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The α-synuclein (α-syn) amyloid fibrils are involved in various neurogenerative diseases. Solid-state NMR (ssNMR) has been showed as a powerful tool to study α-syn aggregates. Here, we report the 1 H, 13 C and 15 N back-bone chemical shifts of a new α-syn polymorph obtained using proton-detected ssNMR spectroscopy under fast (95 kHz) magic-angle spinning conditions. The manual chemical shift assignments were cross-validated using FLYA algorithm. The secondary structural elements of α-syn fibrils were calculated using 13 C chemical shift differences and TALOS software., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The E.coli maltose binding protein (MBP) is a 42.5 kDa molecule widely employed in many biotechnology applications. Because of its molecular size, it has become the main model system for the development of solution NMR methods adapted to large biomolecular targets. Here, we report virtually complete (~ 90%) backbone resonance assignments obtained on a microcrystalline sample of MBP with 1 H-detected solid-state NMR at fast (> 100 kHz) magic-angle spinning. We additionally present the detailed description of the methodology employed for the preparation of the sample and the acquisition and analysis of the NMR spectra. The chemical shifts, obtained with a single uniformly 15 N,  13 C-labelled and fully-protonated sample and about 2 weeks on a 800 MHz NMR spectrometer, have been deposited to the BMRB under the accession number 50089., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Most commonly small outer membrane proteins, possessing between 8 and 12 β-strands, are not involved in transport but fulfill diverse functions such as cell adhesion or binding of ligands. An intriguing exception are the 8-stranded β-barrel proteins of the OmpW family, which are implicated in the transport of small molecules. A representative example is AlkL from Pseudomonas putida GPoI, which functions as a passive importer of hydrophobic molecules. This role is of high interest with respect to both fundamental biological understanding and industrial applications in biocatalysis, since this protein is frequently utilized in biotransformation of alkanes. While the transport function of AlkL is generally accepted, a controversy in the transport mechanism still exists. In order to address this, we are pursuing a structural study of recombinantly produced AlkL reconstituted in lipid bilayers using solid-state NMR spectroscopy. In this manuscript we present 1 H, 13 C and 15 N chemical shift assignments obtained via a suite of 3D experiments employing high magnetic fields (1 GHz and 800 MHz) and the latest magic-angle spinning (MAS) approaches at fast (60-111) kHz rates. We additionally analyze the secondary structure prediction in comparison with those of published structures of homologous proteins.

Purpose: Multidrug resistance-associated proteins (MRPs) mediate the hepatobiliary and renal excretion of many drugs and drug conjugates. The positron emission tomography (PET) tracer 6-bromo-7-[ 11 C]methylpurine is rapidly converted in tissues by glutathione-S-transferases into its glutathione conjugate, and has been used to measure the activity of Abcc1 in the brain and the lungs of mice. Aim of this work was to investigate if the activity of MRPs in excretory organs can be measured with 6-bromo-7-[ 11 C]methylpurine., Procedures: We performed PET scans with 6-bromo-7-[ 11 C]methylpurine in groups of wild-type, Abcc4 (-/-) and Abcc1 (-/-) mice, with and without pre-treatment with the prototypical MRP inhibitor MK571., Results: 6-Bromo-7-[ 11 C]methylpurine-derived radioactivity predominantly underwent renal excretion. In blood, MK571 treatment led to a significant increase in the AUC and a decrease in the elimination rate constant of radioactivity (k elimination,blood ). In the kidneys, there were significant decreases in the rate constant for radioactivity uptake from the blood (k uptake,kidney ), k elimination,kidney , and the rate constant for tubular secretion of radioactivity (k urine ). Experiments in Abcc4 (-/-) mice indicated that Abcc4 contributed to renal excretion of 6-bromo-7-[ 11 C]methylpurine-derived radioactivity., Conclusions: Our data suggest that 6-bromo-7-[ 11 C]methylpurine may be useful to assess the activity of MRPs in the kidneys as well as in other organs (brain, lungs), although further work is needed to identify the MRP subtypes involved in the disposition of 6-bromo-7-[ 11 C]methylpurine-derived radioactivity.

Background: The dismal outcome in children with high-grade brainstem gliomas (BSG) accentuates the need for effective therapeutic strategies. We investigated the role of intensive, including marrow-ablative, chemotherapy regimens in the treatment of young children with newly-diagnosed high-grade BSG., Methods: Between 1991-and-2002, 15 eligible children less than 10 years of age with a diagnosis of high-grade BSG were treated on "Head-Start" I and II protocols (HSI and HSII). Treatment included Induction with 4-5 cycles of one of three intensive chemotherapy regimens followed by Consolidation with one cycle of marrow-ablative chemotherapy (thiotepa, carboplatin and etoposide) with autologous hematopoietic cell rescue (AHCR). Irradiation was required for children over 6 years of age or for those with residual tumor at the end of Consolidation., Results: We had two long-term survivors who were found retrospectively to harbor low-grade glial tumors and thus were not included in the survival analysis. Of the remaining 13 patients, the 1-year event-free (EFS) and overall (OS) survival for these children were 31% (95% CI 9-55%) and 38% (95% CI 14-63%), respectively. Median EFS and OS were 6.6 (95% CI 2.7, 12.7) and 8.7 months (95% CI 6.9, 20.9), respectively. Eight patients developed progressive disease during study treatment (seven during Induction and one at the end of Consolidation). Ten children received focal irradiation, five for residual tumor (three following Induction and two following Consolidation) and five due to disease progression., Conclusions: Children with high-grade BSG did not benefit from this intensive chemotherapy strategy administered prior to irradiation.

Introduction: Epidermal growth factor receptors EGFR and ErbB2 are overexpressed in schwannomas and meningiomas. Preclinical and clinical data indicate that lapatinib, an EGFR/ErbB2 inhibitor, has antitumor activity against vestibular schwannomas in neurofibromatosis type 2 (NF2) patients. Its antitumor activity against meningiomas, however, is unknown., Methods: We conducted a retrospective review of patients with NF2 and progressive vestibular schwannomas treated on a phase 2 clinical trial with lapatinib (NCT00973739). We included patients with at least one volumetrically measurable meningioma (> 0.5 cm 3 ) who received at least five 28-day courses of treatment. Patients received lapatinib 1500 mg daily. Meningioma response was assessed using 3-dimensional MRI volumetrics. Progressive meningioma growth and response were defined as + 20 and - 20% change in tumor volume from baseline, respectively. Off-treatment was defined as any period > 5 months without lapatinib., Results: Eight patients (ages: 20-58 years) who met criteria had 17 evaluable meningiomas with a combined volume of 61.35 cc at baseline, 61.17 cc during treatment, and 108.86 cc (+ 77.44% change) off-treatment, p = 0.0033. Median time on-treatment and off-treatment was 15.5 and 16.7 months, respectively. On-treatment mean and median annualized growth rates were 10.67 and 1.32%, respectively. Off-treatment mean and median annualized growth rates were 20.05 and 10.42%, respectively. The best volumetric response was - 26.1% after 23 months on lapatinib. Two tumors increased > 20% volumetrically on-treatment, compared to eight tumors off-treatment., Conclusions: These data suggest that lapatinib may have growth-inhibitory effects on meningiomas in NF2 patients, and support prospective studies of lapatinib for NF2 patients with progressive meningiomas.

A method for five-dimensional spectral reconstruction of non-uniformly sampled NMR data sets is proposed. It is derived from the previously published signal separation algorithm, with major alterations to avoid unfeasible processing of an entire five-dimensional spectrum. The proposed method allows credible reconstruction of spectra from as little as a few hundred data points and enables sensitive resonance detection in experiments with a high dynamic range of peak intensities. The efficiency of the method is demonstrated on two high-resolution spectra for rapid sequential assignment of intrinsically disordered proteins, namely 5D HN(CA)CONH and 5D (HACA)CON(CO)CONH.

Nuclear magnetic resonance spectroscopy (NMR) can provide a great deal of information about structure and dynamics of biomolecules. The quality of an NMR structure strongly depends on the number of experimental observables and on their accurate conversion into geometric restraints. When distance restraints are derived from nuclear Overhauser effect spectroscopy (NOESY), stereo-specific assignments of prochiral atoms can contribute significantly to the accuracy of NMR structures of proteins and nucleic acids. Here we introduce a series of NOESY-based pulse sequences that can assist in the assignment of chiral CHD methylene protons in random fractionally deuterated proteins. Partial deuteration suppresses spin-diffusion between the two protons of CH2 groups that normally impedes the distinction of cross-relaxation networks for these two protons in NOESY spectra. Three and four-dimensional spectra allow one to distinguish cross-relaxation pathways involving either of the two methylene protons so that one can obtain stereospecific assignments. In addition, the analysis provides a large number of stereospecific distance restraints. Non-uniform sampling was used to ensure optimal signal resolution in 4D spectra and reduce ambiguities of the assignments. Automatic assignment procedures were modified for efficient and accurate stereospecific assignments during automated structure calculations based on 3D spectra. The protocol was applied to calcium-loaded calbindin D9k. A large number of stereospecific assignments lead to a significant improvement of the accuracy of the structure.

The three-dimensional structure determination of RNAs by NMR spectroscopy requires sequential resonance assignment, often hampered by assignment ambiguities and limited dispersion of (1)H and (13)C chemical shifts, especially of C4'/H4'. Here we present a novel through-bond 4D HPCH NMR experiment involving phosphate backbone where C4'-H4' correlations are resolved along the (1)H3'-(31)P spectral planes. The experiment provides high peak resolution and effectively removes ambiguities encountered during assignments. Enhanced peak dispersion is provided by the inclusion of additional (31)P and (1)H3' dimensions and constant-time evolution of chemical shifts. High spectral resolution is obtained by using non-uniform sampling in three indirect dimensions. The experiment fully utilizes the isotopic (13)C-labeling with evolution of C4' carbons. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the C4'-C3' and C4'-C5' homonuclear couplings. Multiple quantum line narrowing is employed to minimize sensitivity loses. The 4D HPCH experiment is verified and successfully applied to a non-coding 34-nt RNA consisting typical structure elements and a 14-nt RNA hairpin capped by cUUCGg tetraloop.

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

A through bond, C4'/H4' selective, "out and stay" type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4'(i)-C4'(i)-C4'(i-1)-H4'(i-1) correlations. The (31)P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4' dimension. The experiment fully utilizes (13)C-labeling of RNA by inclusion of two C4' evolution periods. An additional evolution of H4' is included to further enhance peak resolution. Band selective (13)C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4'-C3' and C4'-C5' homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully (13)C, (15)N-labeled 34-nt hairpin RNA comprising typical structure elements.

Microtubule-associated protein 1B (MAP1B) is a classical high molecular mass microtubule-associated protein expressed at high levels in the brain. It confers specific properties to neuronal microtubules and is essential for neuronal differentiation, brain development and synapse maturation. Misexpression of the protein contributes to the development of brain disorders in humans. However, despite numerous reports demonstrating the importance of MAP1B in regulation of the neuronal cytoskeleton during neurite extension and axon guidance, its mechanism of action is still elusive. Here we focus on the intrinsically disordered microtubule binding domain of the light chain of MAP1B. In order to obtain more detailed structural information about this domain we assigned NMR chemical shifts of backbone and aliphatic side chain atoms.

A 4D (13)C(aromatic),(13)C(ribose)-edited NOESY experiment is introduced to improve sequential assignment of non-coding RNA, often hampered by a limited dispersion of (1)H and (13)C chemical shifts. The (13)C-labeling of RNA is fully utilized by inclusion of two (13)C evolution periods. These dimensions provide enhanced dispersion of resonances in the 4D spectrum. High spectral resolution is obtained using random non-uniform sampling in three indirect dimensions. The autocorrelation peaks are efficiently suppressed using band-selective pulses. Since the dynamic range of observed resonances is significantly decreased, the reconstruction of the 4D spectrum is greatly simplified. The experiment can replace two conventionally sampled 3D NOESY spectra (either ribose-(13)C- or aromatic-(13)C-separated), and remove most ambiguities encountered during sequential walks. The assignment strategy based on a homonuclear and 4D C,C-edited NOESY experiments is proposed and verified on a 34-nt RNA showing typical structure elements.

A band-selective aromatic-aliphatic C,C-edited four-dimensional NOESY experiment is proposed here. Its key advantage is the absence of auto-correlation signals which makes it very attractive for joint use with non-uniform sampling. It is demonstrated here that the sensitivity of the experiment is not significantly affected by utilization of selective pulses (for either aromatic-13C or aliphatic-13C spins). The method was applied to the sample of E32Q mutant of human S100A1 protein, a homodimer of total molecular mass ~20 kDa. High-resolution 4D spectra were obtained from ~1.5 % of sampling points required conventionally. It is shown that superior resolution facilitates unambiguous assignment of observed aliphatic-aromatic cross-peaks. Additionally, the addition of aliphatic-13C dimension enables to resolve peaks with degenerated aliphatic (1)H chemical shifts. All observed cross-peaks were validated against previously determined 3D structure of E32Q mutant of S100A1 protein (PDB 2LHL). The increased reliability of structural constraints obtained from the proposed high-resolution 4D 13C(ali),13C(aro)-edited NOESY can be exploited in the automated protocols of structure determination of proteins.

Endothelial and monocyte-activating polypeptide II (EMAP II) is a cytokine that plays an important role in inflammation, apoptosis and angiogenesis processes in tumour tissues. Structurally, the EMAP II is a 169 amino acid residues long C-terminal domain (residues 147-312) of auxiliary tRNA binding protein p43. In spite of existence in pdb databank of two X-ray structures there are some important aspects of EMAP II cytokine function which are still not fully understood in detail. To obtain information about 3D structure and backbone dynamic processes in solution we perform structure evaluation of human EMAP II cytokine by NMR spectroscopy. The standard approach to sequence-specific backbone assignment using 3D NMR data sets was not successful in our studies and was supplemented by recently developed 4D NMR experiments with random sampling of evolution time space. Here we report the backbone and side chain (1)H, (13)C, and (15)N chemical shifts in solution for recombinant EMAP II cytokine together with secondary structure provided by TALOS + software.

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

Spectra obtained by application of multidimensional Fourier Transformation (MFT) to sparsely sampled nD NMR signals are usually corrupted due to missing data. In the present paper this phenomenon is investigated on simulations and experiments. An effective iterative algorithm for artifact suppression for sparse on-grid NMR data sets is discussed in detail. It includes automated peak recognition based on statistical methods. The results enable one to study NMR spectra of high dynamic range of peak intensities preserving benefits of random sampling, namely the superior resolution in indirectly measured dimensions. Experimental examples include 3D (15)N- and (13)C-edited NOESY-HSQC spectra of human ubiquitin.

Purpose: We evaluated the usefulness of small animal brain positron emission tomography (PET) imaging with the amyloid-beta (A beta) probe 2-(1-{6-[(2-[(18)F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malonitrile ([(18)F]FDDNP) and with 2-deoxy-2-[F-18]fluoro-D-glucose (FDG) for detection and quantification of pathological changes occurring in a transgenic mouse model of Alzheimer's disease (Tg2576 mice)., Procedures: [(18)F]FDDNP (n = 6) and FDG-PET scans (n = 3) were recorded in Tg2576 mice (age 13-15 months) and age-matched wild-type litter mates. Brain volumes of interest were defined by co-registration of PET images with a 3D MOBY digital mouse phantom. Regional [(18)F]FDDNP retention in mouse brain was quantified in terms of the relative distribution volume (DVR) using Logan's graphical analysis with cerebellum as a reference region., Results: Except for a lower maximum brain uptake of radioactivity in transgenic animals, the regional brain kinetics as well as DVR values of [(18)F]FDDNP appeared to be similar in both groups of animals. Also for FDG, regional radioactivity retention was almost identical in the brains of transgenic and control animals., Conclusions: We could not detect regionally increased [(18)F]FDDNP binding and regionally decreased FDG binding in the brains of Tg2576 transgenic versus wild-type mice. However, small group differences in signal might have been masked by inter-animal variability. In addition, technical limitations of the applied method (partial volume effect, spatial resolution) for measurements in such small organs as mouse brain have to be taken into consideration.

Lobular capillary hemangiomas are common, benign, vascular soft-tissue tumors located in the head and neck during the childhood and early adulthood. Report of these lesions in the central nervous system has been anecdotal. The case of one patient treated for spinal cord compression secondary to a capillary hemangioma with elevated proliferation index is presented. A review of the published cases in the English literature is provided, as well as a discussion on our case findings and the management of these lesions in the spinal cord.