Applying FAIR Principles to Plant Phenotypic Data Management in GnpIS.

GnpIS is a data repository for plant phenomics that stores whole field and greenhouse experimental data including environment measures. It allows long-term access to datasets following the FAIR principles: Findable, Accessible, Interoperable, and Reusable, by using a flexible and original approach. It is based on a generic and ontology driven data model and an innovative software architecture that uncouples data integration, storage, and querying. It takes advantage of international standards including the Crop Ontology, MIAPPE, and the Breeding API. GnpIS allows handling data for a wide range of species and experiment types, including multiannual perennial plants experimental network or annual plant trials with either raw data, i.e., direct measures, or computed traits. It also ensures the integration and the interoperability among phenotyping datasets and with genotyping data. This is achieved through a careful curation and annotation of the key resources conducted in close collaboration with the communities providing data. Our repository follows the Open Science data publication principles by ensuring citability of each dataset. Finally, GnpIS compliance with international standards enables its interoperability with other data repositories hence allowing data links between phenotype and other data types. GnpIS can therefore contribute to emerging international federations of information systems.

Mining Plant Genomic and Genetic Data Using the GnpIS Information System.

GnpIS is an information system designed to help scientists working on plants and fungi to decipher the molecular and genetic architecture of trait variations by facilitating the navigation through genetic, genomic, and phenotypic information. The purpose of the present chapter is to illustrate how users can (1) explore datasets from phenotyping experiments in order to build new datasets for studying genotype × environment interactions in traits, (2) browse into the results of other genetic analysis data such as GWAS to generate or check working hypothesis about candidate genes or to identify important alleles and germplasms for breeding programs, and (3) explore the polymorphism in specific area of the genome using InterMine, JBrowse tools embedded in the GnpIS information system.

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases.

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Physical continuity of the perimysium from myofibers to tendons: involvement in lateral force transmission in skeletal muscle.

Advances in muscle physiology suggest that the perimysium plays a role in the transmission of lateral contractile forces. This hypothesis is strongly supported by our recent demonstration of the existence of "Perimysial Junctional Plates" in bovine Flexor carpi radialis muscle [Passerieux, E., Rossignol, R., Chopard, A., Carnino, A., Marini, J.F., Letellier, T., Delage, J.P. 2006. Structural organization of the perimysium in bovine skeletal muscle: junctional plates and associated intracellular subdomains. J. Struct. Biol. 154 (2), 206-216] However, the overall organization of the perimysium collagen network, as well as its continuity and heterogeneity, have still not been described in detail throughout the entire muscle. We used an extension of the standard NaOH digestion technique and scanning electron microscopy to analyze perimysium architecture in bovine Flexor carpi radialis muscle. First, we observed that the perimysium is made of a highly ordered network of collagen fibers, binding the myofibers from tendon to tendon. We identified basic collagen cable structures, characterized by a straight portion (3 cm long) in the direction of the myofibers and a curved terminal portion at 60 degrees. These cables reach the myofiber surface at the level of the previously described "Perimysial Junctional Plates". At a higher level of organization, these cables stick together to form the walls of numerous tubes arranged in a overlapping honeycomb pattern around the myofibers. At the ends of these tubes, the straight portions of the collagen cables ramify in large bundles that merge with the tendons. Taken together, these observations identify four levels of organization in the perimysium: (i) Perimysial Junctional Plates that constitute the focal attachment between the perimysium and the myofibers, (ii) collagen plexi attaching adjacent myofibers, (iii) a loose lattice of large interwoven fibers, and (iv) honeycomb tubes connecting two tendons. This spatial arrangement of the perimysium supports the view of a complex pattern of lateral force transmission from myofibers to tendons and adjacent muscles.

[Mitochondria in anaesthesia and intensive care]. / La mitochondrie: rôles et implications en anesthésie-réanimation.

OBJECTIVE: Mitochondria play a key role in energy metabolism within the cell through the oxidative phosphorylation. They are also involved in many cellular processes like apoptosis, calcium signaling or reactive oxygen species production. The objectives of this review are to understand the interactions between mitochondrial metabolism and anaesthetics or different stress situations observed in ICU and to know the clinical implications. DATA SOURCES: References were obtained from PubMed data bank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) using the following keywords: mitochondria, anaesthesia, anaesthetics, sepsis, preconditioning, ischaemia, hypoxia. DATA SYNTHESIS: Mitochondria act as a pharmacological target for the anaesthetic agents. The effects can be toxic like in the case of the local anaesthetics-induced myotoxicity. On the other hand, beneficial effects are observed in the anaesthetic-induced myocardial preconditioning. Mitochondrial metabolism could be disturbed in many critical situations (sepsis, chronic hypoxia, ischaemia-reperfusion injury). The study of the underlying mechanisms should allow to propose in the future new specific therapeutics.

Physiological diversity of mitochondrial oxidative phosphorylation.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

Structural organization of the perimysium in bovine skeletal muscle: Junctional plates and associated intracellular subdomains.

We analyzed the structural features of the perimysium collagen network in bovine Flexor carpi radialis muscle using various sample preparation methods and microscopy techniques. We first observed by scanning electron microscopy that perimysium formed a regular network of collagen fibers with three hierarchical levels including (i) a loose lattice of large interwoven fibers ramified in (ii) numerous collagen plexi attaching together adjacent myofibers at the level of (iii) specific structures that we call perimysial junctional plates. Second, we looked more closely at the intracellular organization underneath each plate using transmission electron microscopy, immunohistochemistry, and a three-dimensional reconstruction from serial sections. We observed the accumulation of myonuclei arranged in clusters surrounded by a high density of subsarcolemmal mitochondria and the proximity of capillary branches. Third, we analyzed the distribution of these perimysial junctional plates, subsarcolemmal mitochondria, and myonuclei clusters along the myofibers using a statistical analysis of the distances between these structures. This revealed a global colocalization and the existence of adhesion domains between endomysium and perimysium. Taken together, our observations give a better description of the perimysium organization in skeletal muscle, and provide evidence that perimysial junctional plates with associated intracellular subdomains may participate in the lateral transmission of contractile forces as well as mechanosensing.

Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release.

Intracellular amyloid beta-peptide (A beta) accumulation is considered to be a key pathogenic factor in sporadic Alzheimer's disease (AD), but the mechanisms by which it triggers neuronal dysfunction remain unclear. We hypothesized that gradual mitochondrial dysfunction could play a central role in both initiation and progression of sporadic AD. Thus, we analyzed changes in mitochondrial structure and function following direct exposure to increasing concentrations of A beta(1--42) and A beta(25--35) in order to look more closely at the relationships between mitochondrial membrane viscosity, ATP synthesis, ROS production, and cytochrome c release. Our results show the accumulation of monomeric A beta within rat brain and muscle mitochondria. Subsequently, we observed four different and additive modes of action of A beta, which were concentration dependent: (i) an increase in mitochondrial membrane viscosity with a concomitant decrease in ATP/O, (ii) respiratory chain complexes inhibition, (iii) a potentialization of ROS production, and (iv) cytochrome c release.

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Relationships between muscle mitochondrial metabolism and stress-induced corticosterone variations in rats.

In order to determine the effect of chronic and acute stress on muscle mitochondrial metabolism, two strains of rats were selected on the basis of their different hypothalamo-pituitary-adrenal (HPA) axis responses to different stressors [Spontaneous Hypertensive Rats (SHR) and Lewis rats]. For 8 weeks animals were stressed by daily exposure to either a novel environment (SHR: n=16, Lewis: n=16) or forced exercise (SHR: n=16, Lewis: n=16). An unstressed group was left undisturbed (SHR: n=5, Lewis: n=5). Half of the stressed animals (n=32) were submitted to an acute stress (1-h immobilization). The mitochondrial responses of plantaris muscle [cytochrome-c-oxidase (COX), citrate synthase and succinate dehydrogenase activities, the latter two being measured as indices of functional mitochondrial amount] in the presence of different physiological plasma corticosterone (CORT) concentrations were analyzed. The novel environment and forced exercise stress induced different levels of plasma CORT which were negatively correlated with the amount of functional mitochondria in the plantaris muscle. Therefore, a chronic intermittent stress is able to induce an increase in plasma CORT which may be related to deleterious changes in muscle mitochondrial metabolism. Lastly, the acute stress was not associated with a decrease in functional mitochondria but with an increase in COX activity. This suggests that the relationship between CORT and muscle mitochondrial metabolism depends both on the level and duration of endogenous glucocorticoids exposure.

Initiation of DNA replication by DNA polymerases from primers forming a triple helix.

Despite extensive studies on oligonucleotide-forming triple helices, which were discovered in 1957, their possible relevance in the initiation of DNA replication remains unknown. Using sequences forming triple helices, we have developed a DNA polymerisation assay by using hairpin DNA templates with a 3' dideoxynucleotide end and an unpaired 5'-end extension to be replicated. The T7 DNA polymerase successfully elongated nucleotides to the expected size of the template from the primers forming triple helices composed of 9-14 deoxyguanosine-rich residues. The triple helix-forming primer required for this reaction has to be oriented parallel to the homologous sequence of the hairpin DNA template. Substitution of the deoxyguanosine residues by N7 deazadeoxyguanosines in the hairpin of the template prevented primer elongation, suggesting that the formation of a triple helix is a prerequisite for primer elongation. Furthermore, DNA sequencing could be achieved with the hairpin template through partial elongation of the third DNA strand forming primer. The T4 DNA polymerase and the Klenow fragment of DNA polymerase I provided similar DNA elongation to the T7 polymerase-thioredoxin complex. On the basis of published crystallographic data, we show that the third DNA strand primer fits within the catalytic centre of the T7 DNA polymerase, thus underlying this new property of several DNA polymerases which may be relevant to genome rearrangements and to the evolution of the genetic apparatus, namely the DNA structure and replication processes.

Effect of 'binary mitochondrial heteroplasmy' on respiration and ATP synthesis: implications for mitochondrial diseases.

Respiratory-chain-complex subunits in mitochondria are encoded by nuclear or mitochondrial DNA. This property might have profound implications for the phenotypic expression of mutations affecting oxidative phosphorylation complexes. The aim of this paper is to study the importance of the origin of the mutation (nuclear or mitochondrial) on the expression of mitochondrial defects. We have therefore developed theoretical models illustrating three mechanisms of nuclear or mitochondrial DNA mutation giving rise to a deficiency in the respiratory-chain complex: (1) a partial deficiency, homogeneously distributed in all of the mitochondria; (2) a complete deficiency, only affecting some of the mitochondria ('binary mitochondrial heteroplasmy'); and (3) a partial deficiency, affecting only some of the mitochondria. We show that mutations affecting oxidative phosphorylation complexes will be expressed in different ways depending on their origins. Although the expression of nuclear or mitochondrial mutations is evidence of a biochemical threshold, we demonstrate that the threshold value depends on the origin and distribution of the mutation (homogeneous or not) and also on the energy demand of the tissue. This last prediction has been confirmed in an experimental model using hexokinase for the simulation of the energy demand and a variation in mitochondrial concentration. We also emphasize the possible role of 'binary mitochondrial heteroplasmy' in the expression of mitochondrial DNA mutations and thus the importance of the origin of the deficit (mutation) for the diagnosis or therapy of mitochondrial diseases.

Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics.

We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM.

What do mitochondrial diseases teach us about normal mitochondrial functions...that we already knew: threshold expression of mitochondrial defects.

This paper shows how metabolic control analysis (MCA) can help to explain two important features of mitochondrial diseases: (i) the existence of a threshold in the expression of the complex deficiencies on the respiratory flux or on ATP synthesis, i.e. the fact that it is necessary to have a large complex deficiency in order to observe a substantial decrease in these fluxes; (ii) the tissue specificity, i.e. the fact that all tissues are not affected, even if the complex deficiency is present in all of them. We also show the limits of MCA, particularly when considering the in vivo situation. However, MCA offers a new way to consider mitochondrial diseases. The fact that fluxes only slightly change, when a complex is affected, is done at the expense of great changes in intermediate metabolite concentrations; intermediate metabolites situated upstream from the deficient complex are more reduced, leading to a greater generation of free radicals. This could bring an explanation for the diseases observed in conditions where the mitochondrial rate of ATP synthesis is only slightly affected.

Simple models of threshold curves in the expression of inborn errors of metabolism: application to some experimental observations.

The expression of an enzymatic deficiency in a metabolic network can present a biochemical threshold. This threshold can be characterised thus: (1) a low activity of the enzyme can sustain a normal flux, but (2) a minute further decrease of its activity makes the flux collapse. We give simple mathematical models displaying such a behaviour, and we apply the models to some examples of oxidative phosphorylation dependency on respiratory chain complex deficiency.

Modification of mitochondrial metabolism in fibroblasts from mice with a skeletal muscle mutation (muscular dysgenesis). Evidence of embryonic communication between myoblasts and fibroblasts.

Muscle development during embryogenesis is a complex process involving many mechanisms. It requires a close communication among the different cellular types of the muscle, especially the fibroblasts and myoblasts. Indeed, any abnormality in one cell type might influence the differentiation of the other. Thus, any disturbance altering the metabolism of the myoblasts might lead to modifications in the fibroblasts. To study this phenomenon, we used the dysgenic mouse (mdg-"muscular dysgenesis") carrying a homozygous recessive lethal mutation expressed only in skeletal muscle cells. First, we found that fibroblasts isolated from such mutant muscle (and not from mutant skin tissue) and grown in culture exhibited an altered metabolism. Secondly, muscle fibroblasts showed a lower capacity for proliferation. We also observed that respiration and ATP synthesis of dysgenic muscle fibroblasts were deficient, while respiratory chain enzymatic activities were normal. Finally, intracellular [Ca2+] levels of dysgenic fibroblasts are 50% of those of normal fibroblasts. These results support the hypothesis that certain characteristics of fibroblasts are determined by the surrounding cellular environment during embryonic organogenesis, and that such modifications are stable when the fibroblasts are isolated in vitro. Since fibroblast differentiation was disrupted permanently, this suggests, in the case of myopathies, that the modified cells, surrounding the muscle tissue, could contribute to the muscle pathology. Synergistic activities of this type should be considered when studying the course of pathologies in different types of muscle diseases.

Statistical analysis of mitochondrial pathologies in childhood: identification of deficiencies using principal component analysis.

Mitochondrial pathologies are a heterogeneous group of metabolic disorders that are frequently characterized by anomalies of oxidative phosphorylation, especially in the respiratory chain. The identification of these anomalies may involve many investigations, and biochemistry is a main tool. However, considering the whole set of biochemical data, the interpretation of the results by the traditionally used statistical methods remains complex and does not always lead to an unequivocal conclusion about the presence or absence of a respiratory chain defect. This arises from three main problems: (a) the absence of an a priori-defined control population, because the determination of the control values are derived from the whole set of investigated patients, (b) the small size of the population studied, (c) the large number of variables collected, each of which creates a wide variability. To cope with these problems, the principal component analysis (PCA) has been applied to the biochemical data obtained from 35 muscle biopsies of children suspected of having a mitochondrial disease. This analysis makes it possible for each respiratory chain complex to distinguish between different subsets within the whole population (normal, deficient, and, in between, borderline subgroups of patients) and to detect the most discriminating variables. PCA of the data of all complexes together showed that mitochondrial diseases in this population were mainly caused by multiple deficits in respiratory chain complexes. This analysis allows the definition of a new subgroup of newborns, which have high respiratory chain complex activity values. Our results show that the PCA method, which simultaneously takes into account all of the concerned variables, allows the separation of patients into subgroups, which may help clinicians make their diagnoses.

Tissue variation in the control of oxidative phosphorylation: implication for mitochondrial diseases.

Metabolic control analysis has often been used for quantitative studies of the regulation of mitochondrial oxidative phosphorylations (OXPHOS). The main contribution of this work has been to show that the control of mitochondrial metabolic fluxes can be shared among several steps of the oxidative phosphorylation process, and that this distribution can vary according to the steady state and the tissue. However, these studies do not show whether this observed variation in the OXPHOS control is due to the experimental conditions or to the nature of the mitochondria. To find out if there actually exists a tissue variation in the distribution of OXPHOS control coefficients, we determined the control coefficients of seven OXPHOS complexes on the oxygen-consumption flux in rat mitochondria isolated from five different tissues under identical experimental conditions. Thus in this work, only the nature of the mitochondria can be responsible for any variation detected in the control coefficient values between different tissues. The analysis of control coefficient distribution shows two tissue groups: (i) the muscle and the heart, controlled essentially at the level of the respiratory chain; and (ii) the liver, the kidney and the brain, controlled mainly at the phosphorylation level by ATP synthase and the phosphate carrier. We propose that this variation in control coefficient according to the tissue origin of the mitochondria can explain part of the tissue specificity observed in mitochondrial cytopathies.

Threshold effect and tissue specificity. Implication for mitochondrial cytopathies.

Mitochondrial cytopathies present a tissue specificity characterized by the fact that even if a mitochondrial DNA mutation is present in all tissues, only some will be affected and induce a pathology. Several mechanisms have been proposed to explain this phenomenon such as the appearance of a sporadic mutation in a given stem cell during embryogenesis or mitotic segregation, giving different degrees of heteroplasmy in tissues. However, these mechanisms cannot be the only ones involved in tissue specificity. In this paper, we propose an additional mechanism contributing to tissue specificity. It is based on the metabolic expression of the defect in oxidative phosphorylation (OXPHOS) complexes that can present a biochemical threshold. The value of this threshold for a given OXPHOS complex can vary according to the tissue; thus different tissues will display different sensitivities to a defect in an OXPHOS complex. To verify this hypothesis and to illustrate the pathological consequences of the variation in biochemical thresholds, we studied their values for seven OXPHOS complexes in mitochondria isolated from five different rat tissues. Two types of behavior in the threshold curves can be distinguished corresponding to two modes of OXPHOS response to a deficiency. We propose a classification of tissues according to their type of OXPHOS response to a complex deficiency and therefore to their threshold values.

Menkes disease: study of the mitochondrial respiratory chain in three cases.

Mitochondrial oxidative metabolism in three patients with typical Menkes disease was studied. In two cases, a general decrease in all of the respiratory chain complex activities (I, II, III and IV) was observed. However, in the most severe case, these activities were entirely normal. Our results emphasize the diversity of the cellular expression of Menkes disease which can, in some cases, be associated with a mitochondrial encephalomyopathy.