of inductive effects, the intramolecular hydrogen bonding network, and the Coulomb repulsion between protonated ammonium groups. The ytterbium complex of ttham, [Yb(ttham)]Cl(3), is coordinatively frustrated. Due to steric constraints, in addition to the four backbone nitrogen atoms, only three of the four symmetry-equivalent terminal acetamide donors can coordinate simultaneously to the ytterbium ion, and the dangling fourth one exchanges quickly with the other three. The ytterbium complexes of a total of five ligands (ttham, 1bttpam, 4bttpam, 2,2',2''-triaminotriethylaminehexaacetamide (ttaham), and diethylenetriamine-N,N,N',N'',N''-pentaacetamide (dtpam)) were studied with respect to their CEST properties. In solution, all of these complexes have a low symmetry. The presence of multiple magnetically different amide groups in each complex prevents the realization of very high CEST effects. These results nevertheless form an excellent basis for a further optimization of this class of ligands.

MRI contrast agents based on chemical exchange-dependent saturation transfer (CEST), such as Yb(III)DOTAM complexes, are highly suitable for pH mapping. In this paper, the synthesis of Yb(III)DOTAM-functionalized poly(propylene imine) dendrimers is described. The applicability of these dendritic PARACEST MRI agents for pH mapping has been evaluated on a 7 T NMR spectrometer and on a 3 T clinical MRI scanner. As expected, based on the different numbers of exchangeable amide protons, the lowest detectable concentration of the first and third generation dendritic PARACEST agents is by a respective factor of about 4 and 16 lower than that of a mononuclear reference complex. The pH dependence of the CEST effect observed for these compounds depends on the generation of the poly(propylene imine) dendrimer. Upon going to higher generations of the Yb(III)DOTAM-terminated dendrimer, a shift of the maximum CEST effect towards lower pH values was observed. This allows for a fine-tuning of the responsive pH region by varying the dendritic framework.

Multivariate curve resolution (MCR) has been applied to separate pure spectra and pure decay profiles of DOSY NMR data. Given good initial guesses of the pure decay profiles, and combined with the nonlinear least square regression (NLR), MCR can result in good separation of the pure components. Nevertheless, due to the presence of artefacts in experimental data, validation of a MCR model is still necessary. In this paper, the covariance matrix of the residuals (CMR), obtained by postmultiplying the residual matrix with its transpose, is proposed to evaluate the quality of the results of an experimental data set. Plots of the rows of this matrix give a general impression of the covariance in the frequency domain of the residual matrix. Different patterns in the plot indicate possible causes of experimental imperfections. This new criterion can be used as diagnosis in order to improve experimental settings as well as suggest appropriate preprocessing of DOSY NMR data.

Contains fulltext : 57929.pdf (Publisher’s version ) (Open Access) The aim of this study was to further explore the use of magnetic resonance imaging (MRI), magnetic resonance angiography (MRA), and dual-energy X-ray absorptiometry (DEXA) to assess bone formation and blood circulation in a pedicled bone graft substitute. In 14 Wistar rats, initially 10 weeks old, heterogeneous demineralized femur bone matrix implants were wrapped in pedicled adductor thigh muscle flaps. One rat died after surgery. Subsequently, bone formation and maintenance of blood vessel functionality were evaluated in six rats 6 weeks postimplantation by means of in vivo MRI/MRA and postmortem histomorphometry. The other seven rats were left for 12 weeks, whereafter bone formation was evaluated by in vivo DEXA and postmortem histomorphometry. The results demonstrated that after 6 weeks bone formation was present in four of six animals, quantified as 42 (+/-35)% and 25 (+/-19)% by means of MRI and histomorphometry, respectively. MRA was able to show patency of the pedicles of these four rats only, which suggests that the lack of blood supply in the other two rats is the cause of the failure to form bone. In the 12-week group, histology showed increased bone formation without signs of osteolysis, which was quantified histomorphometrically to be as high as 48 (+/-15)%. DEXA failed to show bone formation. It is concluded that in vivo MRI proved to be a reliable method for monitoring ectopic bone formation in a rat model, whereas in vivo DEXA was unable to detect the implants. Furthermore, in vivo MRA proved to be a useful technique for studying the circulation of muscle flaps in this animal model.

Item does not contain fulltext In animal studies of tissue engineering of bone, histology remains the standard for assessing bone formation. As longitudinal studies with this method are feasible only at the cost of large numbers of animals, we looked for an alternative. Therefore, demineralized bone matrix (DBM) and inactivated demineralized bone matrix (iDBM) implants were subcutaneously implanted in a rat. At 1, 3, 5, and 7 weeks postimplantation soft X-ray and magnetic resonance imaging (MRI) were done to monitor bone formation in the implants. At 7 weeks, the animal was killed and the implants were retrieved for histology. Our results showed that in vivo MRI is well suited to assess bone formation larger than 0.5 mm in diameter and to monitor the complete three-dimensional shape of the newly formed bone noninvasively and longitudinally. The MRI results matched well with the histology results obtained at 7 weeks. In contrast, X-ray imaging appeared inappropriate to monitor the bone formation process in DBM.

A novel temperature-sensitive liposomal MRI contrast agent has been developed, which allows drug carrier localization using (1)H CEST with simultaneous quantification of the drug release using (19)F MR imaging in response to a local temperature increase.

DNA junctions are potential intermediates in various important genetic processes, including mutagenesis and recombination. The quantity of research carried out in this area is rapidly increasing. Examples of three-way and four-way junctions are now relatively well characterized and a few common properties have been recognized, of which the most important is the tendency of junctions to fold into one or more coaxially stacked helical conformations or cross-over structures.

The circular DNA decamer 5'-dpCGC-TT-GCG-TT-3' was studied in solution by means of NMR spectroscopy and molecular dynamics in H2O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops is opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5'-GTTC-3' loop with only one remaining (solvent-accessible) hydrogen bond between NH alpha of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5'-CTTG-3' loop remains stable. The two conformers occur in slow equilibrium (rate constant 2-20 s-1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (3JHH, 3JHP, 3JCP) gave detailed information about the local geometry around backbone torsion angles beta, gamma, delta, and epsilon, revealing a relatively high flexibility of the 5'-GTTC-3' loop. The values of the coupling constants are virtually temperature-independent. 'Weakly constrained' molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged overr-3to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S2 factors andr-6averaging of ther-3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5'-GTTC-3' loop. The difference in stability of the 5'-CTTG-3' and 5'-GTTC-3' loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle chi.

In 1964 Holliday postulated the formation of cruciform structures (four-way junctions) in duplex DNA as intermediate in genetic recombination. Since then, many biochemical and biophysical investigations were directed at solving questions concerning structural details of stable four-way junctions. Thus far, NMR spectroscopy played a minor part in these investigations on account of the minimum size of the molecule (expressed as the number of nucleotide residues) that was thought necessary to produce a stable cruciform structure. Indeed, the smallest four-way junction studied thus far by NMR methods was built from four separate DNA strands, each containing 16 nucleotides, a total of 64. Obviously, with such a large structure one runs into assignment problems. We considered the possibility of constructing a stable four-way junction from a single strand of DNA. The underlying idea was to make use of our detailed knowledge of the building principles of stable minihairpin loops. These loops, containing only two nucleotides to bridge the gap between antiparallel strands, are maximally stable in DNA sequences like 5′-d(-C-TT-G-), where C and G form a normal Watson-Crick base pair and the two T residues cross the minor groove to form the minihairpin loop. Three of such miniloops could in principle cap three arms of the cruciform. The fourth arm would have an open end. The problem to be solved is to find the minimum length that is required to insure stability of the three closed arms and of the fourth open arm. We were successful with a structure that has three short stems (four base pairs each) and an open-end stem consisting of eight base pairs, a total of 46 residues. NMR experiments, carried out on this molecule in the presence of Mg2+, showed details of folding which have never been observed before.

A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.