Identification and functional characterization of auxiliary enzymes of the β-oxidation in " Arabidopsis thaliana "

Abstract: The ß-oxidation is the universal pathway that allows living organisms to degrade fatty acids. leading to lipid homeostasis and carbon and energy recovery from the fatty acid molecules. This pathway is centred on four core enzymatic activities sufficient to degrade saturated fatty acids. Additional auxiliary enzymes of the ß-oxidation are necessary for the complete degradation of a larger array of molecules encompassing the unsaturated fatty acids. The main pathways of the ßoxidation of fatty acids have been investigated extensively and auxiliary enzymes are well-known in mammals and yeast. The comparison of the established ß-oxidation systems suggests that the activities that are required to proceed to the full degradation of unsaturated fatty acids are present regardless of the organism and rely on common active site templates. The precise identity of the plant enzymes was unknown. By homology searches in the genome of Arabidopsis thaliana, I identified genes. encoding for proteins that could be orthologous to the yeast or animal auxiliary enzymes Δ 3, Δ 2-enoyl-CoA isomerase, Δ 3,5, Δ 2,4 -dienoyl-CoA isomerase, and type 2 enoyl-CoA hydratase. I established that these genes are expressed in Arabidopsis and that their expression can be correlated to the expression of core ß-oxidation genes. Through the observation of chimeric fluorescent protein fusions, I demonstrated that the identified proteins are localized in the peroxisóme, the only organelle where the ß-oxidation occurs in plants. Enzymatic assays were performed with the partially purified enzymes to demonstrate that the identified enzymes can catalyze the same in vitro reactions as their non-plant orthologs. The activities in vivo of the plant enzymes were demonstrated by heterologous complementation of the corresponding yeast Saccharomyces cerevisiae mutants. The complementation was visualized using the artificial polyhydroxyalkanoate (PHA) production in yeast peroxisomes. The recombinant strains, expressing a Pseudomonas aeruginosa PHA synthase modified for a peroxisomal localization, produce this polymer that serves as a trap for the 3-hydroxyacyl-CoA intermediaries of the ßoxidation and that reflects qualitatively and quantitatively the array of molecules that are processed through the ß-oxidation. This complementation demonstrated the implication of the plant Δ 3, Δ 2-enoyl-CoA isomerases and Δ3,5, Δ2,4-dienoyl-CoA isomerase in the degradation of odd chain position unsaturated fatty acids. The presence of a monofunctional type 2 enoyl-CoA hydratase is a novel in eukaryotes. Downregulation of the corresponding gene expression in an Arabidopsis line, modified to produce PHA in the peroxisome, demonstrated thàt this enzyme participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2Eenoyl-CoA for further degradation through the core ß-oxidation cycle. Résumé: La ß-oxydation est une voie universelle de dégradation des acides gras qui permet aux organismes vivants d'assurer une homéostasie lipidique et de récupérer l'énergie et le carbone contenus dans les acides gras. Le coeur de cette voie est composé de quatre réactions enzymatiques suffisantes à la dégradation des acides gras saturés. La présence des enzymes auxiliaires de la ß-oxydation est nécessaire à la dégradation d'une gamme plus étendue de molécules comprenant les acides gras insaturés. Les voies principales de la ß-oxydation des acides gras ont été étudiées en détail et les enzymes auxiliaires sont déterminées chez les mammifères et la levure. La comparaison entre les systèmes de ß-oxydation connus suggère que les activités requises pour la dégradation complète des acides gras insaturés reposent sur la présence de site actifs similaires. L'identité précise des enzymes auxiliaires chez les plantes était inconnue. En cherchant par homologie dans le génome de la plante modèle Arabidopsis thaliana, j'ai identifié des gènes codant pour des protéines pouvant être orthologues aux enzymes auxiliaires Δ3 Δ2-enoyl-CoA isomérase, Δ 3,5 Δ 2,4-dienoyl-CoA isomérase et enoyl-CoA hydratase de type 2 d'origine fongique ou mammalienne. J'ai établi la corrélation de l'expression de ces gènes dans Arabidopsis avec celle de gènes des enzymes du coeur de la ß-oxydation. En observant des chimères de fusion avec des protéines fluorescentes, j'ai démontré que les protéines identifiées sont localisées dans le péroxysomes, le seul organelle où la ß-oxydation se déroule chez les plantes. Des essais enzymatiques ont été conduits avec ces enzymes partiellement purifiées pour démontrer que les enzymes identifiées sont capables de catalyser in vitro les mêmes réactions que leurs orthologues non végétaux. Les activités des enzymes végétales in vivo ont été .démontrées par complémentation hétérologue des mutants de délétion correspondants de levure Saccharomyces cerevisiae. La visualisation de la complémentation est rendue possible par la synthèse de polyhydroxyalcanoate (PHA) dans les péroxysomes de levure. Les souches recombinantes expriment la PHA synthase de Pseudomonas aeruginosa modifiée pour être localisée dans le péroxysome produisent ce polymère qui sert de piège pour les 3-hydroxyacylCoAs intermédiaires de la ß-oxydation et qui reflète qualitativement et quantitativement la gamme de molécules qui subit la ß-oxydation. Cette complémentation a permis de démontrer que les Δ3, Δ2-enoyl-CoA isomérases, et la Δ3.5, Δ2,4-dienoyl-CoA isomérase végétales sont impliquées dans la dégradation des acides gras insaturés en position impaire. L'enoyl-CoA hydratase de type 2 monofonctionelle est une enzyme nouvelle chez les eucaryotes. La sous-expression du gène correspondant dans une lignée d'Arabidopsis modifiée pour produite du PHA dans le péroxysome a permis de démontrer que cette enzyme participe in vivo à la dégradation des acides gras ayant une double liaison en conformation cis (Z) en position paire.

EEG-based functional networks in schizophrenia.

Schizophrenia is often considered as a dysconnection syndrome in which, abnormal interactions between large-scale functional brain networks result in cognitive and perceptual deficits. In this article we apply the graph theoretic measures to brain functional networks based on the resting EEGs of fourteen schizophrenic patients in comparison with those of fourteen matched control subjects. The networks were extracted from common-average-referenced EEG time-series through partial and unpartial cross-correlation methods. Unpartial correlation detects functional connectivity based on direct and/or indirect links, while partial correlation allows one to ignore indirect links. We quantified the network properties with the graph metrics, including mall-worldness, vulnerability, modularity, assortativity, and synchronizability. The schizophrenic patients showed method-specific and frequency-specific changes especially pronounced for modularity, assortativity, and synchronizability measures. However, the differences between schizophrenia patients and normal controls in terms of graph theory metrics were stronger for the unpartial correlation method.

Systems biology in the development of HIV vaccines.

PURPOSE OF REVIEW: In the present review, we will provide the scientific rationale for applying systems biology to the development of vaccines and particularly HIV vaccines, the predictive power of systems biology on the vaccine immunological profile, the correlation between systems biology and the immunological functional profiles of different candidate vaccines, and the value of systems biology in the selection process of identifying the best-in-class candidate vaccines and in the decision process to move into in-vivo evaluation in clinical trials. RECENT FINDINGS: Systems biology has been recently applied to the characterization of the protective yellow fever vaccine YF17D and of seasonal flu vaccines. This has been instrumental in the identification of the components of the immune response that need to be stimulated by the vaccine in order to generate protective immunity. It is worth noting that a systems biology approach is currently being performed to identify correlates of immune protection of the RV144 Thai vaccine, the only known vaccine that showed modest protection against HIV reacquisition. SUMMARY: Systems biology represents a novel and powerful approach to predict the vaccine immunological profile, to identify the protective components of the immune response, and to help in the selection process of the best-in-class vaccines to move into clinical development.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

Dynamic Modelling of Material Flows and Sustainable Resource Use: Case Studies in Regional Metabolism and Space Life Support Systems

Sustainable resource use is one of the most important environmental issues of our times. It is closely related to discussions on the 'peaking' of various natural resources serving as energy sources, agricultural nutrients, or metals indispensable in high-technology applications. Although the peaking theory remains controversial, it is commonly recognized that a more sustainable use of resources would alleviate negative environmental impacts related to resource use. In this thesis, sustainable resource use is analysed from a practical standpoint, through several different case studies. Four of these case studies relate to resource metabolism in the Canton of Geneva in Switzerland: the aim was to model the evolution of chosen resource stocks and flows in the coming decades. The studied resources were copper (a bulk metal), phosphorus (a vital agricultural nutrient), and wood (a renewable resource). In addition, the case of lithium (a critical metal) was analysed briefly in a qualitative manner and in an electric mobility perspective. In addition to the Geneva case studies, this thesis includes a case study on the sustainability of space life support systems. Space life support systems are systems whose aim is to provide the crew of a spacecraft with the necessary metabolic consumables over the course of a mission. Sustainability was again analysed from a resource use perspective. In this case study, the functioning of two different types of life support systems, ARES and BIORAT, were evaluated and compared; these systems represent, respectively, physico-chemical and biological life support systems. Space life support systems could in fact be used as a kind of 'laboratory of sustainability' given that they represent closed and relatively simple systems compared to complex and open terrestrial systems such as the Canton of Geneva. The chosen analysis method used in the Geneva case studies was dynamic material flow analysis: dynamic material flow models were constructed for the resources copper, phosphorus, and wood. Besides a baseline scenario, various alternative scenarios (notably involving increased recycling) were also examined. In the case of space life support systems, the methodology of material flow analysis was also employed, but as the data available on the dynamic behaviour of the systems was insufficient, only static simulations could be performed. The results of the case studies in the Canton of Geneva show the following: were resource use to follow population growth, resource consumption would be multiplied by nearly 1.2 by 2030 and by 1.5 by 2080. A complete transition to electric mobility would be expected to only slightly (+5%) increase the copper consumption per capita while the lithium demand in cars would increase 350 fold. For example, phosphorus imports could be decreased by recycling sewage sludge or human urine; however, the health and environmental impacts of these options have yet to be studied. Increasing the wood production in the Canton would not significantly decrease the dependence on wood imports as the Canton's production represents only 5% of total consumption. In the comparison of space life support systems ARES and BIORAT, BIORAT outperforms ARES in resource use but not in energy use. However, as the systems are dimensioned very differently, it remains questionable whether they can be compared outright. In conclusion, the use of dynamic material flow analysis can provide useful information for policy makers and strategic decision-making; however, uncertainty in reference data greatly influences the precision of the results. Space life support systems constitute an extreme case of resource-using systems; nevertheless, it is not clear how their example could be of immediate use to terrestrial systems.

Neural substrates of social emotion regulation: a FMRI study on imitation and expressive suppression to dynamic facial signals.

Emotion regulation is crucial for successfully engaging in social interactions. Yet, little is known about the neural mechanisms controlling behavioral responses to emotional expressions perceived in the face of other people, which constitute a key element of interpersonal communication. Here, we investigated brain systems involved in social emotion perception and regulation, using functional magnetic resonance imaging (fMRI) in 20 healthy participants. The latter saw dynamic facial expressions of either happiness or sadness, and were asked to either imitate the expression or to suppress any expression on their own face (in addition to a gender judgment control task). fMRI results revealed higher activity in regions associated with emotion (e.g., the insula), motor function (e.g., motor cortex), and theory of mind (e.g., [pre]cuneus) during imitation. Activity in dorsal cingulate cortex was also increased during imitation, possibly reflecting greater action monitoring or conflict with own feeling states. In addition, premotor regions were more strongly activated during both imitation and suppression, suggesting a recruitment of motor control for both the production and inhibition of emotion expressions. Expressive suppression (eSUP) produced increases in dorsolateral and lateral prefrontal cortex typically related to cognitive control. These results suggest that voluntary imitation and eSUP modulate brain responses to emotional signals perceived from faces, by up- and down-regulating activity in distributed subcortical and cortical networks that are particularly involved in emotion, action monitoring, and cognitive control.

Functional GacS in Pseudomonas DSS73 prevents digestion by Caenorhabditis elegans and protects the nematode from killer flagellates.

The success of biocontrol bacteria in soil depends in part on their ability to escape predation. We explored the interactions between Pseudomonas strain DSS73 and two predators, the nematode Caenorhabditis elegans and the flagellate Cercomonas sp. Growth of the nematode in liquid culture was arrested when it was feeding on DSS73 or a DSS73 mutant (DSS73-15C2) unable to produce the biosurfactant amphisin, whereas a regulatory gacS mutant (DSS73-12H8) that produces no exoproducts supported fast growth of the nematode. The flagellate Cercomonas sp. was able to grow on all three strains. The biosurfactant-deficient DSS73 mutant caused severe dilation of the nematode gut. In three-species systems (DSS73, Cercomonas and C. elegans), the nematodes fed on the flagellates, which in turn grazed the bacteria and the number of C. elegans increased. The flagellates Cercomonas sp. usually kill C. elegans. However, DSS73 protected the nematodes from flagellate killing. Soil microcosms inoculated with six rhizobacteria and grazed by nematodes were colonized more efficiently by DSS73 than similar systems grazed by flagellates or without grazers. In conclusion, our results suggest that C. elegans and DSS73 mutually increase the survival of one another in complex multispecies systems and that this interaction depends on the GacS regulator.

Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts.

Zebrafish and Xenopus have become popular model organisms for studying vertebrate development of many organ systems, including the heart. However, it is not clear whether the single ventricular hearts of these species possess any equivalent of the specialized ventricular conduction system found in higher vertebrates. Isolated hearts of adult zebrafish (Danio rerio) and African toads (Xenopus laevis) were stained with voltage-sensitive dye and optically mapped in spontaneous and paced rhythms followed by histological examination focusing on myocardial continuity between the atrium and the ventricle. Spread of the excitation wave through the atria was uniform with average activation times of 20 +/- 2 and 50 +/- 2 ms for zebrafish and Xenopus toads, respectively. After a delay of 47 +/- 8 and 414 +/- 16 ms, the ventricle became activated first in the apical region. Ectopic ventricular activation was propagated significantly more slowly (total ventricular activation times: 24 +/- 3 vs. 14 +/- 2 ms in zebrafish and 74 +/- 14 vs. 35 +/- 9 ms in Xenopus). Although we did not observe any histologically defined tracts of specialized conduction cells within the ventricle, there were trabecular bands with prominent polysialic acid-neural cell adhesion molecule staining forming direct myocardial continuity between the atrioventricular canal and the apex of the ventricle; i.e., the site of the epicardial breakthrough. We thus conclude that these hearts are able to achieve the apex-to-base ventricular activation pattern observed in higher vertebrates in the apparent absence of differentiated conduction fascicles, suggesting that the ventricular trabeculae serve as a functional equivalent of the His-Purkinje system.

A random linear functional approach to efficiency bounds

This paper suggests a method for obtaining efficiency bounds in models containing either only infinite-dimensional parameters or both finite- and infinite-dimensional parameters (semiparametric models). The method is based on a theory of random linear functionals applied to the gradient of the log-likelihood functional and is illustrated by computing the lower bound for Cox's regression model

Functional independence within the self-memory system: new insights from two cases of developmental amnesia.

Neuropsychological and neuroimaging data suggest that the self-memory system can be fractionated into three functionally independent systems processing personal information at several levels of abstraction, including episodic memories of one's life (episodic autobiographical memory, EAM), semantic knowledge of facts about one's life (semantic autobiographical memory, SAM), and semantic knowledge of one's personality [conceptual self, (CS)]. Through the study of two developmental amnesic patients suffering of neonatal brain injuries, we explored how the different facets of the self-memory system develop when growing up with bilateral hippocampal atrophy. Neuropsychological evaluations showed that both of them suffered from dramatic episodic learning disability with no sense of recollection (Remember/Know procedure), whereas their semantic abilities differed, being completely preserved (Valentine) or not (Jocelyn). Magnetic resonance imaging, including quantitative volumetric measurements of the hippocampus and adjacent (entorhinal, perirhinal, and temporopolar) cortex, showed severe bilateral atrophy of the hippocampus in both patients, with additional atrophy of adjacent cortex in Jocelyn. Exploration of EAM and SAM according to lifetime periods covering the entire lifespan (TEMPAu task, Piolino et al., 2009) showed that both patients had marked impairments in EAM, as they lacked specificity, details and sense of recollection, whereas SAM was completely normal in Valentine, but impaired in Jocelyn. Finally, measures of patients' CS (Tennessee Self-Concept Scale, Fitts and Warren, 1996), checked by their mothers, were generally within normal range, but both patients showed a more positive self-concept than healthy controls. These two new cases support a modular account of the medial-temporal lobe with episodic memory and recollection depending on the hippocampus, and semantic memory and familiarity on adjacent cortices. Furthermore, they highlight developmental episodic and semantic functional independence within the self-memory system suggesting that SAM and CS may be acquired without episodic memories.

Neuroendocrine regulation and central dopamine (DA) systems in physical and psychological stress

Physical and psychological stress cause different patterns of changes in the fluorescence intensity of nigral and tuberoinfundibular DA neurons which point to changes in neuronal activity. In order to investigate possible interactions between alpha-MSH (alpha-melanotropin) and DA systems in stress, systemic and intraventricular injections of antiserum against alpha-MSH were made. The functional state of DA neurons was assessed by histochemical microfluorimetry and hormone levels were measured by radioimmunossay. Antiserum against alpha-MSH was found to affect the functional state of DA neurons, but only thorugh the intravenous route. Under physical stress i.v. injection of antiserum against alpha-MSH was accompanied by elevated levels of activity of the DA neurons of the substantia nigra. An intraventricular injection of the same antiserum was ineffective. In psychological stress, an effect was again seen only after intravenous injection of antiserum against alpha-MSH. In this situation, the activity in DA cell groups of the substantia nigra, ventral tegmental area and tubero-infundibular system was increased after antiserum injection. Possible influences from manipulations were checked; certain effects which depended upon experimental situation were noted. Our data suggest a modulatory influence of circulating alpha-MSH on the functional state of central DA systems.

Predicting human resting-state functional connectivity from structural connectivity

In the cerebral cortex, the activity levels of neuronal populations are continuously fluctuating. When neuronal activity, as measured using functional MRI (fMRI), is temporally coherent across 2 populations, those populations are said to be functionally connected. Functional connectivity has previously been shown to correlate with structural (anatomical) connectivity patterns at an aggregate level. In the present study we investigate, with the aid of computational modeling, whether systems-level properties of functional networks-including their spatial statistics and their persistence across time-can be accounted for by properties of the underlying anatomical network. We measured resting state functional connectivity (using fMRI) and structural connectivity (using diffusion spectrum imaging tractography) in the same individuals at high resolution. Structural connectivity then provided the couplings for a model of macroscopic cortical dynamics. In both model and data, we observed (i) that strong functional connections commonly exist between regions with no direct structural connection, rendering the inference of structural connectivity from functional connectivity impractical; (ii) that indirect connections and interregional distance accounted for some of the variance in functional connectivity that was unexplained by direct structural connectivity; and (iii) that resting-state functional connectivity exhibits variability within and across both scanning sessions and model runs. These empirical and modeling results demonstrate that although resting state functional connectivity is variable and is frequently present between regions without direct structural linkage, its strength, persistence, and spatial statistics are nevertheless constrained by the large-scale anatomical structure of the human cerebral cortex.

Pronounced species divergence in corticospinal tract reorganization and functional recovery after lateralized spinal cord injury favors primates.

Experimental and clinical studies suggest that primate species exhibit greater recovery after lateralized compared to symmetrical spinal cord injuries. Although this observation has major implications for designing clinical trials and translational therapies, advantages in recovery of nonhuman primates over other species have not been shown statistically to date, nor have the associated repair mechanisms been identified. We monitored recovery in more than 400 quadriplegic patients and found that functional gains increased with the laterality of spinal cord damage. Electrophysiological analyses suggested that corticospinal tract reorganization contributes to the greater recovery after lateralized compared with symmetrical injuries. To investigate underlying mechanisms, we modeled lateralized injuries in rats and monkeys using a lateral hemisection, and compared anatomical and functional outcomes with patients who suffered similar lesions. Standardized assessments revealed that monkeys and humans showed greater recovery of locomotion and hand function than did rats. Recovery correlated with the formation of corticospinal detour circuits below the injury, which were extensive in monkeys but nearly absent in rats. Our results uncover pronounced interspecies differences in the nature and extent of spinal cord repair mechanisms, likely resulting from fundamental differences in the anatomical and functional characteristics of the motor systems in primates versus rodents. Although rodents remain essential for advancing regenerative therapies, the unique response of the primate corticospinal tract after injury reemphasizes the importance of primate models for designing clinically relevant treatments.

CNDOL: A fast and reliable method for the calculation of electronic properties of very large systems. Applications to retinal binding pocket in rhodopsin and gas phase porphine.

Very large molecular systems can be calculated with the so called CNDOL approximate Hamiltonians that have been developed by avoiding oversimplifications and only using a priori parameters and formulas from the simpler NDO methods. A new diagonal monoelectronic term named CNDOL/21 shows great consistency and easier SCF convergence when used together with an appropriate function for charge repulsion energies that is derived from traditional formulas. It is possible to obtain a priori molecular orbitals and electron excitation properties after the configuration interaction of single excited determinants with reliability, maintaining interpretative possibilities even being a simplified Hamiltonian. Tests with some unequivocal gas phase maxima of simple molecules (benzene, furfural, acetaldehyde, hexyl alcohol, methyl amine, 2,5 dimethyl 2,4 hexadiene, and ethyl sulfide) ratify the general quality of this approach in comparison with other methods. The calculation of large systems as porphine in gas phase and a model of the complete retinal binding pocket in rhodopsin with 622 basis functions on 280 atoms at the quantum mechanical level show reliability leading to a resulting first allowed transition in 483 nm, very similar to the known experimental value of 500 nm of "dark state." In this very important case, our model gives a central role in this excitation to a charge transfer from the neighboring Glu(-) counterion to the retinaldehyde polyene chain. Tests with gas phase maxima of some important molecules corroborate the reliability of CNDOL/2 Hamiltonians.