Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Natural genetic variation of root system architecture from Arabidopsis to Brachypodium: towards adaptive value.

Root system architecture is a trait that displays considerable plasticity because of its sensitivity to environmental stimuli. Nevertheless, to a significant degree it is genetically constrained as suggested by surveys of its natural genetic variation. A few regulators of root system architecture have been isolated as quantitative trait loci through the natural variation approach in the dicotyledon model, Arabidopsis. This provides proof of principle that allelic variation for root system architecture traits exists, is genetically tractable, and might be exploited for crop breeding. Beyond Arabidopsis, Brachypodium could serve as both a credible and experimentally accessible model for root system architecture variation in monocotyledons, as suggested by first glimpses of the different root morphologies of Brachypodium accessions. Whether a direct knowledge transfer gained from molecular model system studies will work in practice remains unclear however, because of a lack of comprehensive understanding of root system physiology in the native context. For instance, apart from a few notable exceptions, the adaptive value of genetic variation in root system modulators is unknown. Future studies should thus aim at comprehensive characterization of the role of genetic players in root system architecture variation by taking into account the native environmental conditions, in particular soil characteristics.

RecA-DNA helical filaments in genetic recombination.

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.

A pathogen-specific epitope inserted into recombinant secretory immunoglobulin A is immunogenic by the oral route.

Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F beta strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses.

Characterization of Fas (Apo-1, CD95)-Fas ligand interaction.

The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Ab initio modeling and molecular dynamics simulation of the alpha 1b-adrenergic receptor activation.

This work describes the ab initio procedure employed to build an activation model for the alpha 1b-adrenergic receptor (alpha 1b-AR). The first version of the model was progressively modified and complicated by means of a many-step iterative procedure characterized by the employment of experimental validations of the model in each upgrading step. A combined simulated (molecular dynamics) and experimental mutagenesis approach was used to determine the structural and dynamic features characterizing the inactive and active states of alpha 1b-AR. The latest version of the model has been successfully challenged with respect to its ability to interpret and predict the functional properties of a large number of mutants. The iterative approach employed to describe alpha 1b-AR activation in terms of molecular structure and dynamics allows further complications of the model to allow prediction and interpretation of an ever-increasing number of experimental data.

Cost-effectiveness of low-molecular-weight heparin for secondary prophylaxis of cancer-related venous thromboembolism.

Although extended secondary prophylaxis with low-molecular-weight heparin was recently shown to be more effective than warfarin for cancer-related venous thromboembolism, its cost-effectiveness compared to traditional prophylaxis with warfarin is uncertain. We built a decision analytic model to evaluate the clinical and economic outcomes of a 6-month course of low-molecular-weight heparin or warfarin therapy in 65-year-old patients with cancer-related venous thromboembolism. We used probability estimates and utilities reported in the literature and published cost data. Using a US societal perspective, we compared strategies based on quality-adjusted life-years (QALYs) and lifetime costs. The incremental cost-effectiveness ratio of low-molecular-weight heparin compared with warfarin was 149,865 dollars/QALY. Low-molecular-weight heparin yielded a quality-adjusted life expectancy of 1.097 QALYs at the cost of 15,329 dollars. Overall, 46% (7108 dollars) of the total costs associated with low-molecular-weight heparin were attributable to pharmacy costs. Although the low-molecular-weigh heparin strategy achieved a higher incremental quality-adjusted life expectancy than the warfarin strategy (difference of 0.051 QALYs), this clinical benefit was offset by a substantial cost increment of 7,609 dollars. Cost-effectiveness results were sensitive to variation of the early mortality risks associated with low-molecular-weight heparin and warfarin and the pharmacy costs for low-molecular-weight heparin. Based on the best available evidence, secondary prophylaxis with low-molecular-weight heparin is more effective than warfarin for cancer-related venous thromboembolism. However, because of the substantial pharmacy costs of extended low-molecular-weight heparin prophylaxis in the US, this treatment is relatively expensive compared with warfarin.

Ant genomics sheds light on the molecular regulation of social organization.

Ants are powerful model systems for the study of cooperation and sociality. In this review, we discuss how recent advances in ant genomics have contributed to our understanding of the evolution and organization of insect societies at the molecular level.

Stem cell transplantation in brain tumors: a new field for molecular imaging?

Neural stem cells have been proposed as a new and promising treatment modality in various pathologies of the central nervous system, including malignant brain tumors. However, the underlying mechanism by which neural stem cells target tumor areas remains elusive. Monitoring of these cells is currently done by use of various modes of molecular imaging, such as optical imaging, magnetic resonance imaging and positron emission tomography, which is a novel technology for visualizing metabolism and signal transduction to gene expression. In this new context, the microenvironment of (malignant) brain tumors and the blood-brain barrier gains increased interest. The authors of this review give a unique overview of the current molecular-imaging techniques used in different therapeutic experimental brain tumor models in relation to neural stem cells. Such methods for molecular imaging of gene-engineered neural stem/progenitor cells are currently used to trace the location and temporal level of expression of therapeutic and endogenous genes in malignant brain tumors, closing the gap between in vitro and in vivo integrative biology of disease in neural stem cell transplantation.

Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis.

Background. Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. Methods. S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. Results. We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. Conclusions. This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

Identification and characterization of novel molecular mechanisms involved in initiation and progression of myelination

Schwann cells synthesize a large amount of membrane that form a specialized structure called myelin that surrounds axons and facilitate the transmission of electrical signal along neurons in peripheral nervous system (PNS). Previous studies demonstrated that both Schwann cell differentiation and de-differentiation (in the situation of a nerve injury or demyelinating disease) are regulated by cell-intrinsic regulators including several transcription factors. In particular, the de-differentiation of mature Schwann cells is driven by the activation of multiple negative regulators of myelination including Sox2, c-Jun, Notch and Pax3, all usually expressed in immature Schwann cells and suppressed at the onset of myelination. In order to identify new regulators of myelination involved in the development of the PNS, we analyzed the gene-expression profiling data from developing PNS and from three models of demyelinating neuropathies. This analysis led to the identification of Sox4, a member of the Sox family of transcription factors, as a potential candidate. To characterize the molecular function of Sox4 in PNS, we generated two transgenic lines of mice, which overexpress Sox4 specifically in Schwann cells. Detailed analysis of these mice showed that the overexpression of Sox4 in Schwann cells causes a delay in progression of myelination between post-natal day 2 (P2) and P5. Our in vitro analysis suggested that Sox4 cDNA can be overexpressed while the protein translation is tightly regulated. Interestingly, we observed that Sox4 protein is stabilized in nerves of the CMT4C mouse, a model of the human neuropathy. We therefore crossed Sox4 transgenic mice with CMT4C mice and we observed that Sox4 overexpression exacerbated the neuropathy phenotype in these mice. While recognized as being crucial for the normal function of both neurons and myelinating glial cells, the processes that regulate the beginning of myelination and the nature of the neuro-glial cross-talk remains mostly unknown. In order to gain insight into the molecular pathways involved in the interactions between neurons and associated glial cells, we developed a neuron-glia co-culture system based on microfluidic chambers and successfully induced myelination in this system by ascorbic acid. Importantly, we observed that in addition to acting on Schwann cells, ascorbic acid also modulate neuronal/axonal NRG1/ErbB2-B3 signalling. The experimental setting used in our study thus allowed us to discover a novel phenomena of propagation for myelination in vitro. The further characterization of this event brought us to identify other compounds able to induce myelination: ADAMs secretases inhibitor GM6001 and cyclic-AMP. The results generated during my thesis project are therefore not only important for the advancement of our understanding of how the PNS works, but may also potentially help to develop new therapies aiming at improvement of PNS myelination under disease conditions. - Les cellules de Schwann synthétisent une grande quantité de membrane formant une structure spécialisée appelée myéline qui entoure les axones et facilite la transmission du signal électrique le long des neurones du système nerveux périphérique (SNP). Des études antérieures ont démontré que la différenciation et la dédifférenciation des cellules de Schwann (dans la situation d'une lésion nerveuse ou d'une maladie démyélinisante) sont régulées par des régulateurs cellulaires intrinsèques, incluant plusieurs facteurs de transcription. En particulier, la dédifférenciation des cellules de Schwann matures est contrôlée par l'activation de plusieurs régulateurs négatifs de la myélinisation dont Sox2, c-Jun, Notch et Pax3, tous habituellement exprimés dans des cellules de Schwann immatures et supprimés au début de la myélinisation. Afin d'identifier de nouveaux régulateurs de myélinisation impliqués dans le développement du SNP, nous avons analysé le profil d'expression génique durant le développement du SNP ainsi que dans trois modèles de neuropathies démyélinisantes. Cette analyse a mené à l'identification de Sox4, un membre de la famille des facteurs de transcription Sox, comme étant un candidat potentiel. Dans le but de caractériser la fonction moléculaire de Sox4 dans le SNP, nous avons généré deux lignées transgéniques de souris qui surexpriment Sox4 spécifiquement dans les cellules de Schwann. L'analyse détaillée de ces souris a montré que la surexpression de Sox4 dans les cellules de Schwann provoque un retard dans la progression de la myélinisation entre le jour postnatal 2 (P2) et P5. Notre analyse in vitro a suggéré que l'ADNc de Sox4 peut être surexprimé alors que la traduction des protéines est quand à elle étroitement régulée. De façon intéressante, nous avons observé que la protéine Sox4 est stabilisée dans les nerfs des souris CMT4C, un modèle de neuropathie humaine. Nous avons donc croisé les souris transgéniques Sox4 avec des souris CMT4C et avons observé que la surexpression de Sox4 exacerbe le phénotype de neuropathie chez ces souris. Bien que reconnus comme étant cruciaux pour le fonctionnement normal des neurones et des cellules gliales myélinisantes, les processus qui régulent le début de la myélinisation ainsi que la nature des interactions neurone-glie restent largement méconnus. Afin de mieux comprendre les mécanismes moléculaires impliqués dans les interactions entre les neurones et les cellules gliales leur étant associés, nous avons développé un système de co-culture neurone-glie basé sur des chambres microfluidiques et y avons induit avec succès la myélinisation avec de l'acide ascorbique. Étonnamment, nous avons remarqué que, en plus d'agir sur les cellules de Schwann, l'acide ascorbique module également la voie de signalisation neuronale/axonale NRG1/ErbB2-B3. Le protocole expérimental utilisé dans notre étude a ainsi permis de découvrir un nouveau phénomène de propagation de la myélinisation in vitro. La caractérisation plus poussée de ce phénomène nous a menés à identifier d'autres composés capables d'induire la myélinisation: L'inhibiteur de sécrétases ADAMs GM6001 et l'AMP cyclique. Les résultats obtenus au cours de mon projet de thèse ne sont donc pas seulement importants pour l'avancement de notre compréhension sur la façon dont le SNP fonctionne, mais peuvent aussi potentiellement aider à développer de nouvelles thérapies visant à l'amélioration de la myélinisation du SNP dans des conditions pathologiques.

A higher mutational burden in females supports a "female protective model" in neurodevelopmental disorders.

Increased male prevalence has been repeatedly reported in several neurodevelopmental disorders (NDs), leading to the concept of a "female protective model." We investigated the molecular basis of this sex-based difference in liability and demonstrated an excess of deleterious autosomal copy-number variants (CNVs) in females compared to males (odds ratio [OR] = 1.46, p = 8 × 10(-10)) in a cohort of 15,585 probands ascertained for NDs. In an independent autism spectrum disorder (ASD) cohort of 762 families, we found a 3-fold increase in deleterious autosomal CNVs (p = 7 × 10(-4)) and an excess of private deleterious single-nucleotide variants (SNVs) in female compared to male probands (OR = 1.34, p = 0.03). We also showed that the deleteriousness of autosomal SNVs was significantly higher in female probands (p = 0.0006). A similar bias was observed in parents of probands ascertained for NDs. Deleterious CNVs (>400 kb) were maternally inherited more often (up to 64%, p = 10(-15)) than small CNVs < 400 kb (OR = 1.45, p = 0.0003). In the ASD cohort, increased maternal transmission was also observed for deleterious CNVs and SNVs. Although ASD females showed higher mutational burden and lower cognition, the excess mutational burden remained, even after adjustment for those cognitive differences. These results strongly suggest that females have an increased etiological burden unlinked to rare deleterious variants on the X chromosome. Carefully phenotyped and genotyped cohorts will be required for identifying the symptoms, which show gender-specific liability to mutational burden.