Targeted Compound Selection with Increased Sensitivity in C-Enriched Biological and Environmental Samples Using C-DREAMTIME in Both High-Field and Low-Field NMR.

Chemical characterization of complex mixtures by Nuclear Magnetic Resonance (NMR) spectroscopy is challenging due to a high degree of spectral overlap and inherently low sensitivity. Therefore, NMR experiments that reduce overlap and increase signal intensity hold immense potential for the analysis of mixtures such as biological and environmental media. Here, we introduce a C version of DREAMTIME (Designed Refocused Excitation And Mixing for Targets In Vivo and Mixture Elucidation) NMR, which, when analyzing C-enriched materials, allows the user to selectively detect only the compound(s) of interest and remove all other peaks in a C spectrum.

Evaluation of double-tuned single-sided planar microcoils for the analysis of small C enriched biological samples using H- C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences.

Comprehensive Multiphase NMR of whole organisms: A complementary tool to NMR.

Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e.

Inverse or direct detect experiments and probes: Which are "best" for in-vivo NMR research of C enriched organisms?

In-vivo Nuclear Magnetic Resonance (NMR) spectroscopy is a unique and powerful approach for understanding sublethal toxicity, recovery, and elucidating a contaminant's toxic mode of action. However, magnetic susceptibility distortions caused by the organisms, along with sample complexity, lead to broad and overlapping 1D NMR spectra. As such, 2D NMR in combination with C enrichment (to increase signal) is a requirement for metabolite assignment and monitoring using high field in-vivo flow based NMR.

NMR assignment of the in vivo daphnia magna metabolome.

Daphnia (freshwater fleas) are among the most widely used organisms in regulatory aquatic toxicology/ecology, while their recent listing as an NIH model organism is stimulating research for understanding human diseases and processes. Daphnia are small enough to fit inside high field NMR spectrometers and can be kept alive indefinitely using flow systems that deliver food and oxygen. As such, in vivo NMR holds the potential to monitor when/if environmental stress is occurring, understand "why" chemicals are toxic (biochemical pathways impacted and toxic-mode-of-action), and differentiate between a temporary flux response (i.

Understanding the Fate of Environmental Chemicals Inside Living Organisms: NMR-Based C Isotopic Suppression Selects Only the Molecule of Interest within C-Enriched Organisms.

In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as (water fleas) and (freshwater shrimps) are commonly C-enriched to increase the signal in NMR experiments. A key goal of in vivo NMR is to monitor how molecules (nutrients, contaminants, or drugs) are metabolized.

Selective Amino Acid-Only in Vivo NMR: A Powerful Tool To Follow Stress Processes.

In vivo NMR of small C-enriched aquatic organisms is developing as a powerful tool to detect and explain toxic stress at the biochemical level. Amino acids are a very important category of metabolites for stress detection as they are involved in the vast majority of stress response pathways. As such, they are a useful proxy for stress detection in general, which could then be a trigger for more in-depth analysis of the metabolome.

Assessing the potential of quantitative 2D HSQC NMR in C 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 H-C 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.

The new magnetic diagnostics in the WEST tokamak.

The WEST tokamak consists of a major upgrade of the superconducting medium size tokamak Tore Supra aiming at testing ITER divertor components. Such modification has required rebuilding a full set of magnetic diagnostics. The project was started in 2013 and completed in 2016.

Partially C-labeled mouse tissue as reference for LC-MS based untargeted metabolomics.

The inclusion of stable isotope-labeled reference standards in the sample is an established method for the detection and relative quantification of metabolic features in untargeted metabolomics. In order to quantify as many metabolites as possible, the reference should ideally include the same metabolites in their stable isotope-labeled form as the sample under investigation. We present here an attempt to use partially C-labeled mouse material as internal standard for relative metabolite quantification of mouse and human samples in untargeted metabolomics.

Development and Application of a Low-Volume Flow System for Solution-State in Vivo NMR.

In vivo nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique, since it allows samples to be analyzed in their natural, unaltered state, criteria paramount for living organisms. In this study, a novel continuous low-volume flow system, suitable for in vivo NMR metabolomics studies, is demonstrated. The system allows improved locking, shimming, and water suppression, as well as allowing the use of trace amounts of expensive toxic contaminants or low volumes of precious natural environmental samples as stressors.

Stable isotope compounds - production, detection, and application.

Stable isotopes are used in wide fields of application from natural tracers in biology, geology and archeology through studies of metabolic fluxes to their application as tracers in quantitative proteomics and structural biology. We review the use of stable isotopes of biogenic elements (H, C, N, O, S, Mg, Se) with the emphasis on hydrogen and its heavy isotope deuterium. We will discuss the limitations of enriching various compounds in stable isotopes when produced in living organisms.

Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model.

Comprehensive multiphase NMR applied to a living organism.

Comprehensive multiphase (CMP) NMR is a novel technology that integrates all the hardware from solution-, gel- and solid-state into a single NMR probe, permitting all phases to be studied in intact samples. Here comprehensive multiphase (CMP) NMR is used to study all components in a living organism for the first time. This work describes 4 new scientific accomplishments summarized as: (1) CMP-NMR is applied to a living animal, (2) an effective method to deliver oxygen to the organisms is described which permits longer studies essential for in-depth NMR analysis in general, (3) a range of spectral editing approaches are applied to fully differentiate the various phases solutions (metabolites) through to solids (shell) (4) C isotopic labelling and multidimensional NMR are combined to provide detailed assignment of metabolites and structural components .

Identification of aquatically available carbon from algae through solution-state NMR of whole (13)C-labelled cells.

Green algae and cyanobacteria are primary producers with profound impact on food web functioning. Both represent key carbon sources and sinks in the aquatic environment, helping modulate the dissolved organic matter balance and representing a potential biofuel source. Underlying the impact of algae and cyanobacteria on an ecosystem level is their molecular composition.

Autotrophic production of stable-isotope-labeled arginine in Ralstonia eutropha strain H16.

With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong P(cbbL) promoter of the R.

Incidental findings in paranasal sinuses and mastoid cells: a cross-sectional magnetic resonance imaging (MRI) study in a pediatric radiology department.

Purpose: Misdiagnosis of "sinusitis" is still frequent in children, although mucosal swelling in the paranasal sinuses of children has been reported as a common incidental finding. Recent radiological publications on the problem are rare. We prospectively evaluated the mucosal thickening in the paranasal sinuses and mastoid cells of children who underwent MRI of the head for reasons other than sinusitis or mastoiditis.

SILAC zebrafish for quantitative analysis of protein turnover and tissue regeneration.

Defective tissue regeneration is thought to contribute to several human diseases, including neurodegenerative disorders, heart failure and various lung diseases. Boosting the regenerative capacity has been suggested a possible therapeutic approach. Methods to metabolically label newly synthesized proteins in vivo with stable isotopic forms of amino acids holds promise for the study of protein turnover and tissue regeneration that are fundamental to the sustained life of all organisms.

Stable isotope metabolic labeling with a novel N-enriched bacteria diet for improved proteomic analyses of mouse models for psychopathologies.

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic.

DNA melting by RNA polymerase at the T7A1 promoter precedes the rate-limiting step at 37 degrees C and results in the accumulation of an off-pathway intermediate.

The formation of a transcriptionally active complex by RNA polymerase involves a series of short-lived structural intermediates where protein conformational changes are coupled to DNA wrapping and melting. We have used time-resolved KMnO(4) and hydroxyl-radical X-ray footprinting to directly probe conformational signatures of these complexes at the T7A1 promoter. Here we demonstrate that DNA melting from m12 to m4 precedes the rate-limiting step in the pathway and takes place prior to the formation of full downstream contacts.

The tree-edit-distance, a measure for quantifying neuronal morphology.

The shape of neuronal cells strongly resembles botanical trees or roots of plants. To analyze and compare these complex three-dimensional structures it is important to develop suitable methods. We review the so called tree-edit-distance known from theoretical computer science and use this distance to define dissimilarity measures for neuronal cells.

Conformational heterogeneity in RNA polymerase observed by single-pair FRET microscopy.

Kinetic, structural, and single-molecule transcription measurements suggest that RNA polymerase can adopt many different conformations during elongation. We have measured the geometry of the DNA and RNA in ternary elongation complexes using single-pair fluorescence resonance energy transfer. Six different synthetic transcription elongation complexes were constructed from DNA containing an artificial transcription bubble, an RNA primer, and core RNA polymerase from Escherichia coli.

Structural changes in the vascular bundles of light-exposed and shaded spruce needles suffering from Mg deficiency and ozone pollution.

The correlation between structural changes of the vascular bundles and needle yellowing was examined for needles of damaged spruce (Picea abies (L.) Karst.) growing at a Mg-deficient and ozone polluted mountain site in the Central Black Forest (840m a.