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The OpenSky Network is a non-profit association that crowdsources the global collection of live air traffic control data broadcast by aircraft and makes them available to researchers. OpenSky's data have been used by over 100 academic groups in the past 5 years, with popular research applications ranging from improved weather forecasting to climate analysis. With the COVID-19 outbreak, the demand for live and historic aircraft flight data has surged. Researchers around the world use air traffic data to comprehend the spread of the pandemic and analyse the effects of the global containment measures on economies, climate and other systems. With this work, we present a comprehensive air traffic dataset, derived and enriched from the full OpenSky data and made publicly available for the first time (Olive et al., 2020; https://doi.org/10.5281/zenodo.3931948, last access: 9 February 2021). It spans all flights seen by the network's more than 3500 members between 1 January 2019 and 1 July 2020. The archive is being updated every month and for the first 18 months includes 41 900 660 flights, from 160 737 aircraft, which were seen to frequent 13 934 airports in 127 countries.

The use of wireless signals for the purposes of localization enables a host of applications relating to the determination and verification of the positions of network participants ranging from radar to satellite navigation. Consequently, this has been a longstanding interest of theoretical and practical research in mobile networks and many solutions have been proposed in the scientific literature. However, it is hard to assess the performance of these in the real world and, more importantly, to compare their advantages and disadvantages in a controlled scientific manner. With this work, we attempt to improve the current state of art methodology in localization research and to place it on a solid scientific grounding for future investigations. Concretely, we developed LocaRDS, an open reference data set of real-world crowdsourced flight data featuring more than 222 million measurements from over 50 million transmissions recorded by 323 sensors. We demonstrate how we can verify the quality of LocaRDS measurements so that it can be used to test, analyze and directly compare different localization methods. Finally, we provide an example implementation for the aircraft localization problem and a discussion of possible metrics for use with LocaRDS.

Physical or chemical interactions between drug product (DP) components can occur during manufacturing and/or upon storage; and may alter DP shelf life and performance. In this work a new Powder X-ray Diffraction (PXRD) peak was observed in DP under accelerated storage conditions. Due to the complex drug product matrix (including API, polymer, fillers, super disintegrant and lubricant), it was challenging to pinpoint the component(s) responsible for the new peak. In addition to PXRD, other orthogonal techniques including Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS), Solid State Nuclear Magnetic Resonance (SSNMR) and Infrared (IR) spectroscopy were employed in this investigation to understand the root cause mechanistically. Specifically, multi nuclei SSNMR ( 1 H, 23 Na, 13 C) was instrumental in delineating the components of the matrix. We identified the root cause to be an acid base reaction occurring in the DP, whereby sodium ion in sodium stearyl fumarate (SSF) is replaced by proton leading to SSF form conversion. We also identified commercially available SSF to be a hydrate that can dehydrate to an anhydrous form upon heating. In general, the same techniques can be used to investigate interactions of any multi component solid dosage forms., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I 320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand β6 of the Pdx1 (βα) 8 -barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

A crystalline phase of the pharmaceutical compound ronacaleret hydrochloride is studied by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and single-crystal X-ray diffraction. The crystal structure is determined to contain two independent cationic molecules and chloride anions in the asymmetric unit, which combine with the covalent structure of the molecule to yield complex SSNMR spectra. Experimental approaches based on dipolar correlation, chemical shift tensor analysis, and quadrupolar interaction analysis are employed to obtain detailed information about this phase. Density functional theory (DFT) calculations are used to predict chemical shielding and electric field gradient (EFG) parameters for comparison with experiment. (1)H SSNMR experiments performed at 16.4 T using magic-angle spinning (MAS) and homonuclear dipolar decoupling provide information about hydrogen bonding and molecular connectivity that can be related to the crystal structure. (19)F and (13)C assignments for the Z' = 2 structure are obtained using DFT calculations, (19)F homonuclear dipolar correlation, and (13)C-(19)F heteronuclear dipolar correlation experiments. (35)Cl MAS experiments at 16.4 T observe two chlorine sites that are assigned using calculated chemical shielding and EFG parameters. SSNMR dipolar correlation experiments are used to extract (1)H-(13)C, (1)H-(15)N, (1)H-(19)F, (13)C-(19)F, and (1)H-(35)Cl through-space connectivity information for many positions of interest. The results allow for the evaluation of the performance of a suite of SSNMR experiments and computational approaches as applied to a complex but typical pharmaceutical solid phase.

A number of superconducting electron cyclotron resonance (ECR) ion sources use gyrotrons at either 24 or 28 GHz for ECR heating. In these systems, the microwave power is launched into the plasma using the TE01 circular waveguide mode. This is fundamentally different and may be less efficient than the typical rectangular, linearly polarized TE10 mode used for launching waves at lower frequencies. To improve the 28 GHz microwave coupling in VENUS, a TE01-HE11 mode conversion system has been built to test launching HE11 microwave power into the plasma chamber. The HE11 mode is a quasi-Gaussian, linearly polarized mode, which should couple strongly to the plasma electrons. The mode conversion is done in two steps. First, a 0.66 m long "snake" converts the TE01 mode to the TE11 mode. Second, a corrugated circular waveguide excites the HE11 mode, which is launched directly into the plasma chamber. The design concept draws on the development of similar devices used in tokamaks and stellerators. The first tests of the new coupling system are described below.

Recently the Versatile ECR for NUclear Science (VENUS) ion source was engaged in a 60-day long campaign to deliver high intensity (48)Ca(11+) beam to the 88-Inch Cyclotron. As the first long term use of VENUS for multi-week heavy-element research, new methods were developed to maximize oven to target efficiency. First, the tuning parameters of VENUS for injection into the cyclotron proved to be very different than those used to tune VENUS for maximum beam output of the desired charge state immediately following its bending magnet. Second, helium with no oxygen support gas was used to maximize the efficiency. The performance of VENUS and its low temperature oven used to produce the stable requested 75 eμA of (48)Ca(11+) beam current was impressive. The consumption of (48)Ca in VENUS using the low temperature oven was checked roughly weekly, and was found to be on average 0.27 mg/h with an ionization efficiency into the 11+ charge state of 5.0%. No degradation in performance was noted over time. In addition, with the successful operation of VENUS the 88-Inch cyclotron was able to extract a record 2 pμA of (48)Ca(11+), with a VENUS output beam current of 219 eμA. The paper describes the characteristics of the VENUS tune used for maximum transport efficiency into the cyclotron as well as ongoing efforts to improve the transport efficiency from VENUS into the cyclotron. In addition, we briefly present details regarding the recent successful repair of the cryostat vacuum system.

Chemical imaging using confocal Raman microscopy is a useful analytical tool in drug development because of its ability to spatially image active ingredients and excipients in dosage forms and relate their distribution to product performance. While Raman spectra are highly specific for individual components of a formulation, most Raman microscopic mapping experiments require extensive experimental time. Laser wavelengths in the near-infrared range are used to suppress fluorescence but reduce sensitivity because of the inverse quadratic dependence of Raman scattering on laser wavelength. Compact, simple ultraviolet (UV) laser designs now allow for confocal UV Raman microscopy to be performed using a versatile instrument also capable of conventional Raman microscopy and epifluorescence imaging analyses. This study presents the results of UV Raman microscopy analyses using 266 nm laser irradiation of four pharmaceutical compositions of interest, including two types of tablets containing low doses of active ingredients (in the 0.2% w/w range), an amorphous dispersion containing 1% w/w of a small molecule drug, and an enteric coated layered peptide formulation. Resonance Raman enhancements are observed for four of the active ingredients studied in these formulations. The spectroscopic properties of the materials used in this study are also assessed by diffuse reflectance UV-visible spectroscopy, fluorescence spectroscopy, and conventional bulk Fourier transform Raman spectroscopy using 1064 nm laser irradiation. Confocal UV Raman microscopy was found to offer good sensitivity and allowed for rapid microscopic mapping of drugs and excipients at low concentrations in pharmaceutical formulations.

The solubility of drug molecules can often be improved through preparation and delivery of cyclodextrin (CD) inclusion complexes. These drug-oligosaccharide complexes can be prepared in solution and converted to the solid state via methods such as lyophilization and spray-drying, or they can be prepared directly from solids by a variety of methods. The development of drug-CD complexes as solids allows for potential advantages in dosage form design, such as the preparation of layered formulations, and it also can yield improvements in chemical and physical stability. 2D solid-state NMR (SSNMR) methods provide a direct way to probe drug-CD interactions in solid complexes through dipolar interactions between nuclei within the drug and CD molecules. In this study, 2D heteronuclear and homonuclear correlation SSNMR experiments involving (1)H, (13)C, (19)F, and (31)P nuclei are used to demonstrate the inclusion of drug within the CD cavity in a variety of powder samples. To illustrate the general applicability of the SSNMR approach presented, examples are shown for the drugs diflunisal, adefovir dipivoxil, voriconazole, dexamethasone, and prednisolone in complexes with α-CD, β-CD, and sulfobutylether-substituted β-CD. The quantitative analysis of included and free drug fractions in a solid drug-CD complex using SSNMR is also demonstrated. On the basis of these results, general approaches to the characterization of these materials using SSNMR are proposed.

Solid-state (13)C, (19)F, and (15)N magic angle spinning NMR studies of Form I of atorvastatin calcium are reported, including chemical shift tensors of all resolvable carbon sites and fluorine sites. The complete (13)C and (19)F chemical shift assignments are given based on an extensive analysis of (13)C-(1)H HETCOR and (13)C-(19)F HETCOR results. The solid-state NMR data indicate that the asymmetric unit of this material contains two atorvastatin molecules. A possible structure of Form I of atorvastatin calcium (ATC-I), derived from solid-state NMR data and density functional theory calculations of various structures, is proposed for this important active pharmaceutical ingredient (API).

The 28 GHz Ion Source VENUS (versatile ECR for nuclear science) is back in operation after the superconducting sextupole leads were repaired and a fourth cryocooler was added. VENUS serves as an R&D device to explore the limits of electron cyclotron resonance source performance at 28 GHz with its 10 kW gryotron and optimum magnetic fields and as an ion source to increase the capabilities of the 88-Inch Cyclotron both for nuclear physics research and applications. The development and testing of ovens and sputtering techniques cover a wide range of applications. Recent experiments on bismuth demonstrated stable operation at 300 eμA of Bi(31+), which is in the intensity range of interest for high performance heavy-ion drivers such as FRIB (Facility for Rare Isotope Beams). In addition, the space radiation effects testing program at the cyclotron relies on the production of a cocktail beam with many species produced simultaneously in the ion source and this can be done with a combination of gases, sputter probes, and an oven. These capabilities are being developed with VENUS by adding a low temperature oven, sputter probes, as well as studying the RF coupling into the source.

Solid-state NMR (SSNMR) can provide detailed structural information about amorphous solid dispersions of pharmaceutical small molecules. In this study, the ability of SSNMR experiments based on dipolar correlation, spin diffusion, and relaxation measurements to characterize the structure of solid dispersions is explored. Observation of spin diffusion effects using the 2D (1)H-(13)C cross-polarization heteronuclear correlation (CP-HETCOR) experiment is shown to be a useful probe of association between the amorphous drug and polymer that is capable of directly proving glass solution formation. Dispersions of acetaminophen and indomethacin in different polymers are examined using this approach, as well as (1)H double-quantum correlation experiments to probe additional structural features. (1)H-(19)F CP-HETCOR serves a similar role for fluorinated drug molecules such as diflunisal in dispersions, providing a rapid means to prove the formation of a glass solution. Phase separation is detected using (13)C, (19)F, and (23)Na-detected (1)H T(1) experiments in crystalline and amorphous solid dispersions that contain small domains. (1)H T(1) measurements of amorphous nanosuspensions of trehalose and dextran illustrate the ability of SSNMR to detect domain size effects in dispersions that are not glass solutions via spin diffusion effects. Two previously unreported amorphous solid dispersions involving up to three components and containing voriconazole and telithromycin are analyzed using these experiments to demonstrate the general applicability of the approach.

4,7-Dihydro-1H-tricyclopenta[def,jkl,pqr]triphenylene (sumanene) and indeno[1,2,3-cd]fluoranthene (indenofluoranthene) are structural moieties related to Buckminsterfullerene (C(60)). As such, understanding their structural characteristics is of great interest because of the insight they shed upon C(60). Hence, solid-state NMR (ssNMR) and ab initio quantum mechanical calculations with Gaussian03 are used in order to understand and to better characterize the molecular conformation and properties of sumanene and indenofluoranthene. Sumanene has bowl shaped curvature in its natural conformation and indenofluoranthene is planar in its natural conformation, which led us to examine how altering the curvature affects the chemical shifts in relation to those of C(60). Using X-ray structures of both sumanene and indenofluoranthene as our starting model, we calculate the energy and chemical shielding tensors and compare these data with those collected utilizing the (13)C ssNMR FIREMAT experiment. We define curvature of sumanene and indenofluoranthene using the pi-orbital axis vector (POAV) pyramidalization angle (theta(p)). We calculate the energy of varying conformations of indenofluoranthene versus their theta(p) associated with each deformed conformation.

This paper describes the recent development and commissioning of a pepper-pot emittance meter at the Lawrence Berkeley National Laboratory (LBNL). It is based on a potassium bromide (KBr) scintillator screen in combination with a charged coupled device camera. Pepper-pot scanners record the full four-dimensional transverse phase space emittances which are particularly interesting for electron cyclotron resonance ion sources. The strengths and limitations of evaluating emittances using optical pepper-pot scanners are described and systematic errors induced by the optical data acquisition system will be presented. Light yield tests of KBr exposed to different ion species and first emittance measurement data using ion beams extracted from the 6.4 GHz LBNL electron cyclotron resonance ion source are presented and discussed.

Introduction: The Glasgow Coma Scale (GCS) is the most widely used scoring system for comatose patients in intensive care. Limitations of the GCS include the impossibility to assess the verbal score in intubated or aphasic patients, and an inconsistent inter-rater reliability. The FOUR (Full Outline of UnResponsiveness) score, a new coma scale not reliant on verbal response, was recently proposed. The aim of the present study was to compare the inter-rater reliability of the GCS and the FOUR score among unselected patients in general critical care. A further aim was to compare the inter-rater reliability of neurologists with that of intensive care unit (ICU) staff., Methods: In this prospective observational study, scoring of GCS and FOUR score was performed by neurologists and ICU staff on 267 consecutive patients admitted to intensive care., Results: In a total of 437 pair wise ratings the exact inter-rater agreement for the GCS was 71%, and for the FOUR score 82% (P = 0.0016); the inter-rater agreement within a range of +/- 1 score point for the GCS was 90%, and for the FOUR score 92% (P = ns.). The exact inter-rater agreement among neurologists was superior to that among ICU staff for the FOUR score (87% vs. 79%, P = 0.04) but not for the GCS (73% vs. 73%). Neurologists and ICU staff did not significantly differ in the inter-rater agreement within a range of +/- 1 score point for both GCS (88% vs. 93%) and the FOUR score (91% vs. 88%)., Conclusions: The FOUR score performed better than the GCS for exact inter-rater agreement, but not for the clinically more relevant agreement within the range of +/- 1 score point. Though neurologists outperformed ICU staff with regard to exact inter-rater agreement, the inter-rater agreement of ICU staff within the clinically more relevant range of +/- 1 score point equalled that of the neurologists. The small advantage in inter-rater reliability of the FOUR score is most likely insufficient to replace the GCS, a score with a long tradition in intensive care.

The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.

Pyridoxal 5'-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C(p)) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C(p) is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A "swap mutant" in which the complete alphaN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via alphaN and catalytic activation of the glutaminase are linked processes.

The (1)H-(13)C solid-state NMR heteronuclear correlation (HETCOR) experiment is demonstrated to provide shift assignments in certain powders that have two or more structurally independent molecules in the unit cell (i.e. multiple molecules per asymmetric unit). Although this class of solids is often difficult to characterize using other methods, HETCOR provides both the conventional assignment of shifts to molecular positions and associates many resonances with specific molecules in the asymmetric unit. Such assignments facilitate conformational characterization of the individual molecules of the asymmetric unit and the first such characterization solely from solid-state NMR data is described. HETCOR offers advantages in sensitivity over prior methods that assign resonances in the asymmetric unit by (13)C-(13)C correlations and therefore allows shorter average analysis times in natural abundance materials. The (1)H-(13)C analysis is demonstrated first on materials with known shift assignments from INADEQUATE data (santonin and Ca(OAc)(2) phase I) to verify the technique and subsequently is extended to a pair of unknown solids: (+)-catechin and Ca(OAc)(2) phase II. Sufficient sensitivity and resolution is achieved in the spectra to provide assignments to one of the specific molecules of the asymmetric unit at over 54% of the sites.

Two biosynthetic routes for the synthesis of pyridoxal 5'-phosphate (PLP), the biologically active compound of vitamin B6, have been characterized. The major pathway leads to direct formation of PLP from a pentasaccharide and a trisaccharide and is operative in plants, fungi, protozoa, and bacteria. This reaction is catalyzed by a single glutamine amidotransferase enzyme complex consisting of a pyridoxal synthase, termed Pdx1, and a glutaminase, termed Pdx2. In this complex, Pdx2 generates ammonia from L-glutamine and supplies it to Pdx1 for incorporation into PLP. The glutaminase activity of Pdx2 requires the presence of Pdx1 in a heteromeric complex, previously characterized by a crystallographic three-dimensional (3D) structure determination. Here, we give a thermodynamic description of complex formation of Bacillus subtilis PLP synthase in the absence or presence of L-glutamine. We show that L-glutamine directly affects the tightness of the protein complex, which exhibits dissociation constants of 6.9 and 0.3 microM in its absence and presence, respectively (at 25 degrees C). This result relates to the positioning of L-glutamine on the heterodimer interface as seen in the 3D structure. In an analysis of the temperature dependence of the enthalpy, negative heat capacity changes (deltaCp) agree with a protein interface governed by hydrophobic interactions. The measured heat capacity change is also a function of L-glutamine, with a negative deltaCp in the presence of L-glutamine and a more negative one in its absence. These findings suggest that L-glutamine not only affects the strength of complex formation but also determines the forces involved in complex formation, with regard to different relative contributions of hydrophobic and hydrophilic interactions.

Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.

The principal (13)C chemical-shift values for the pi-[TCNE](2)(2-) dimer anion within an array of counterions have been measured to understand better the electronic structure of these atypical chemical species in several related TCNE-based structures. The structure of pi-[TCNE](2)(2-) is unusual as it contains two very long C-C bond lengths (ca. 2.9 Angstroms) between the two monomeric units and has been found to exist as a singlet state, suggestive of a (1)A(1g) (b(2u)(2)b(1g)(0)) electronic configuration. A systematic study of several oxidation states of [TCNE](n) (n = 0, 1-, 2-) was conducted to determine how the NMR chemical-shift tensor values change as a function of electronic structure and to understand the interactions that lead to spin-pairing of the monomer units. The density functional theory (DFT) calculated nuclear shielding tensors are correlated with the experimentally determined principal chemical-shift values. Such theoretical methods provide information on the tensor magnitudes and orientations of their principal tensor components with respect to the molecular frame. Both theoretical and experimental ethylenic chemical-shielding tensors reveal high sensitivity in the component, delta(perpendicular), lying in the monomer molecular plane and perpendicular to the pi-electron plane. This largest shift dependence on charge density is observed to be about -111 ppm/e(-) for delta(perpendicular). The component in the molecular plane but parallel to the central C=C bond, delta(parallel), exhibits a sensitivity of approximately -43 ppm/e(-). However, the out-of-plane component delta'(perpendicular) shows a minimal dependence of -2.6 ppm/e(-) on the oxidation state (n) of [TCNE](n). These relative values support the claim that it is changes within the ethylenic pi-electrons and not the sigma-electrons that best account for the dramatic variations in bonding and shift tensors in this series of compounds. Concerning the intraion bonding, relatively weak Wiberg bond orders between the two monomeric components of the dimer correlate with the long bonds linking the two [TCNE(*)](-) monomers. The chemical-shift tensors for the cyano group, compared to the ethylene shifts, exhibit a reduced sensitivity on the TCNE oxidation state. The experimental principal chemical-shift components agree (within typical errors) with the calculated quantum mechanical shieldings used to correlate the bonding. The embedded ion model (EIM) was used to investigate the typically large electrostatic lattice potential in these ionic materials. Chemical-shielding principal values calculated with the EIM model differ from experiment by +/-3.82 ppm on average, whereas in the absence of an electrostatic field model, the experimental and theoretical results agree by +/-4.42 ppm, which is only a modest increase in error considering the overall ionic magnitudes associated with the tensor variations. Apparently, the effects of the sizable long-range electrostatic fields cancel when the shifts are computed because of lattice symmetry.

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.

A new sensitive 2D isotropic-anisotropic separation experiment that utilizes stroboscopic phase encoding in the evolution dimension (SPEED) under magic angle sample spinning is presented. This 2D experiment consists of a train of 2N - 1 pi pulses that are applied over 2N rotor periods. The pi pulse train effectively reduces the apparent spinning speed in the evolution dimension by a factor of 1 / (2N) from the mechanical spinning speed. Thus, problems commonly associated with magic angle turning such as stable slow spinning, different matching and TPPM proton decoupling conditions are avoided. Data replication similar to the five pi replicated magic angle turning (FIREMAT) and pseudo 2D sideband suppression (P2DSS) experiments transfers resolution from the acquisition dimension to the evolution dimension. Hence, large spectral windows with good digital resolution are obtained with a few evolution increments. Here, slow spinning sideband patterns are extracted from the replicated 2D dataset with TIGER processing. Nevertheless, 2D Fourier transformation is also applicable. The extracted sideband patterns are identical to magic angle turning sideband pattern allowing for easy extraction of principal shift components. Accurate (13)C principal shift components are obtained for 3-methylglutaric acid using SPEED and FIREMAT experiments to validate the method. Furthermore, SPEED spectra for calcium acetate and alpha santonin are reported to show the wide applicability of this new experiment.

The determination of backbone conformations in powdered peptides using 13C and 15N shift tensor information is explored. The 13C and 15N principal shift values in natural abundance 13C and 15N melanostatin (L-Pro-L-Leu-Gly amide) are measured using the FIREMAT technique. Furthermore, the orientation of the C-N bond in the 13C shift principal axis system for the backbone carbons is obtained from the presence of the 13C-14N dipolar coupling. The Ramachandran angles for the title compound are obtained from solid-state NMR data by comparing the experimentally determined shift tensor information to systematic theoretical shielding calculations on N-formyl-L-amino acid-amide models. The effects of geometry optimization and neglect of intermolecular interactions on the theoretical shielding values in the model compounds are investigated. The sets of NMR derived Ramachandran angles are assembled in a set of test structures that are compared to the available single-crystal X-ray structure. Shift tensor calculations on the test structures and the X-ray structure are used to further assess the importance of intermolecular interactions when the shift tensor is used as a structural probe in powdered peptides.

EIM (embedded ion method), cluster, combined EIM/cluster, and isolated molecule (13)C and (15)N chemical shielding and quadrupolar coupling constant (QCC) calculations at the B3LYP level with D95**, D95++**, 6-311G**, and 6-311+G** basis sets were done on the amino acids l-alanine, l-asparagine monohydrate, and l-histidine monohydrate monohydrochloride and on the two polymorphs α and γ glycine. The intermolecular interactions that are present in the amino acid crystals are accounted for in the EIM calculations by a finite array of point charges calculated from Ewald lattice sums and in the cluster calculations by a shell of neighboring molecules or molecular fragments. The combined EIM/cluster calculations utilize a cluster of molecules inside an EIM point charge array. The theoretical (13)C and (15)N principal shielding values for the amino acids studied are compared to the experimental principal shift values. In addition, theoretical CN bond orientations in the chemical shift principal axis system (PAS) are compared to the experimental orientations obtained from (13)C-(14)N dipolar couplings. The theoretical QCC at the nitrogen positions are compared to experimental (14)N QCC principal values reported in the literature. The carbon and nitrogen theoretical chemical shielding, the C-N orientations, and the QCCs from the ab initio calculations show improved agreement with the experimental values when the intermolecular interactions are accounted for by EIM or cluster calculations. The EIM (13)C shielding calculations are found to give better agreement with the experimental values than cluster (13)C shielding calculations. However, to achieve good agreement between the theoretical (14)N QCC and the (15)N principal shielding values with the respective experimental values, both intermolecular electrostatic and covalent interactions have to be included explicitly in the EIM/cluster calculations.

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed., ((c) 2002 Elsevier Science (USA).)

Two of the three conformational polymorphs of dimethyl-3,6-dichloro-2,5-dihydroxyterephthalate are studied by solid-state NMR techniques. The structural differences between the polymorphs have previously been studied by X-ray. In these two polymorphs named white and yellow due to their color, the major structural difference is the torsional angle between the ester group and the aromatic ring. The yellow form has a dihedral angle of 4 degrees between the plane of the aromatic ring and the plane of the ester group, while the white form has two different molecules per unit cell with dihedral angles of 70 degrees and 85 degrees. This change greatly affects the conjugation in the pi-electronic system. In addition, there are differences in the hydrogen-bonding patterns, with the white form having intermolecular hydrogen bonds and the yellow form having intramolecular hydrogen bonds. In this work, the carbon isotropic chemical shift values and the chlorine electric field gradient (EFG) tensor information are extracted from the (13)C MAS spectra, and the principal values of the chemical shift tensors of the carbons are obtained from 2D FIREMAT experiments. Quantum chemical calculations of the chemical shift tensor data as well as the EFG tensor are performed at the HF and DFT levels of theory on individual molecules and on stacks of three molecules to account for the important intermolecular interactions in the white form. The differences between the spectral data on the two polymorphs are discussed in terms of the known electronic and structural differences.