Assessing the potential of quantitative 2D HSQC NMR in 13C enriched living organisms.

In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.

Dissolution Trapping of Carbon Dioxide in Heterogeneous Aquifers.

The geologic architecture in sedimentary reservoirs affects the behavior of density-driven flow and the dispersion of CO2-rich brine. The spatial organization and connectivity of facies types play an important role. Low-permeability facies may suppress fingering and reduce vertical spreading, but may also increase transverse mixing. This is more pronounced when geologic structures create preferential flow pathways through connected facies types. We perform high-resolution simulations of three-dimensional (3D) heterogeneous formations whose connectivity cannot be represented in two-dimensional models consistent with percolation theory. This work focuses on the importance of 3D facies-based heterogeneity and connectivity on advection-diffusion transport of dissolved CO2. Because the dissolution of CO2 and the subsequent density increase of brine are the driving force for gravitational instabilities, we model the phase behavior with the accurate cubic-plus-association equation-of-state, which accounts for the self-association of polar water molecules and the cross-association between CO2 and water. Our results elucidate how the spatial organization of facies affects the dynamics of CO2 convective mixing. Scaling relations for the evolution of a global dispersion-width provide insights that can be universally applied. The results suggest that the long-term evolution and scaling of dispersion are surprisingly similar for homogeneous and (binary and multiscale) heterogeneous porous media.

Next-generation heteronuclear decoupling for high-field biomolecular NMR spectroscopy.

Ultra-high-field NMR spectroscopy requires an increased bandwidth for heteronuclear decoupling, especially in biomolecular NMR applications. Composite pulse decoupling cannot provide sufficient bandwidth at practical power levels, and adiabatic pulse decoupling with sufficient bandwidth is compromised by sideband artifacts. A novel low-power, broadband heteronuclear decoupling pulse is presented that generates minimal, ultra-low sidebands. The pulse was derived using optimal control theory and represents a new generation of decoupling pulses free from the constraints of periodic and cyclic sequences. In comparison to currently available state-of-the-art methods this novel pulse provides greatly improved decoupling performance that satisfies the demands of high-field biomolecular NMR spectroscopy.

The Fantastic Four: A plug 'n' play set of optimal control pulses for enhancing NMR spectroscopy.

We present highly robust, optimal control-based shaped pulses designed to replace all 90° and 180° hard pulses in a given pulse sequence for improved performance. Special attention was devoted to ensuring that the pulses can be simply substituted in a one-to-one fashion for the original hard pulses without any additional modification of the existing sequence. The set of four pulses for each nucleus therefore consists of 90° and 180° point-to-point (PP) and universal rotation (UR) pulses of identical duration. These 1ms pulses provide uniform performance over resonance offsets of 20kHz ((1)H) and 35kHz ((13)C) and tolerate reasonably large radio frequency (RF) inhomogeneity/miscalibration of ±15% ((1)H) and ±10% ((13)C), making them especially suitable for NMR of small-to-medium-sized molecules (for which relaxation effects during the pulse are negligible) at an accessible and widely utilized spectrometer field strength of 600MHz. The experimental performance of conventional hard-pulse sequences is shown to be greatly improved by incorporating the new pulses, each set referred to as the Fantastic Four (Fanta4).

Optimal control design of band-selective excitation pulses that accommodate relaxation and RF inhomogeneity.

Existing optimal control protocols for mitigating the effects of relaxation and/or RF inhomogeneity on broadband pulse performance are extended to the more difficult problem of designing robust, refocused, frequency selective excitation pulses. For the demanding case of T(1) and T(2) equal to the pulse length, anticipated signal losses can be significantly reduced while achieving nearly ideal frequency selectivity. Improvements in performance are the result of allowing residual unrefocused magnetization after applying relaxation-compensated selective excitation by optimized pulses (RC-SEBOPs). We demonstrate simple pulse sequence elements for eliminating this unwanted residual signal.

New strategies for designing robust universal rotation pulses: application to broadband refocusing at low power.

Optimizing pulse performance often requires a compromise between maximizing signal amplitude and minimizing spectral phase errors. We consider methods for the de novo design of universal rotation pulses, applied specifically but not limited to refocusing pulses. Broadband inversion pulses that rotate all magnetization components 180° about a given fixed axis are necessary for refocusing and mixing in high-resolution NMR spectroscopy. The relative merits of various methodologies for generating pulses suitable for broadband refocusing are considered. The de novo design of 180° universal rotation pulses (180(UR)(°)) using optimal control can provide improved performance compared to schemes which construct refocusing pulses as composites of existing pulses. The advantages of broadband universal rotation by optimized pulses (BURBOP) are most evident for pulse design that includes tolerance to RF inhomogeneity or miscalibration. Nearly ideal refocusing is possible over a resonance offset range of ± 170% relative to the nominal pulse B(1) field, concurrent with tolerance to B(1) inhomogeneity/miscalibration of ± 33%. We present new modifications of the optimal control algorithm that incorporate symmetry principles (S-BURBOP) and relax conservative limits on peak RF pulse amplitude for short time periods that pose no threat to the probe. We apply them to generate a set of low-power 180(BURBOP)(°) pulses suitable for widespread use in (13)C spectroscopy on the majority of available probes. A quantitative measure for the reduced spectral phase error provided by these symmetry principles is also derived. For pulses designed according to this symmetry, refocusing phase errors are virtually eliminated upon application of EXORCYCLE or an equivalent G-180(S-BURBOP)(°)-G gradient sandwich, independent of resonance offset and RF inhomogeneity. The magnitude of the refocused component is not significantly compromised in achieving such ideal phase performance.

Design and application of robust rf pulses for toroid cavity NMR spectroscopy.

We present robust radio frequency (rf) pulses that tolerate a factor of six inhomogeneity in the B1 field, significantly enhancing the potential of toroid cavity resonators for NMR spectroscopic applications. Both point-to-point (PP) and unitary rotation (UR) pulses were optimized for excitation, inversion, and refocusing using the gradient ascent pulse engineering (GRAPE) algorithm based on optimal control theory. In addition, the optimized parameterization (OP) algorithm applied to the adiabatic BIR-4 UR pulse scheme enabled ultra-short (50 µs) pulses with acceptable performance compared to standard implementations. OP also discovered a new class of non-adiabatic pulse shapes with improved performance within the BIR-4 framework. However, none of the OP-BIR4 pulses are competitive with the more generally optimized UR pulses. The advantages of the new pulses are demonstrated in simulations and experiments. In particular, the DQF COSY result presented here represents the first implementation of 2D NMR spectroscopy using a toroid probe.

Optimal control design of pulse shapes as analytic functions.

Representing NMR pulse shapes by analytic functions is widely employed in procedures for optimizing performance. Insights concerning pulse dynamics can be applied to the choice of appropriate functions that target specific performance criteria, focusing the solution search and reducing the space of possible pulse shapes that must be considered to a manageable level. Optimal control theory can accommodate significantly larger parameter spaces and has been able to tackle problems of much larger scope than more traditional optimization methods. However, its numerically generated pulses, as currently constructed, do not readily incorporate the capabilities of particular functional forms, and the pulses are not guaranteed to vary smoothly in time, which can be a problem for faithful implementation on older hardware. An optimal control methodology is derived for generating pulse shapes as simple parameterized functions. It combines the benefits of analytic and numerical protocols in a single powerful algorithm that both complements and enhances existing optimization strategies.

Linear phase slope in pulse design: application to coherence transfer.

Using optimal control methods, robust broadband excitation pulses can be designed with a defined linear phase dispersion. Applications include increased bandwidth for a given pulse length compared to equivalent pulses requiring no phase correction, selective pulses, and pulses that mitigate the effects of relaxation. This also makes it possible to create pulses that are equivalent to ideal hard pulses followed by an effective evolution period. For example, in applications, where the excitation pulse is followed by a constant delay, e.g. for the evolution of heteronuclear couplings, part of the pulse duration can be absorbed in existing delays, significantly reducing the time overhead of long, highly robust pulses. We refer to the class of such excitation pulses with a defined linear phase dispersion as ICEBERG pulses (Inherent Coherence Evolution optimized Broadband Excitation Resulting in constant phase Gradients). A systematic study of the dependence of the excitation efficiency on the phase dispersion of the excitation pulses is presented, which reveals surprising opportunities for improved pulse sequence performance.

Optimal control design of excitation pulses that accommodate relaxation.

An optimal control algorithm for mitigating the effects of T(1) and T(2) relaxation during the application of long pulses is derived. The methodology is applied to obtain broadband excitation that is not only tolerant to RF inhomogeneity typical of high resolution probes, but is relatively insensitive to relaxation effects for T(1) and T(2) equal to the pulse length. The utility of designing pulses to produce specific phase in the final magnetization is also presented. The results regarding relaxation and optimized phase are quite general, with many potential applications beyond the specific examples presented here.

The features of Drosophila core promoters revealed by statistical analysis.

BACKGROUND: Experimental investigation of transcription is still a very labor- and time-consuming process. Only a few transcription initiation scenarios have been studied in detail. The mechanism of interaction between basal machinery and promoter, in particular core promoter elements, is not known for the majority of identified promoters. In this study, we reveal various transcription initiation mechanisms by statistical analysis of 3393 nonredundant Drosophila promoters. RESULTS: Using Drosophila-specific position-weight matrices, we identified promoters containing TATA box, Initiator, Downstream Promoter Element (DPE), and Motif Ten Element (MTE), as well as core elements discovered in Human (TFIIB Recognition Element (BRE) and Downstream Core Element (DCE)). Promoters utilizing known synergetic combinations of two core elements (TATA_Inr, Inr_MTE, Inr_DPE, and DPE_MTE) were identified. We also establish the existence of promoters with potentially novel synergetic combinations: TATA_DPE and TATA_MTE. Our analysis revealed several motifs with the features of promoter elements, including possible novel core promoter element(s). Comparison of Human and Drosophila showed consistent percentages of promoters with TATA, Inr, DPE, and synergetic combinations thereof, as well as most of the same functional and mutual positions of the core elements. No statistical evidence of MTE utilization in Human was found. Distinct nucleosome positioning in particular promoter classes was revealed. CONCLUSION: We present lists of promoters that potentially utilize the aforementioned elements/combinations. The number of these promoters is two orders of magnitude larger than the number of promoters in which transcription initiation was experimentally studied. The sequences are ready to be experimentally tested or used for further statistical analysis. The developed approach may be utilized for other species.

Functional characterization of core promoter elements: the downstream core element is recognized by TAF1.

Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase II-transcribed genes. The downstream core element (DCE) was discovered in the human beta-globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). We show here that the DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF(II)250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex.

Promoter classifier: software package for promoter database analysis.

Promoter Classifier is a package of seven stand-alone Windows-based C++ programs allowing the following basic manipulations with a set of promoter sequences: (i) calculation of positional distributions of nucleotides averaged over all promoters of the dataset; (ii) calculation of the averaged occurrence frequencies of the transcription factor binding sites and their combinations; (iii) division of the dataset into subsets of sequences containing or lacking certain promoter elements or combinations; (iv) extraction of the promoter subsets containing or lacking CpG islands around the transcription start site; and (v) calculation of spatial distributions of the promoter DNA stacking energy and bending stiffness. All programs have a user-friendly interface and provide the results in a convenient graphical form. The Promoter Classifier package is an effective tool for various basic manipulations with eukaryotic promoter sequences that usually are necessary for analysis of large promoter datasets. The program Promoter Divider is described in more detail as a representative component of the package.

Computational technique for improvement of the position-weight matrices for the DNA/protein binding sites.

Position-weight matrices (PWMs) are broadly used to locate transcription factor binding sites in DNA sequences. The majority of existing PWMs provide a low level of both sensitivity and specificity. We present a new computational algorithm, a modification of the Staden-Bucher approach, that improves the PWM. We applied the proposed technique on the PWM of the GC-box, binding site for Sp1. The comparison of old and new PWMs shows that the latter increase both sensitivity and specificity. The statistical parameters of GC-box distribution in promoter regions and in the human genome, as well as in each chromosome, are presented. The majority of commonly used PWMs are the 4-row mononucleotide matrices, although 16-row dinucleotide matrices are known to be more informative. The algorithm efficiently determines the 16-row matrices and preliminary results show that such matrices provide better results than 4-row matrices.

Synergy of human Pol II core promoter elements revealed by statistical sequence analysis.

MOTIVATION: The subject of our paper is bioinformatics analysis of the distinguishing features of human promoter DNA sequences, in particular of synergetic combinations of core promoter elements therein. We suppose that specific scenarios of transcription initiation are essentially related to various particular implementations of the interaction of basal transcription machinery with promoter DNA, depending on the presence and mutual positioning of core promoter elements. RESULTS: In addition to the combinations of core promoter elements previously experimentally confirmed [TATA box and Initiator (Inr), Downstream Promoter Element (DPE) and Inr, and TFIIB recognition element (BRE) and TATA box] we propose other alternate synergetic combinations: BRE and Inr, BRE and DPE, and TATA and DPE with respective models. The suggestion is based on a high statistical significance of the alternate combinations in promoters, comparable with the significance of the known combinations. We also present arguments that the BRE element is statistically more important than previously thought, and suggest possible mechanisms of action of the core elements in the promoters with multiple transcription start sites. CONTACT: ioschikhes-1@medctr.osu.edu SUPPLEMENTARY INFORMATION: Supplementary information is available at http://bmi.osu.edu/~ilya/synergy/Gershenzon_SuppMat-R.pdf.