Comparison of order parameters (<i>S²</i>) computed by NMRdyn (open circles) and relax [5] (crosses) for a relaxation analysis on micelle-bound neuropeptide Y [8].

<p>The original set of relaxation data used was downloaded from the Biological Magnetic Resonance Bank <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003820#pone.0003820-Ulrich1" target="_blank">[20]</a>. <i>S²</i> describes the flexibility of a given residue, with a value close to 1 indicative of high local order, and is one of the most informative parameters from a relaxation analysis. The agreement between the results from NMRdyn and relax validates NMRdyn's implementation.</p

Model-free models and associated parameters.

<p>Model-free models and associated parameters.</p

Flowchart describing the operation of NMRdyn.

<p>Panel A shows that the system being studied may be a monomeric protein or may include oligomers. The types of parameters that describe the dynamics of the system are labeled. In the monomeric case, relaxation data are usually measured at different magnetic field strengths and at one concentration, for a series of n nuclei (typically the backbone amide nitrogen for each amino acid, or backbone/sidechain ¹³C labeled sites) as shown in panel B. To study self-association, data at multiple concentrations are required. Relaxation data, molecular parameters, and physical constants are used as input into NMRdyn. Panel C (left side) shows that in the case of a monomeric protein, NMRdyn performs a ‘classical’ analysis, where the AIC value is minimized until it and all microdynamic parameters converge with model optimization and model selection performed at each minimization step. For studies of protein self-association (Panel C, right side), a grid-search approach can be applied, resulting in a set of microdynamic parameters describing the monomeric protein and the oligomer.</p

Fixed stoichiometry analysis of kalata B1, the prototypical cyclotide, assuming a monomer-tetramer equilibrium.

<p>NMRdyn was used to perform a search over different association constants. The overall Akaike's Information Criteria (AIC) score was used to judge the goodness of the fit, with the aim of obtaining the minimum AIC score. The results indicate that an association constant of approximately 3×10⁷ M⁻³ can be used to describe the formation of the kalata B1 tetramer in solution.</p

Impacts of the Callipyge Mutation on Ovine Plasma Metabolites and Muscle Fibre Type

<div><p>The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.</p></div

Metabolic pathways involved in postnatal lamb development.

<p>Metabolites indentified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone-0099726-t002" target="_blank">Table 2</a> are summarized according to their occurrence in metabolic pathways as annotated by KEGG metabolic pathways. ↓, metabolites decreasing with age (blue); ↑, metabolites increasing with age (red). TMAO, trimethylamine N-oxide; HCHO, formaldehyde; CH₄, methane. The reactions contained in the boxed section are likely to be derived from rumen microbial metabolism, except for reactions denoted by **, which occur in mammalian tissues <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726-Asatoor1" target="_blank">[63]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726-Yeung1" target="_blank">[84]</a>, and reactions denoted by *, which can occur both in mammalian tissues or the gut microbiome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726-Zhang1" target="_blank">[85]</a>. This representation of the data recognises that plasma metabolites report the combined metabolic activities of all major tissues as well as the rumen microbiome.</p

Comparison of relative concentrations of metabolites in plasma from Callipyge (N^matC^pat) and normal (N^matN^pat) lambs at 12 weeks of age^a.

a<p>Data were obtained by using relative intensities of plasma metabolites in 16 N^matC^pat and 21 N^matN^pat samples which were collected at 12 weeks of age from a total of 37 animals. Concentrations of metabolites identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726.s005" target="_blank">Table S1</a>, but not listed in this table, did not change significantly.</p>b<p>VIP, Variable Importance in the Projection. Variables with VIP >1 were considered significant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726-Eriksson1" target="_blank">[52]</a>.</p>c<p>Fold changes of levels of plasma metabolites in N^matC^pat lambs compared with the levels in N^matN^pat lambs.</p>d<p>Labeled <i>P</i> values were obtained from Mann–Whitney U test and other <i>P</i> values were determined by Student’s <i>t</i> test.</p>e<p>BH <i>P_adj</i>, significance levels of metabolites after Benjamini-Hochberg multiple testing correction.</p>f,g<p>Mean ± one standard deviation, <i>n</i> = 21 and 16, respectively.</p>h<p>Unidentified metabolites.</p

Metabolic pathway analysis.

<p>The metabolic pathways are represented as circles according to their scores from enrichment (vertical axis) and topology analyses (pathway impact, horizontal axis) using MetaboAnalyst 2.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726-Xia1" target="_blank">[56]</a>. Darker circle colors indicate more significant changes of metabolites in the corresponding pathway. The size of the circle corresponds to the pathway impact score and is correlated with the centrality of the involved metabolites. The metabolic pathways involved in age differences are shown in panels A and B, and the pathways perturbed due to the Callipyge mutation are summarized in panels C and D. Panels A and C were generated using the <i>Homo sapiens</i> library, while the <i>Bos taurus</i> library was selected for production of panels B and D, respectively. Pathways were annotated by numbering when the <i>P</i> values calculated from the enrichment analysis were <0.05. The annotated pathways include: 1, Aminoacyl-tRNA biosynthesis; 2, Arginine and proline metabolism; 3, Biotin metabolism; 4, Cyanoamino acid metabolism; 5, D-Glutamine and D-glutamate metabolism; 6, Galactose metabolism; 7, Glycine, serine and threonine metabolism; 8, Lysine degradation; 9, Methane metabolism; 10, Nitrogen metabolism; 11, Synthesis and degradation of ketone bodies. The color of each metabolic pathway is related to the <i>P</i> value obtained from enrichment analysis and its size represents the fold enrichment score <i>i.e.</i> −ln(<i>P</i>).</p

Changes in relative concentrations of key metabolites in lamb plasma during postnatal development^a.

a<p>Data were obtained by using relative intensities of plasma metabolites in 73 samples, comprising 36 (14 N^matC^pat+22 N^matN^pat) and 37 (16 N^matC^pat+21 N^matN^pat) plasma samples collected at 8 and 12 weeks of age, respectively. Concentrations of metabolites identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726.s005" target="_blank">Table S1</a>, but not listed in this table, did not change significantly.</p>b<p>VIP, Variable Importance in the Projection. Variables with VIP >1 were considered significant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099726#pone.0099726-Eriksson1" target="_blank">[52]</a>.</p>c<p>Fold changes of metabolites in plasma at 12 weeks compared with 8 weeks of age.</p>d<p>Labeled <i>P</i> values were obtained from Mann–Whitney U test and other <i>P</i> values were determined by Student’s <i>t</i> test.</p>e<p>BH <i>P_adj</i>, significance levels of metabolites after Benjamini-Hochberg multiple testing correction.</p>f,g<p>Mean ± one standard deviation, <i>n</i> = 36 and 37, respectively.</p>h<p>TMAO, trimethylamine N-oxide.</p>i<p>Unidentified metabolites.</p

Myofibre characteristics of muscle affected (<i>semimembranosus</i> and <i>semitendinosus</i>) and not affected (<i>supraspinatus</i>) by the Callipyge mutation, determined using myosin heavy chain (MHC) immunohistochemistry in normal (N^matN^pat) and Callipyge (N^matC^pat) lambs at 11 weeks of age.

a<p>Significant <i>P</i> values (<i>P</i><0.05) are labeled as *.</p>b<p>CSA, cross-sectional area.</p>c<p>Type 1, type 1 myosin heavy chain (MHC) slow twitch oxidative fibres; Type 2A, type 2A MHC fast oxidative-glycolytic fibres; Type 2X, type 2X MHC fast twitch glycolytic fibres; Type 2C, type 1–type 2A intermediate fibres; Type 2AX, type 2A–type 2X intermediate fibres.</p