[Emergency medicine in the German Maritime Search and Rescue Service-Evaluation of medical emergencies in the North Sea and Baltic Sea over 2 years]. / Notfallmedizin in der Deutschen Gesellschaft zur Rettung Schiffbrüchiger ­ Auswertung medizinischer Notfälle auf der Nord- und Ostsee über 2 Jahre.

BACKGROUND: The logistic peculiarities of an emergency maritime location and the frequent additional threat of accidental hypothermia mean that the treatment of medical emergencies at sea are particularly demanding. This article describes the characteristics of emergency medical missions of the German Maritime Search and Rescue Service (DGzRS) as the main provider of non-helicopter-based medical maritime rescue on the seas off the coasts of Germany. MATERIAL AND METHODS: A retrospective analysis of all missions by the DGzRS in 2017 and 2018 was carried out. The data and times of the missions as well as the severity of the diseases of the patients (graduated using the NACA score) were evaluated and exemplarily compared to those of a medical emergency ambulance service from the City of Lübeck. RESULTS: In a total of 182 medical missions 224 patients were treated. The mission units of the DGzRS needed a mean time of 30 ± 21 min up to arrival and 43 ± 30 min for rescue, treatment and transport. In 63 missions the patients were accompanied by an emergency physician, who was brought in from the ground rescue service in 44 missions. Due to the waiting time for boarding of the additional personnel, the departure in 26 missions was delayed by an average of 18 ± 7 min. The average severity of the disease in the maritime rescue was significantly higher than in the emergency medical service of Lübeck but the number of resuscitations and fatalities were comparable. CONCLUSION: Although the severity of medical emergencies on the seas off the coasts of Germany was high, the emergency physicians frequently arrived with a considerable delay. There is an urgent need for an effective support of the DGzRS by medical personnel specifically trained for maritime missions.

Paramagnetic-iterative relaxation matrix approach: extracting PRE-restraints from NOESY spectra for 3D structure elucidation of biomolecules.

Paramagnetic relaxation enhancement (PRE) can be used to determine long-range distance restraints in biomolecules. The PREs are typically determined by analysis of intensity differences in HSQC experiments of paramagnetic and diamagnetic spin labels. However, this approach requires both isotope- and spin-labelling. Herein, we report a novel method to evaluate NOESY intensities in the presence of a paramagnetic moiety to determine PRE restraints. The advantage of our approach over HSQC-based approaches is the increased number of available signals without the need for isotope labelling. NOESY intensities affected by a paramagnetic center were evaluated during a structure calculation within the paramagnetic iterative relaxation matrix approach (P-IRMA). We applied P-IRMA to a 14-mer RNA with a known NMR solution structure, which allowed us to assess the quality of the PRE restraints. To this end, three different spin labels have been attached at different positions of the 14-mer to test the influence of flexibility on the structure calculation. Structural disturbances introduced by the spin label have been evaluated by chemical shift analysis. Furthermore, the impact of P-IRMA on the quality of the structure bundles were tested by intentionally leaving out available diamagnetic restraints. Our analyses show that P-IRMA is a powerful tool to refine RNA structures for systems that are insufficiently described by using only diamagnetic restraints.

Impact of spin label rigidity on extent and accuracy of distance information from PRE data.

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, UreidoU-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromatic 13C bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Çm is slightly better than the data quality for the flexible UreidoTEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).

Light-induced antibiotic release from a coumarin-caged compound on the ultrafast timescale.

A synthesis route for puromycin caged with the photo-responsive 7-diethylaminocoumarinyl protecting group carbamate was developed. The inactivation and recovery of the cytotoxic effect of puromycin was tested with a XTT cell viability assay. The uncaging mechanism was studied by ultrafast transient absorption spectroscopy and by time-correlated single photon counting. The combination of these results with quantum-chemical calculations provided detailed insights in dynamics upon excitation. Interestingly, a change of the dipole moment due to structural rearrangements of the amino moiety led to an intermolecular charge transfer on the picosecond time-scale. IR measurements marked the successful uncaging via the release of CO2, resulting from the carbamate linker. This decarboxylation constituted the rate-limiting step of the uncaging reaction and occurred on the subsecond timescale. DEACM-puromycin, thus, represents an efficient photo-activatable antibiotic for in-cell applications.

(19)F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy.

In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus (19)F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5'-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the (19)F isotope. The thermal stability of the (19)F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a (1)H,(15)N-HSQC allow the identification of Watson-Crick base paired uridine signals and the (19)F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of (19)F-labeling even for sizeable RNAs in the range of 70 nucleotides.

NMR-based Fragment Screening in a Minimum Sample but Maximum Automation Mode.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms of the macrodomain of SARS-CoV-2 non-structural protein 3b.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Detection of osteoarthritis using acoustic emission analysis.

Osteoarthritis (OA) of the knee is a widespread disease, often resulting in pain, restricted mobility and a reduction of activities and participation. Initial studies gave hints that Acoustic Emission Analysis (AEA) is capable of detecting early changes in cartilage structure. However, up to date no in vivo validation studies have been conducted. A prospective pilot study was conducted to investigate this diagnostic capability and the accuracy of the AEA, using magnetic resonance imaging (MRI) as a reference standard. Additionally, potential factors influencing false positive or negative results were studied. Twenty-eight patients, receiving MRI due to discomfort of the knee, were examined with AEA. Sensitivity was 0.92 for the whole knee and 0.86 to 1 for different parts of the knee. The specificity was 0.7 and 0.59 to 0.78, respectively. Confidence intervals varied between 0 and 0.33 for sensitivity and 0.1 and 0.24 for specificity. The diagnostic accuracy of the AEA was shown to be good to very good. However, because of the relatively small number of patients involved, interpretation of the data should be handled with care. Future studies with greater sample sizes have to be conducted to confirm the results of this investigation.

Main-chain dynamics of a partially folded protein: 15N NMR relaxation measurements of hen egg white lysozyme denatured in trifluoroethanol.

15N NMR relaxation measurements have been used to study the dynamic behaviour of the main-chain of hen lysozyme in a partially folded state, formed in a 70% (v/v) trifluoroethanol (TFE)/30% water mixture at 37 degrees C and pH 2. This state is characterised by helical secondary structure in the absence of extensive tertiary interactions. The NMR relaxation data were interpreted by mapping of spectral density functions and by derivation of segmental as well as global order parameters. The results imply that the dynamics of lysozyme in TFE can, at least for the great majority of residues, be adequately described by internal motions which are superimposed on all overall isotropic tumbling of the molecule. Although the dynamic behaviour shows substantial variations along the polypeptide chain, it correlates well with the conformational preferences identified in the TFE state by other NMR parameters. Segments of the polypeptide chain which are part of persistent helical structures are highly restricted in their motion (S2 > 0.8 , with effective internal correlation times tau(e) < 200 ps) but are also found to experience conformational exchange on a millisecond timescale. Regions which are stabilised in less persistent helical structure possess greater flexibility (0.6 < S2 < 0.8, 200 ps < tau(e) < 1 ns) and those which lack defined conformational preferences are highly flexible (S2 < 0.6, tau(e) approximately 1 ns). The dynamic behaviour of the main-chain was found to be correlated with other local features of the polypeptide chain, including hydrophobicity and the position of the disulphide bridges. Despite the absence of extensive tertiary interactions, preferential stabilisation of native-like secondary structure by TFE results in a pattern of main-chain dynamics which is similar to that of the native state.

Analysis of main chain torsion angles in proteins: prediction of NMR coupling constants for native and random coil conformations.

Using a data base of 85 high resolution protein crystal structures the distributions of main chain torsion angles, both in secondary structure and in coil regions where no secondary structure is present, have been analysed. These torsion angle distributions have been used to predict NMR homonuclear and heteronuclear coupling constants for residues in secondary structure using known Karplus relationships. For alpha helices, 3(10) helices and beta strands mean predicted 3JHN alpha coupling constants are 4.8, 5.6 and 8.5 Hz, respectively. These values differ significantly from those expected for the ideal phi angles (3.9, 3.0 and 8.9 Hz; phi = -57 degrees, -49 degrees, -139 degrees for alpha and 3(10) helices and beta strands (antiparallel), respectively) in regular secondary structure, but agree well with available experimental NMR data for nine proteins. The crystallographic data set has also been used to provide a basis for interpreting coupling constants measured for peptides and denatured proteins. Using a model for a random coil, in which all residues adopt distributions of phi, psi angles equivalent to those seen for residues in the coil regions of native folded proteins, predicted 3JHN alpha values for different residue types have been found to range from 5.9 Hz and 6.1 Hz for glycine and alanine, respectively, to 7.7 Hz for valine. A good correlation has been found between the predicted 3JHN alpha coupling constants for this model and experimental values for a set of peptides that other evidence suggest are highly unstructured. For other peptides, however, deviations from the predictions of the model are clear and provide evidence for additional interactions within otherwise disordered states. The values of homonuclear and heteronuclear coupling constants derived from the protein data base listed here therefore provide a basis not only for analysing the secondary structure of native proteins in solution but for assessing and interpreting the extent of structure present in peptides and non-native states of proteins.

Side-chain conformations in an unfolded protein: chi1 distributions in denatured hen lysozyme determined by heteronuclear 13C, 15N NMR spectroscopy.

Using a 13C and 15N-labelled sample, multi-dimensional heteronuclear NMR techniques have been carried out to characterise hen lysozyme denatured in 8 M urea at pH 2.0. The measurement of 3J(C',Cgamma) and 3J(N,Cgamma) coupling constants has enabled side-chain chi1 torsion angle populations to be probed in the denatured polypeptide chain. Analysis of the coupling constant data has allowed the relative populations of the three staggered rotamers about chi1 to be defined for 51 residues. The amino acids can broadly be divided into five classes that show differing side-chain conformational preferences in the denatured state. These range from a strong preference for the -60 degrees chi1 rotamer for methionine and leucine (74-79 % population) to a favouring of the +60 degrees chi1 rotamer for threonine (67 % population). The differences in behaviour reflect the steric and electrostatic characteristics of the side-chains concerned. A close agreement is seen between the chi1 populations calculated from the experimental coupling constant data and predictions from the statistical model for a random coil that uses the chi1 torsion angle distributions in a data base of native protein structures. Short-range interactions therefore dominate in determining the local conformational properties of side-chains in a denatured protein. Deviations are, however, observed for many of the aromatic residues involved in hydrophobic clusters within the denatured protein. For these residues the effects of additional non-local interactions in the clusters presumably play a major role in determining the chi1 preferences.

A refined solution structure of hen lysozyme determined using residual dipolar coupling data.

A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H-15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low-energy calculated structures has an average backbone RMSD of 0.50+/-0.13A to the mean structure and of 1.49+/-0.10A to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation-dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.

New methylene specific experiments for the measurement of scalar spin-spin coupling constants between protons attached to 13C.

New two- and three-dimensional NMR methods are proposed for the measurement of 3J(H, H) coupling constants between two adjacent methylene moieties. The new experiment, which is based on a combination of the E.COSY principle and double/zero quantum heteronuclear spectroscopy, has been applied to diaceton-glucose and to the protein rhodniin. The coupling constants of CH-CH2 groups have been compared with those obtained from a HCCH-E.COSY experiment to check the reliability of the results. An analysis of the coupling constants derived by comparison between experimental and simulated spectra is presented. Simulations were done with the program wtest considering fully correlated dipolar relaxation. Side-chain conformations in amino acids with adjacent methylene groups can be determined by the new experiment.

Non-destructive and non-invasive observation of friction and wear of human joints and of fracture initiation by acoustic emission.

Quality control in orthopaedic diagnostics according to DIN EN ISO 9000ff requires methods of non-destructive process control, which do not harm the patient by radiation or by invasive examinations. To obtain an improvement in health economy, quality-controlled and non-destructive measurements have to be introduced into the diagnostics and therapy of human joints and bones. A non-invasive evaluation of the state of wear of human joints and of the cracking tendency of bones is, as of today's point of knowledge, not established. The analysis of acoustic emission signals allows the prediction of bone rupture far below the fracture load. The evaluation of dry and wet bone samples revealed that it is possible to conclude from crack initiation to the bone strength and thus to predict the probability of bone rupture.

[Acoustic emission analysis of human bones within the scope of clinical diagnosis]. / Schallemissionsanalyse an menschlichen Knochen im Rahmen der klinischen Diagnostik.

Fractures occurring in human bones produce an acoustic signal, analysis of which permits an evaluation of its source. In the industrial setting acoustic emission analysis (AEA) is used to non-invasively monitor the function of stressed technical systems or parts of systems. During servicing and monitoring of technical systems, acoustic signals emitted by cracks or material deformation are located with the aid of a few acoustic sensors and evaluated for risk-identification purposes. With appropriate technology, therefore, both cortical and trabecular bone can be monitored by acoustic emission analysis. A search is currently ongoing for suitable acoustic technology capable of assessing the extent and location of bone defects and predicting associated risks of fractures occurring. In the present study a system for the measurement and analysis of acoustic emission is described which permits the measurement and analysis of acoustic signals obtained from processed and fresh human and porcine femora. In slightly modified form this system was then used to assess the type and extent of acoustic emission obtained from explanted human femora exposed to cyclical torsional loading until fracture occurred.

Electrochemical and crystal structural analysis of alpha- and dehydro-alpha-lapachones.

Lapachones are pharmaceutically active compounds generating reactive oxygen species. The crystal structure and redox behaviour of the title lapachones, derivates of quinones, were determined by X-ray diffraction and cyclovoltametric measurements. The observed results were compared with beta-lapachone.

Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment.

Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca(2+) from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca(2+) to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca(2+) binding was investigated by means of a Ca(2+)-sensitive electrode. In all cases, Ca(2+) binds with low affinity; the half-maximum binding constant K(1/2) ranges from 6 to 16 mM. The binding stoichiometry between Ca(2+) ions and carboxylic groups is approximately 1:1; an exception is GARP2, where a binding stoichiometry of approximately 1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca(2+) binding.