MHC Class II Transactivator Is an In Vivo Regulator of Osteoclast Differentiation and Bone Homeostasis Co-opted From Adaptive Immunity.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo. © 2014 American Society for Bone and Mineral Research.

Pleiotropy and the low cost of individual traits promote cooperation.

The evolution of cooperation is thought to be promoted by pleiotropy, whereby cooperative traits are coregulated with traits that are important for personal fitness. However, this hypothesis faces a key challenge: what happens if mutation targets a cooperative trait specifically rather than the pleiotropic regulator? Here, we explore this question with the bacterium Pseudomonas aeruginosa, which cooperatively digests complex proteins using elastase. We empirically measure and theoretically model the fate of two mutants-one missing the whole regulatory circuit behind elastase production and the other with only the elastase gene mutated-relative to the wild-type (WT). We first show that, when elastase is needed, neither of the mutants can grow if the WT is absent. And, consistent with previous findings, we show that regulatory gene mutants can grow faster than the WT when there are no pleiotropic costs. However, we find that mutants only lacking elastase production do not outcompete the WT, because the individual cooperative trait has a low cost. We argue that the intrinsic architecture of molecular networks makes pleiotropy an effective way to stabilize cooperative evolution. Although individual cooperative traits experience loss-of-function mutations, these mutations may result in weak benefits, and need not undermine the protection from pleiotropy.

HIV-1 drug resistance transmission networks in southwest Switzerland.

To determine viral subtypes and resistance mutations to antiretroviral treatment (ART) in untreated HIV-1 acutely infected subjects from Southwest Switzerland. Clinical samples were obtained from the HIV primary infection cohort from Lausanne. Briefly, pol gene was amplified by nested PCR and sequenced to generate a 1?kb sequence spanning protease and reverse transcriptase key protein regions. Nucleotide sequences were used to assess viral genotype and ART resistance mutations. Blood specimens and medical information were obtained from 30 patients. Main viral subtypes corresponded to clade B, CRF02_AG, and F1. Resistant mutations to PIs consisted of L10V and accessory mutations 16E and 60E present in all F1 clades. The NNRTI major resistant mutation 103N was detected in all F1 viruses and in other 2 clades. Additionally, we identified F1 sequences from other 6 HIV infected and untreated individuals from Southwest Switzerland, harboring nucleotide motifs and resistance mutations to ART as observed in the F1 strains from the cohort. These data reveal a high transmission rate (16.6%) for NNRTI resistant mutation 103N in a cohort of HIV acute infection. Three of the 5 resistant strains were F1 clades closely related to other F1 isolates from HIV-1 infection untreated patients also coming from Southwest Switzerland. Overall, we provide strong evidence towards an HIV-1 resistant transmission network in Southwest Switzerland. These findings have relevant implications for the local molecular mapping of HIV-1 and future ART surveillance studies in the region.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le résultat d'une lésion ou d'un dysfonctionnement du système nerveux. Les symptômes qui suivent la douleur neuropathique sont sévères et leur traitement inefficace. Une meilleure approche thérapeutique peut être proposée en se basant sur les mécanismes pathologiques de la douleur neuropathique. Lors d'une lésion périphérique une douleur neuropathique peut se développer et affecter le territoire des nerfs lésés mais aussi les territoires adjacents des nerfs non-lésés. Une hyperexcitabilité des neurones apparaît au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle épinière. Le but de ce travail consiste à mettre en évidence les modifications moléculaires associées aux nocicepteurs lésés et non-lésés au niveau des DRG et des laminae I et II de la corne dorsale, là où l'information nociceptive est intégrée. Pour étudier les changements moléculaires liés à la douleur neuropathique nous utilisons le modèle animal d'épargne du nerf sural (spared nerve injury model, SNI) une semaine après la lésion. Pour la sélection du tissu d'intérêt nous avons employé la technique de la microdissection au laser, afin de sélectionner une sous-population spécifique de cellules (notamment les nocicepteurs lésés ou non-lésés) mais également de prélever le tissu correspondant dans les laminae superficielles. Ce travail est couplé à l'analyse à large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons étudié les courants électriques et les propriétés biophysiques des canaux sodiques (Na,,ls) dans les neurones lésés et non-lésés des DRG. Aussi bien dans le système nerveux périphérique, entre les neurones lésés et non-lésés, qu'au niveau central avec les aires recevant les projections des nocicepteurs lésés ou non-lésés, l'analyse du transcriptome montre des différences de profil d'expression. En effet, nous avons constaté des changements transcriptionnels importants dans les nocicepteurs lésés (1561 gènes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 gènes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-lésés (60 gènes), mais important dans leurs laminae de projection (459 gènes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observé que plusieurs processus biologiques sont modifiés. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les mécanismes de réponse au stress sont affectés. Par contre dans les neurones non-lésés seuls les processus biologiques liés au métabolisme et au développement sont modifiés. Au niveau de la corne dorsale de la moelle, nous avons observé des modifications importantes des processus immuno-inflammatoires dans l'aire affectée par les nerfs lésés et des changements associés à l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-lésés. L'analyse approfondie des canaux sodiques a démontré plusieurs changements d'expression, principalement dans les neurones lésés. Les analyses fonctionnelles n'indiquent aucune différence entre les densités de courant tétrodotoxine-sensible (TTX-S) dans les neurones lésés et non-lésés même si les niveaux d'expression des ARNm des sous-unités TTX-S sont modifiés dans les neurones lésés. L'inactivation basale dépendante du voltage des canaux tétrodotoxine-insensible (TTX-R) est déplacée vers des potentiels positifs dans les cellules lésées et non-lésées. En revanche la vitesse de récupération des courants TTX-S et TTX-R après inactivation est accélérée dans les neurones lésés. Ces changements pourraient être à l'origine de l'altération de l'activité électrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En résumé, ces résultats suggèrent l'existence de mécanismes différenciés affectant les neurones lésés et les neurones adjacents non-lésés lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle épinière qui surviennent après lésion sont probablement intégrés différemment selon la perception de signaux des neurones périphériques lésés ou non-lésés. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqués dans la genèse et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied functionál properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Dynamic simulation of regulatory networks using SQUAD.

BACKGROUND: The ambition of most molecular biologists is the understanding of the intricate network of molecular interactions that control biological systems. As scientists uncover the components and the connectivity of these networks, it becomes possible to study their dynamical behavior as a whole and discover what is the specific role of each of their components. Since the behavior of a network is by no means intuitive, it becomes necessary to use computational models to understand its behavior and to be able to make predictions about it. Unfortunately, most current computational models describe small networks due to the scarcity of kinetic data available. To overcome this problem, we previously published a methodology to convert a signaling network into a dynamical system, even in the total absence of kinetic information. In this paper we present a software implementation of such methodology. RESULTS: We developed SQUAD, a software for the dynamic simulation of signaling networks using the standardized qualitative dynamical systems approach. SQUAD converts the network into a discrete dynamical system, and it uses a binary decision diagram algorithm to identify all the steady states of the system. Then, the software creates a continuous dynamical system and localizes its steady states which are located near the steady states of the discrete system. The software permits to make simulations on the continuous system, allowing for the modification of several parameters. Importantly, SQUAD includes a framework for perturbing networks in a manner similar to what is performed in experimental laboratory protocols, for example by activating receptors or knocking out molecular components. Using this software we have been able to successfully reproduce the behavior of the regulatory network implicated in T-helper cell differentiation. CONCLUSION: The simulation of regulatory networks aims at predicting the behavior of a whole system when subject to stimuli, such as drugs, or determine the role of specific components within the network. The predictions can then be used to interpret and/or drive laboratory experiments. SQUAD provides a user-friendly graphical interface, accessible to both computational and experimental biologists for the fast qualitative simulation of large regulatory networks for which kinetic data is not necessarily available.

Genetic removal of tri-unsaturated fatty acids suppresses developmental and molecular phenotypes of an Arabidopsis tocopherol-deficient mutant. Whole-body mapping of malondialdehyde pools in a complex eukaryote.

Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.

Molecular crime and cellular punishment: active detoxification of misfolded and aggregated proteins in the cell by the chaperone and protease networks.

Labile or mutation-sensitised proteins may spontaneously convert into aggregation-prone conformations that may be toxic and infectious. This hazardous behavior, which can be described as a form of "molecular criminality", can be actively counteracted in the cell by a network of molecular chaperone and proteases. Similar to law enforcement agents, molecular chaperones and proteases can specifically identify, apprehend, unfold and thus neutralize "criminal" protein conformers, allowing them to subsequently refold into harmless functional proteins. Irreversibly damaged polypeptides that have lost the ability to natively refold are preferentially degraded by highly controlled ATP-consuming proteases. Damaged proteins that escape proteasomal degradation can also be "incarcerated" into dense amyloids, "evicted" from the cell, or internally "exiled" to the lysosome to be hydrolysed and recycled. Thus, remarkable parallels exist between molecular and human forms of criminality, as well as in the cellular and social responses to various forms of crime. Yet, differences also exist: whereas programmed death is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom chosen by lawless societies. Significantly, there is no cellular equivalent for the role of familial care and of education in general, which is so crucial to the proper shaping of functional persons in the society. Unlike in the cell, humanism introduces a bias against radical solutions such as capital punishment, favouring crime prevention, reeducation and social reinsertion of criminals.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

EEG-based functional brain networks: does the network size matter?

Functional connectivity in human brain can be represented as a network using electroencephalography (EEG) signals. These networks--whose nodes can vary from tens to hundreds--are characterized by neurobiologically meaningful graph theory metrics. This study investigates the degree to which various graph metrics depend upon the network size. To this end, EEGs from 32 normal subjects were recorded and functional networks of three different sizes were extracted. A state-space based method was used to calculate cross-correlation matrices between different brain regions. These correlation matrices were used to construct binary adjacency connectomes, which were assessed with regards to a number of graph metrics such as clustering coefficient, modularity, efficiency, economic efficiency, and assortativity. We showed that the estimates of these metrics significantly differ depending on the network size. Larger networks had higher efficiency, higher assortativity and lower modularity compared to those with smaller size and the same density. These findings indicate that the network size should be considered in any comparison of networks across studies.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Advanced mapping of environmental data: Geostatistics, Machine Learning and Bayesian Maximum Entropy

This book combines geostatistics and global mapping systems to present an up-to-the-minute study of environmental data. Featuring numerous case studies, the reference covers model dependent (geostatistics) and data driven (machine learning algorithms) analysis techniques such as risk mapping, conditional stochastic simulations, descriptions of spatial uncertainty and variability, artificial neural networks (ANN) for spatial data, Bayesian maximum entropy (BME), and more.

Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Application de la modélisation moléculaire à de nouvelles approches thérapeutiques du cancer [Application of molecular modeling to new therapeutic cancer approaches].

Recent progress in the experimental determination of protein structures allow to understand, at a very detailed level, the molecular recognition mechanisms that are at the basis of the living matter. This level of understanding makes it possible to design rational therapeutic approaches, in which effectors molecules are adapted or created de novo to perform a given function. An example of such an approach is drug design, were small inhibitory molecules are designed using in silico simulations and tested in vitro. In this article, we present a similar approach to rationally optimize the sequence of killer T lymphocytes receptors to make them more efficient against melanoma cells. The architecture of this translational research project is presented together with its implications both at the level of basic research as well as in the clinics.