A novel deconvolution method for modeling UDP-N-acetyl-D-glucosamine biosynthetic pathways based on 13C mass isotopologue profiles under non-steady-state conditions

<p>Abstract</p> <p>Background</p> <p>Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of ¹³C isotopologue data for UDP-<it>N</it>-acetyl-<smcaps>D</smcaps>-glucosamine (UDP-GlcNAc) synthesized from [U-¹³C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes.</p> <p>Results</p> <p>We utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of ¹³C incorporation from [U-¹³C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. ¹³C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional ¹³C enrichment in these subunits against experimental data. The reproducibility and robustness of the deconvolution were verified by replicate experiments, extensive statistical analyses, and cross-validation against NMR data.</p> <p>Conclusions</p> <p>This computational approach revealed the relative fluxes through the different biosynthetic pathways of UDP-GlcNAc, which comprises simultaneous sequential and parallel reactions, providing new insight into the regulation of UDP-GlcNAc levels and <it>O</it>-linked protein glycosylation. This is the first such analysis of UDP-GlcNAc dynamics, and the approach is generally applicable to other complex metabolites comprising distinct metabolic subunits, where sufficient numbers of isotopologues can be unambiguously resolved and accurately measured.</p

Derangement of body representation in complex regional pain syndrome: report of a case treated with mirror and prisms

Perhaps the most intriguing disorders of body representation are those that are not due to primary disease of brain tissue. Strange and sometimes painful phantom limb sensations can result from loss of afference to the brain; and Complex Regional Pain Syndrome (CRPS)—the subject of the current report—can follow limb trauma without pathology of either the central or peripheral nervous system. This enigmatic and vexing condition follows relatively minor trauma, and can result in enduring misery and a useless limb. It manifests as severe pain, autonomic dysfunction, motor disability and ‘neglect-like’ symptoms with distorted body representation. For this special issue on body representation we describe the case of a patient suffering from CRPS, including symptoms suggesting a distorted representation of the affected limb. We report contrasting effects of mirror box therapy, as well as a new treatment—prism adaptation therapy—that provided sustained pain relief and reduced disability. The benefits were contingent upon adapting with the affected limb. Other novel observations suggest that: (1) pain may be a consequence, not the cause, of a disturbance of body representation that gives rise to the syndrome; (2) immobilisation, not pain, may precipitate this reorganisation of somatomotor circuits in susceptible individuals; and (3) limitation of voluntary movement is neither due to pain nor to weakness but, rather, to derangement of body representation which renders certain postures from the repertoire of hand movements inaccessible

Systematisation of spatial uncertainties for comparison between a MR and a CT-based radiotherapy workflow for prostate treatments

<p>Abstract</p> <p>Background</p> <p>In the present work we compared the spatial uncertainties associated with a MR-based workflow for external radiotherapy of prostate cancer to a standard CT-based workflow. The MR-based workflow relies on target definition and patient positioning based on MR imaging. A solution for patient transport between the MR scanner and the treatment units has been developed. For the CT-based workflow, the target is defined on a MR series but then transferred to a CT study through image registration before treatment planning, and a patient positioning using portal imaging and fiducial markers.</p> <p>Methods</p> <p>An "open bore" 1.5T MRI scanner, Siemens Espree, has been installed in the radiotherapy department in near proximity to a treatment unit to enable patient transport between the two installations, and hence use the MRI for patient positioning. The spatial uncertainty caused by the transport was added to the uncertainty originating from the target definition process, estimated through a review of the scientific literature. The uncertainty in the CT-based workflow was estimated through a literature review.</p> <p>Results</p> <p>The systematic uncertainties, affecting all treatment fractions, are reduced from 3-4 mm (1Sd) with a CT based workflow to 2-3 mm with a MR based workflow. The main contributing factor to this improvement is the exclusion of registration between MR and CT in the planning phase of the treatment.</p> <p>Conclusion</p> <p>Treatment planning directly on MR images reduce the spatial uncertainty for prostate treatments.</p

Fully automated high-quality NMR structure determination of small 2H-enriched proteins

Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with 15N- and 13C-edited NOESY data obtained with a perdeuterated 15N, 13C-enriched 13CH3 methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR

Inter-assessor reliability of practice based biomechanical assessment of the foot and ankle

Background There is no consensus on which protocols should be used to assess foot and lower limb biomechanics in clinical practice. The reliability of many assessments has been questioned by previous research. The aim of this investigation was to (i) identify (through consensus) what biomechanical examinations are used in clinical practice and (ii) evaluate the inter-assessor reliability of some of these examinations. Methods Part1: Using a modified Delphi technique 12 podiatrists derived consensus on the biomechanical examinations used in clinical practice. Part 2: Eleven podiatrists assessed 6 participants using a subset of the assessment protocol derived in Part 1. Examinations were compared between assessors. Results Clinicians choose to estimate rather than quantitatively measure foot position and motion. Poor inter-assessor reliability was recorded for all examinations. Intra-class correlation coefficient values (ICC) for relaxed calcaneal stance position were less than 0.23 and were less than 0.14 for neutral calcaneal stance position. For the examination of ankle joint dorsiflexion, ICC values suggest moderate reliability (less than 0.61). The results of a random effects ANOVA highlight that participant (up to 5.7°), assessor (up to 5.8°) and random (upto 5.7°) error all contribute to the total error (up to 9.5° for relaxed calcaneal stance position, up to 10.7° for the examination of ankle joint dorsiflexion). Kappa Fleiss values for categorisation of first ray position and mobility were less than 0.05 and for limb length assessment less than 0.02, indicating slight agreement. Conclusion Static biomechanical assessment of the foot, leg and lower limb is an important protocol in clinical practice, but the key examinations used to make inferences about dynamic foot function and to determine orthotic prescription are unreliable

ICAR: endoscopic skull‐base surgery

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Three individuals, three stories, three burials from medieval Trondheim, Norway

This article presents the life stories of three individuals who lived in Trondheim, Norway, dur- ing the 13th century. Based on skeletal examinations, facial reconstructions, genetic analy- ses, and stable oxygen isotope analyses, the birthplace, mobility, ancestry, pathology, and physical appearance of these people are presented. The stories are discussed within the relevant historical context. These three people would have been ordinary citizens, without any privileges out of the ordinary, which makes them quite rare in the academic literature. Through the study of individuals one gets a unique look into the Norwegian medieval society