Toll-Interacting Protein Modulates Gut Flora Composition, Epithelial Barrier Integrity and Susceptibility to Colitis

Toll-like receptor ( TLR) s ignals are key to maintaining hostmicrobial i nteractions. T he T oll-interacting-protein (Tollip) is a ubiquitously-expressed inhibitor of inflammasome a nd TLR signaling. W e hypothesized that T ollip might control g ut homeostasis. G enetic ablation of T ollip d id not lead to spontaneous colitis b ut h ad d ramatic c onsequences on t he intestinal expression of the α-defensin cryptidin 4 and the C-type lectin R EGIIIβ. These c hanges were associated with intestinal dysbiosis a nd e nhanced colonization b y segmented filamentous bacteria - a k ey p ro-inflammatory component of the microbiota. Tollip deficiency increased susceptibility to dextran sulfate sodium (DSS) colitis and aggravated chronic Th17-driven colitis in IL-10-/- mice. Flora d epletion w ith a ntibiotics in T ollip-/- mice w as not sufficient to restore DSS colitis susceptibility and deletion of Tollip in n on-hematopoietic c ells using bone-marrow chimeras w as sufficient to increase s usceptibility t o DSS colitis. After D SS administration, we o bserved several e pithelial defects i n Tollip-/- mice including early tight junctions disruption, increased epithelial apoptosis, and increased intestinal permeability. Overall, our data show that T ollip significantly impacts intestinal h omeostasis by controlling b acterial ecology and intestinal r esponse to chemical and immunological stresses.

Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.

Mutation of the nuclear receptor peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARbeta/delta-null mutant embryos. While very little is known at present about the pathway governed by PPARbeta/delta in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARbeta/delta-null embryos is found in the giant cell layer. PPARbeta/delta activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARbeta/delta is silenced. Conversely, exposure of Rcho-1 cells to a PPARbeta/delta agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARbeta/delta activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARbeta/delta also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARbeta/delta-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARbeta/delta in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARbeta/delta agonist as therapeutic agents of broad application.

Characterization of insulators and barrier elements for gene therapy

Gene transfer that relies on integrating vectors often suffers from epigenetic or regulatory effects that influence the expression of the therapeutic gene and=or of cellular genes located near the vector integration site in the chromosome. Insulator elements act to block gene activation by enhancers, while chromatin domain boundary or barrier sequences prevent gene-silencing effects. At present, the modes of action of insulator and barriers are poorly understood, and their use in the context of gene therapies remains to be documented. Using combinations of reporter genes coding for indicator fluorescent proteins, we constructed assay systems that allow the quantification of the insulator or barrier activities of genetic elements in individual cells. This presentation will illustrate how these assay systems were used to identify short DNA elements that insulate nearby genes from activation by viral vector elements, and=or that block the propagation of a silent chromatin structure that leads to gene silencing. We will show that some barrier elements do not merely block repressive effects, but that they can act to stabilize and sustain transgene expression. We will illustrate that this may be beneficial when transgenes are introduced into stem or precursor cells using non-viral vectors, where later differentiation may lead to the silencing of the therapeutic gene. We will show that these elements can be used to maintain efficient transgene expression upon the differentiation of murine precursor cells towards myofibers, in a model of cell therapy for muscle dystrophies.

Predicting stroke through genetic risk functions: the CHARGE Risk Score Project.

BACKGROUND AND PURPOSE: Beyond the Framingham Stroke Risk Score, prediction of future stroke may improve with a genetic risk score (GRS) based on single-nucleotide polymorphisms associated with stroke and its risk factors. METHODS: The study includes 4 population-based cohorts with 2047 first incident strokes from 22,720 initially stroke-free European origin participants aged ≥55 years, who were followed for up to 20 years. GRSs were constructed with 324 single-nucleotide polymorphisms implicated in stroke and 9 risk factors. The association of the GRS to first incident stroke was tested using Cox regression; the GRS predictive properties were assessed with area under the curve statistics comparing the GRS with age and sex, Framingham Stroke Risk Score models, and reclassification statistics. These analyses were performed per cohort and in a meta-analysis of pooled data. Replication was sought in a case-control study of ischemic stroke. RESULTS: In the meta-analysis, adding the GRS to the Framingham Stroke Risk Score, age and sex model resulted in a significant improvement in discrimination (all stroke: Δjoint area under the curve=0.016, P=2.3×10(-6); ischemic stroke: Δjoint area under the curve=0.021, P=3.7×10(-7)), although the overall area under the curve remained low. In all the studies, there was a highly significantly improved net reclassification index (P<10(-4)). CONCLUSIONS: The single-nucleotide polymorphisms associated with stroke and its risk factors result only in a small improvement in prediction of future stroke compared with the classical epidemiological risk factors for stroke.

A receptor-like kinase mutant with absent endodermal diffusion barrier displays selective nutrient homeostasis defects.

The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)--major players of endodermal differentiation--into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane.

The optimal dividend barrier in the Gamma-Omega model

In the traditional actuarial risk model, if the surplus is negative, the company is ruined and has to go out of business. In this paper we distinguish between ruin (negative surplus) and bankruptcy (going out of business), where the probability of bankruptcy is a function of the level of negative surplus. The idea for this notion of bankruptcy comes from the observation that in some industries, companies can continue doing business even though they are technically ruined. Assuming that dividends can only be paid with a certain probability at each point of time, we derive closed-form formulas for the expected discounted dividends until bankruptcy under a barrier strategy. Subsequently, the optimal barrier is determined, and several explicit identities for the optimal value are found. The surplus process of the company is modeled by a Wiener process (Brownian motion).

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Ly49D engagement on T lymphocytes induces TCR-independent activation and CD8 effector functions that control tumor growth.

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.

Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.

Accurate calculations of intermolecular interaction energies using explicitly correlated coupled cluster wave functions and a dispersion-weighted MP2 method.

Explicitly correlated coupled-cluster calculations of intermolecular interaction energies for the S22 benchmark set of Jurecka, Sponer, Cerny, and Hobza (Chem. Phys. Phys. Chem. 2006, 8, 1985) are presented. Results obtained with the recently proposed CCSD(T)-F12a method and augmented double-zeta basis sets are found to be in very close agreement with basis set extrapolated conventional CCSD(T) results. Furthermore, we propose a dispersion-weighted MP2 (DW-MP2) approximation that combines the good accuracy of MP2 for complexes with predominately electrostatic bonding and SCS-MP2 for dispersion-dominated ones. The MP2-F12 and SCS-MP2-F12 correlation energies are weighted by a switching function that depends on the relative HF and correlation contributions to the interaction energy. For the S22 set, this yields a mean absolute deviation of 0.2 kcal/mol from the CCSD(T)-F12a results. The method, which allows obtaining accurate results at low cost, is also tested for a number of dimers that are not in the training set.

Consequences of modified lipid and glucose metabolism on peripheral nervous system structure and function

284 million people worldwide suffered from type 2 diabetes mellitus (T2DM) in 2010, which will, in approximately half of them, lead to the development of diabetic peripheral neuropathy (DPN). Although DPN is the most common complication of diabetes mellitus and the leading cause of non-traumatic amputations its pathophysiology is still poorly understood. To get more insight into the molecular mechanism underlying DPN in T2DM, I used a rodent model of T2DM, the db/db mice.¦ln vivo electrophysiological recordings of diabetic animals indicated that in addition to reduced nerve conduction velocity db/db mice also present increased nerve excitability. Further ex vivo evaluation of the electrophysiological properties of db/db nerves clearly established a presence of the peripheral nerve hyperexcitability (PNH) phenotype in diabetic animals. Using pharmacological inhibitors we demonstrated that PNH is mostly mediated by the decreased activity of Kv1 channels. ln agreement with these data 1 observed that the diabetic condition led to a reduced presence of the Kv1.2 subunits in juxtaparanodal regions of db/db peripheral nerves whereas its mANA and protein expression levels were not affected. Lmportantly, I confirmed a loss of juxtaparanodal Kv1.2 subunits in nerve biopsies from type 2 diabetic patients. Together these observations indicate that the type 2 diabetic condition leads to potassium-channel mediated changes of nerve excitability thus identifying them as potential drug targets to treat sorne of the DPN related symptoms.¦Schwann cells ensheath and isolate peripheral axons by the production of myelin, which consists of lipids and proteins in a ratio of 2:1. Peripheral myelin protein 2 (= P2, Pmp2 or FABP8) was originally described as one of the most abundant myelin proteins in the peripheral nervous system. P2, which is a member of the fatty acid binding protein (FABP) family, is a 14.8 kDa cytosolic protein expressed on the cytoplasmic side of compact myelin membranes. As indicated by their name, the principal role of FABPs is thought to be the binding and transport of fatty acids.¦To study its role in myelinating glial cells I have recently generated a complete P2 knockout mouse model (P2-/-). I confirmed the loss of P2 in the sciatic nerve of P2-/- mice at the mRNA and protein level. Electrophysiological analysis of the adult (P56) mutant mice revealed a mild but significant reduction in the motor nerve conduction velocity. lnterestingly, this functional change was not accompanied by any detectable alterations in general myelin structure. However, I have observed significant alterations in the mRNA expression level of other FABPs, predominantly FABP9, in the PNS of P2-/- mice as compared to age-matched P2+/+ mice indicating a role of P2 in the glial myelin lipid metabolism.¦Le diabète de type 2 touche 284 million de personnes dans le monde en 2010 et son évolution conduit dans la moitié des cas à une neuropathie périphérique diabétique. Bien que la neuropathie périphérique soit la complication la plus courante du diabète pouvant conduire jusqu'à l'amputation, sa physiopathologie est aujourd'hui encore mal comprise. Dans le but d'améliorer les connaissances moléculaires expliquant les mécanismes de la neuropathie liée au diabète de type 2, j'ai utilisé un modèle murin du diabète de type 2, les souris db/db.¦ln vivo, les enregistrements éléctrophysiologiques des animaux diabétiques montrent qu'en plus d'une diminution de la vitesse de conduction nerveuse, les souris db/db présentent également une augmentation de l'excitabilité nerveuse. Des mesures menées Ex­ vivo ont montré l'existence d'un phénotype d'hyperexcitabilité sur les nerfs périphériques isolés d'animaux diabétiques. Grâce à l'utilisation d'inhibiteurs pharmacologiques, nous avons pu démontrer que l'hyperexcitabilité démontrée était due à une réduction d'activité des canaux Kv1. En accord avec ces données, j'ai observé qu'une situation de diabète conduisait à une diminution des canaux Kv1.2 aux régions juxta-paranodales des nerfs périphériques db/db, alors que l'expression du transcrit et de la protéine restait stable. J'ai également confirmé l'absence de canaux Kv1.2 aux juxta-paranoeuds de biopsies de nerfs de patients diabétiques. L'ensemble de ces observations montrent que les nerfs périphériques chez les patients atteints de diabète de type 2 est due à une diminution des canaux potassiques rapides juxtaparanodaux les identifiant ainsi comme des cibles thérapeutiques potentielles.¦Les cellules de Schwann enveloppent et isolent les axones périphériques d'une membrane spécialisée, la myéline, composée de deux fois plus de lipides que de protéines. La protéine P2 (Pmp2 "peripheral myelin protein 2" ou FABP8 "fatty acid binding protein") est l'une des protéines les plus abondantes au système nerveux périphérique. P2 appartient à la famille de protéines FABP liant et transportant les acides gras et est une protéine cytosolique de 14,8 kDa exprimée du côté cytoplasmique de la myéline compacte.¦Afin d'étudier le rôle de P2 dans les cellules de Schwann myélinisantes, j'ai généré une souris knockout (P2-/-). Après avoir validé l'absence de transcrit et de protéine P2 dans les nerfs sciatiques P2-/-, des mesures électrophysiologiques ont montré une réduction modérée mais significative de la vitesse de conduction du nerf moteur périphérique. Il est important de noter que ces changements fonctionnels n'ont pas pu être associés à quelconque changement dans la structure de la myéline. Cependant, j'ai observé dans les nerfs périphériques P2-/-, une altération significative du niveau d'expression d'ARNm d'autres FABPs et en particulier FABP9. Ce dernier résultat démontre l'importance du rôle de la protéine P2 dans le métabolisme lipidique de la myéline.

Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster.

BACKGROUND AND AIMS: The study of local adaptation in plant reproductive traits has received substantial attention in short-lived species, but studies conducted on forest trees are scarce. This lack of research on long-lived species represents an important gap in our knowledge, because inferences about selection on the reproduction and life history of short-lived species cannot necessarily be extrapolated to trees. This study considers whether the size for first reproduction is locally adapted across a broad geographical range of the Mediterranean conifer species Pinus pinaster. In particular, the study investigates whether this monoecious species varies genetically among populations in terms of whether individuals start to reproduce through their male function, their female function or both sexual functions simultaneously. Whether differences among populations could be attributed to local adaptation across a climatic gradient is then considered. METHODS: Male and female reproduction and growth were measured during early stages of sexual maturity of a P. pinaster common garden comprising 23 populations sampled across the species range. Generalized linear mixed models were used to assess genetic variability of early reproductive life-history traits. Environmental correlations with reproductive life-history traits were tested after controlling for neutral genetic structure provided by 12 nuclear simple sequence repeat markers. KEY RESULTS: Trees tended to reproduce first through their male function, at a size (height) that varied little among source populations. The transition to female reproduction was slower, showed higher levels of variability and was negatively correlated with vegetative growth traits. Several female reproductive traits were correlated with a gradient of growth conditions, even after accounting for neutral genetic structure, with populations from more unfavourable sites tending to commence female reproduction at a lower individual size. CONCLUSIONS: The study represents the first report of genetic variability among populations for differences in the threshold size for first reproduction between male and female sexual functions in a tree species. The relatively uniform size at which individuals begin reproducing through their male function probably represents the fact that pollen dispersal is also relatively invariant among sites. However, the genetic variability in the timing of female reproduction probably reflects environment-dependent costs of cone production. The results also suggest that early sex allocation in this species might evolve under constraints that do not apply to other conifers.

Genome-wide transposon insertion scanning of environmental survival functions in the polycyclic aromatic hydrocarbon degrading bacterium Sphingomonas wittichii RW1.

Sphingomonas wittichii RW1 is a dibenzofuran and dibenzodioxin-degrading bacterium with potentially interesting properties for bioaugmentation of contaminated sites. In order to understand the capacity of the microorganism to survive in the environment we used a genome-wide transposon scanning approach. RW1 transposon libraries were generated with around 22 000 independent insertions. Libraries were grown for an average of 50 generations (five successive passages in batch liquid medium) with salicylate as sole carbon and energy source in presence or absence of salt stress at -1.5 MPa. Alternatively, libraries were grown in sand with salicylate, at 50% water holding capacity, for 4 and 10 days (equivalent to 7 generations). Library DNA was recovered from the different growth conditions and scanned by ultrahigh throughput sequencing for the positions and numbers of inserted transposed kanamycin resistance gene. No transposon reads were recovered in 579 genes (10% of all annotated genes in the RW1 genome) in any of the libraries, suggesting those to be essential for survival under the used conditions. Libraries recovered from sand differed strongly from those incubated in liquid batch medium. In particular, important functions for survival of cells in sand at the short term concerned nutrient scavenging, energy metabolism and motility. In contrast to this, fatty acid metabolism and oxidative stress response were essential for longer term survival of cells in sand. Comparison to transcriptome data suggested important functions in sand for flagellar movement, pili synthesis, trehalose and polysaccharide synthesis and putative cell surface antigen proteins. Interestingly, a variety of genes were also identified, interruption of which cause significant increase in fitness during growth on salicylate. One of these was an Lrp family transcription regulator and mutants in this gene covered more than 90% of the total library after 50 generations of growth on salicylate. Our results demonstrate the power of genome-wide transposon scanning approaches for analysis of complex traits.

The psychostimulant modafinil enhances gap junctional communication in cortical astrocytes.

Sleep-wake cycle is characterized by changes in neuronal network activity. However, for the last decade there is increasing evidence that neuroglial interaction may play a role in the modulation of sleep homeostasis and that astrocytes have a critical impact in this process. Interestingly, astrocytes are organized into communicating networks based on their high expression of connexins, which are the molecular constituents of gap junction channels. Thus, neuroglial interactions should also be considered as the result of the interplay between neuronal and astroglial networks. Here, we investigate the effect of modafinil, a wakefulness-promoting agent, on astrocyte gap junctional communication. We report that in the cortex modafinil injection increases the expression of mRNA and protein of connexin 30 but not those of connexin 43, the other major astroglial connexin. These increases are correlated with an enhancement of intercellular dye coupling in cortical astrocytes, which is abolished when neuronal activity is silenced by tetrodotoxin. Moreover, gamma-hydroxybutyric acid, which at a millimolar concentration induces sleep, has an opposite effect on astroglial gap junctions in an activity-independent manner. These results support the proposition that astroglia may play an important role in complex physiological brain functions, such as sleep regulation, and that neuroglial networking interaction is modified during sleep-wake cycle. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.