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.

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.

Distinct profiles of cytotoxic granules in memory CD8 T cells correlate with functions, differentiation stage, and antigen exposure

RAPPORT DE SYNTHÈSE : Les profils des granules cytotoxiques des cellules T CD8 mémoires sont corrélés à la fonction, à leur état de différentiation et à l'exposition à l'antigène. Les lymphocytes T-CD8 cytotoxiques exercent leur fonction antivirale et antitumorale surtout par la sécrétion des granules cytotoxiques. En général, ce sont l'activité de dégranulation et les granules cytotoxiques (contenant perforine et différentes granzymes) qui définissent les lymphocytes T-CD8 cytotoxiques. Dans cette étude, nous avons investigué l'expression de granzyme K par cytométrie en flux, en comparaison avec l'expression de granzyme A, granzyme B et de perforine. L'expression des granules cytotoxiques a été déterminée dans lymphocytes T-CD8 qui étaient spécifiques pour des différents virus, en particulier spécifique pour le virus d'influenza (flu), le virus Ebstein Barr (EBV), le virus de cytomégalie (CMV) et le virus de l'immunodéficience humaine (HIV). Nous avons observé une dichotomie entre l'expression du granzyme K et de la perforine dans les lymphocytes T-CD8 qui étaient spécifiques aux virus mentionnés. Les profils des lymphocytes T-CD8 spécifiques à flu étaient positifs soit pour granzyme A et granzyme K soit pour le granzyme K seul, mais dans l'ensemble négatifs pour perforine et granzyme B. Les cellules spécifiques à CMV étaient dans la plupart positives pour perforine, granzyme B et A, mais négatives pour le granzyme K. Les cellules spécifiques à EBV et HIV étaient dans la majorité positives pour granzyme A, B et K, et dans la moitié des cas négatives pour la perforine. Nous avons également analysé, selon les marqueurs de mémoire de CD45 et CD127, les profils de différentiation cellulaire: Les cellules avec les granules cytotoxiques contenant exclusivement le granzyme K, étaient associées à un état de différentiation précoce. Au contraire, les protéines cytolytiques perforine, granzyme A et B, correspondent à une différentiation avancée. En outre, les protéines perforine et granzyme B, mais pas les granzymes A et K, sont corrélées à une activité cytotoxique. Finalement, des changements dans l'exposition d'antigène in vitro et in vivo suivant une infection primaire d' HIV ou une vaccination modulent le profil de granules cytotoxiques. Ces résultats nous permettent d'étendre la compréhension de la relation entre les différents profils de granules cytotoxiques des lymphocytes T-CD8 et leur fonction, leur état de différentiation et l'exposition à l'antigène.

Tissue-specific opposing functions of the inflammasome adaptor ASC in the regulation of epithelial skin carcinogenesis.

A chronic inflammatory microenvironment favors tumor progression through molecular mechanisms that are still incompletely defined. In inflammation-induced skin cancers, IL-1 receptor- or caspase-1-deficient mice, or mice specifically deficient for the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in myeloid cells, had reduced tumor incidence, pointing to a role for IL-1 signaling and inflammasome activation in tumor development. However, mice fully deficient for ASC were not protected, and mice specifically deficient for ASC in keratinocytes developed more tumors than controls, suggesting that, in contrast to its proinflammatory role in myeloid cells, ASC acts as a tumor-suppressor in keratinocytes. Accordingly, ASC protein expression was lost in human cutaneous squamous cell carcinoma, but not in psoriatic skin lesions. Stimulation of primary mouse keratinocytes or the human keratinocyte cell line HaCaT with UVB induced an ASC-dependent phosphorylation of p53 and expression of p53 target genes. In HaCaT cells, ASC interacted with p53 at the endogenous level upon UVB irradiation. Thus, ASC in different tissues may influence tumor growth in opposite directions: it has a proinflammatory role in infiltrating cells that favors tumor development, but it also limits keratinocyte proliferation in response to noxious stimuli, possibly through p53 activation, which helps suppressing tumors.

Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals.

Biological interpretation of genome-wide association studies using predicted gene functions.

The main challenge for gaining biological insights from genetic associations is identifying which genes and pathways explain the associations. Here we present DEPICT, an integrative tool that employs predicted gene functions to systematically prioritize the most likely causal genes at associated loci, highlight enriched pathways and identify tissues/cell types where genes from associated loci are highly expressed. DEPICT is not limited to genes with established functions and prioritizes relevant gene sets for many phenotypes.

The retinoid-related orphan receptor RORα promotes keratinocyte differentiation via FOXN1.

RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer.

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.

Neuroendocrine, metabolic, and immune functions during the acute phase response of inflammatory stress in monosodium L-glutamate-damaged, hyperadipose male rat.

In rats, neonatal treatment with monosodium L-glutamate (MSG) induces several metabolic and neuroendocrine abnormalities, which result in hyperadiposity. No data exist, however, regarding neuroendocrine, immune and metabolic responses to acute endotoxemia in the MSG-damaged rat. We studied the consequences of MSG treatment during the acute phase response of inflammatory stress. Neonatal male rats were treated with MSG or vehicle (controls, CTR) and studied at age 90 days. Pituitary, adrenal, adipo-insular axis, immune, metabolic and gonadal functions were explored before and up to 5 h after single sub-lethal i.p. injection of bacterial lipopolysaccharide (LPS; 150 microg/kg). Our results showed that, during the acute phase response of inflammatory stress in MSG rats: (1) the corticotrope-adrenal, leptin, insulin and triglyceride responses were higher than in CTR rats, (2) pro-inflammatory (TNFalpha) cytokine response was impaired and anti-inflammatory (IL-10) cytokine response was normal, and (3) changes in peripheral estradiol and testosterone levels after LPS varied as in CTR rats. These data indicate that metabolic and neroendocrine-immune functions are altered in MSG-damaged rats. Our study also suggests that the enhanced corticotrope-corticoadrenal activity in MSG animals could be responsible, at least in part, for the immune and metabolic derangements characterizing hypothalamic obesity.

Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

Macrophages from Crohn's disease patients exhibit deficient pro-repair functions

BACKGROUND: We previously reported that myeloid cells can induce mucosal healing in a mouse model of acute colitis. Promotion of mucosal repair is becoming a major goal in the treatment of Crohn's disease. Our aim in this study is to investigate the pro-repair function of myeloid cells in healthy donor (HD) and Crohn's disease patients (CD). METHODS: Peripheral blood mononuclear cells (PBMC) from HD and CD patients were isolated from blood samples by Ficoll density gradient. Monocytic CD14+ cells were positively selected by Macs procedure and then differentiated ex-vivo into macrophages (Mφ). The repair function of PBMC, CD14+ monocytic cells and macrophages were evaluated in an in vitro wound healing assay. RESULTS: PBMC and CD14+ myeloid cells from HD and CD were not able to repair at any tested cell concentration. Remarkably, HD Mφ were able to induce wound healing only at high concentration (105 added Mφ), but, if activated with heat killed bacteria, they were able to repair even at very low concentration. On the contrary, not activated CD Mφ were not able to promote healing at any rate, but this function was restored upon activation. CONCLUSION: We showed that CD Mφ in their steady state, unlike HD Mφ, are defective in promoting wound healing. Our results are in keeping with the current theory of CD as an innate immunodeficiency. Defective Mφ may be responsible to the mucosal repair defects in CD patients and to the subsequent chronic activation of the adaptive immune response.