Copy number variants, diseases and gene expression.

Copy number variation (CNV) has recently gained considerable interest as a source of genetic variation likely to play a role in phenotypic diversity and evolution. Much effort has been put into the identification and mapping of regions that vary in copy number among seemingly normal individuals in humans and a number of model organisms, using bioinformatics or hybridization-based methods. These have allowed uncovering associations between copy number changes and complex diseases in whole-genome association studies, as well as identify new genomic disorders. At the genome-wide scale, however, the functional impact of CNV remains poorly studied. Here we review the current catalogs of CNVs, their association with diseases and how they link genotype and phenotype. We describe initial evidence which revealed that genes in CNV regions are expressed at lower and more variable levels than genes mapping elsewhere, and also that CNV not only affects the expression of genes varying in copy number, but also have a global influence on the transcriptome. Further studies are warranted for complete cataloguing and fine mapping of CNVs, as well as to elucidate the different mechanisms by which they influence gene expression.

Plac8 is required for white adipocyte differentiation in vitro and cell number control in vivo.

Plac8 belongs to an evolutionary conserved family of proteins, mostly abundant in plants where they control fruit weight through regulation of cell number. In mice, Plac8 is expressed both in white and brown adipose tissues and we previously showed that Plac8(-/-) mice develop late-onset obesity, with abnormal brown fat differentiation and reduced thermogenic capacity. We also showed that in brown adipocytes, Plac8 is an upstream regulator of C/EBPβ expression. Here, we first assessed the role of Plac8 in white adipogenesis in vitro. We show that Plac8 is induced early after induction of 3T3-L1 adipocytes differentiation, a process that is prevented by Plac8 knockdown; similarly, embryonic fibroblasts obtained from Plac8 knockout mice failed to form adipocytes upon stimulation of differentiation. Knockdown of Plac8 in 3T3-L1 was associated with reduced expression of C/EBPβ, Krox20, and Klf4, early regulators of the white adipogenic program, and we show that Plac8 could transactivate the C/EBPβ promoter. In vivo, we show that absence of Plac8 led to increased white fat mass with enlarged adipocytes but reduced total number of adipocytes. Finally, even though Plac8(-/-) mice showed impaired thermogenesis due to brown fat dysfunction, this was not associated with changes in glycemia or plasma free fatty acid and triglyceride levels. Collectively, these data indicate that Plac8 is an upstream regulator of C/EBPβ required for adipogenesis in vitro. However, in vivo, Plac8 is dispensable for the differentiation of white adipocytes with preserved fat storage capacity but is required for normal fat cell number regulation.

CCL8 and the Immune Control of Cytomegalovirus in Organ Transplant Recipients.

Monitoring of cytomegalovirus cell-mediated immunity is a promising tool for the refinement of preventative and therapeutic strategies posttransplantation. Typically, the interferon-γ response to T cell stimulation is measured. We evaluated a broad range of cytokine and chemokines to better characterize the ex vivo host-response to CMV peptide stimulation. In a cohort of CMV viremic organ transplant recipients, chemokine expression-specifically CCL8 (AUC 0.849 95% CI 0.721-0.978; p = 0.003) and CXCL10 (AUC 0.841, 95% CI 0.707-0.974; p = 0.004)-was associated with control of viral replication. In a second cohort of transplant recipients at high-risk for CMV, the presence of a polymorphism in the CCL8 promoter conferred an increased risk of viral replication after discontinuation of antiviral prophylaxis (logrank hazard ratio 3.6; 95% CI 2.077-51.88). Using cell-sorting experiments, we determined that the primary cell type producing CCL8 in response to CMV peptide stimulation was the monocyte fraction. Finally, in vitro experiments using standard immunosuppressive agents demonstrated a dose-dependent reduction in CCL8 production. Chemokines appear to be important elements of the cell-mediated response to CMV infection posttransplant, as here suggested for CCL8, and translation of this knowledge may allow for the tailoring and improvement of preventative strategies.

Infectiologie et postulats de Koch: tourner la page [Infectious diseases and Koch's postulates: turning the page].

The thoracolumbar junctional region (T10-L1) of the spine is a transitional zone, where more than half of the thoracic and lumbar fractures occur. In this presentation the origin of the pathoanatomical changes in the thoracolumbar junctional region of the spine is discussed in view of the previous studies. These studies refer to a torsional force contributing to the formation of the degenerative changes, especially in the facet joints. Degenerative changes anteriorly and posteriorly do not concur in the thoracolumbar junctional region. Only a weak concurrence is found between disc degeneration and spondylosis, which refer to differences in their pathomechanisms. A strong concurrence between the degenerative changes at different levels, especially anteriorly, reflects factors causing overall degeneration in the thoracolumbar junctional region.

Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis

Epstein-Barr virus (EBV) has been associated with multiple sclerosis (MS), however, most studies examining the relationship between the virus and the disease have been based on serologies, and if EBV is linked to MS, CD8+ T cells are likely to be involved as they are important both in MS pathogenesis and in controlling viruses. We hypothesized that valuable information on the link between MS and EBV would be ascertained from the study of frequency and activation levels of EBV-specific CD8+ T cells in different categories of MS patients and control subjects. We investigated EBV-specific cellular immune responses using proliferation and enzyme linked immunospot assays, and humoral immune responses by analysis of anti-EBV antibodies, in a cohort of 164 subjects, including 108 patients with different stages of MS, 35 with other neurological diseases and 21 healthy control subjects. Additionally, the cohort were all tested against cytomegalovirus (CMV), another neurotropic herpes virus not convincingly associated with MS, nor thought to be deleterious to the disease. We corrected all data for age using linear regression analysis over the total cohorts of EBV- and CMV-infected subjects. In the whole cohort, the rate of EBV and CMV infections were 99% and 51%, respectively. The frequency of IFN-gamma secreting EBV-specific CD8+ T cells in patients with clinically isolated syndrome (CIS) was significantly higher than that found in patients with relapsing-remitting MS (RR-MS), secondary-progressive MS, primary-progressive MS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and our assays, the more intense was the EBV-specific CD8+ T-cell response. Confirming the above results, we found that EBV-specific CD8+ T-cell responses decreased in 12/13 patients with CIS followed prospectively for 1.0 +/- 0.2 years. In contrast, there was no difference between categories for EBV-specific CD4+ T cell, or for CMV-specific CD4+ and CD8+ T-cell responses. Anti-EBV-encoded nuclear antigen-1 (EBNA-1)-specific antibodies correlated with EBV-specific CD8+ T cells in patients with CIS and RR-MS. However, whereas EBV-specific CD8+ T cells were increased the most in early MS, EBNA-1-specific antibodies were increased in early as well as in progressive forms of MS. Our data show high levels of CD8+ T-cell activation against EBV--but not CMV--early in the course of MS, which support the hypothesis that EBV might be associated with the onset of this disease.

Evaluation of steroidomics by liquid chromatography hyphenated to mass spectrometry as a powerful analytical strategy for measuring human steroid perturbations.

This review presents the evolution of steroid analytical techniques, including gas chromatography coupled to mass spectrometry (GC-MS), immunoassay (IA) and targeted liquid chromatography coupled to mass spectrometry (LC-MS), and it evaluates the potential of extended steroid profiles by a metabolomics-based approach, namely steroidomics. Steroids regulate essential biological functions including growth and reproduction, and perturbations of the steroid homeostasis can generate serious physiological issues; therefore, specific and sensitive methods have been developed to measure steroid concentrations. GC-MS measuring several steroids simultaneously was considered the first historical standard method for analysis. Steroids were then quantified by immunoassay, allowing a higher throughput; however, major drawbacks included the measurement of a single compound instead of a panel and cross-reactivity reactions. Targeted LC-MS methods with selected reaction monitoring (SRM) were then introduced for quantifying a small steroid subset without the problems of cross-reactivity. The next step was the integration of metabolomic approaches in the context of steroid analyses. As metabolomics tends to identify and quantify all the metabolites (i.e., the metabolome) in a specific system, appropriate strategies were proposed for discovering new biomarkers. Steroidomics, defined as the untargeted analysis of the steroid content in a sample, was implemented in several fields, including doping analysis, clinical studies, in vivo or in vitro toxicology assays, and more. This review discusses the current analytical methods for assessing steroid changes and compares them to steroidomics. Steroids, their pathways, their implications in diseases and the biological matrices in which they are analysed will first be described. Then, the different analytical strategies will be presented with a focus on their ability to obtain relevant information on the steroid pattern. The future technical requirements for improving steroid analysis will also be presented.

Oral insecticidal activity of root colonizing Pseudomonas fluorescens CHA0: a biocontrol agent with potential to control plant pests and diseases

The application of microbial biocontrol agents for the control of fungal plant diseases and plant insect pests is a promising approach in the development of environmentally benign pest management strategies. The ideal biocontrol organism would be a bacterium or a fungus with activity against both, insect pests and fungal pathogens. Here we demonstrate the oral insecticidal activity of the root colonizing Pseudomonas fluorescens CHA0, which is so far known for its capacity to efficiently suppress fungal plant pathogens. Feeding assays with CHA0-sprayed leaves showed that this strain displays oral insecticidal activity and is able to efficiently kill larvae of three important insect pests. We further show data indicating that the Fit insect toxin produced by CHA0 and also metabolites controlled by the global regulator GacA contribute to oral insect toxicity.

Report of the ECCO workshop on anti-TNF therapy failures in inflammatory bowel diseases: biological roles and effects of TNF and TNF antagonists.

This second section of the first ECCO pathogenesis workshop on anti-TNF therapy failures in inflammatory bowel diseases addresses the biological roles of TNFα and the effects and mechanisms of action of TNFα antagonists. Mechanisms underlying their failure, including induction of TNF-independent inflammatory pathways and phenomena of paradoxical inflammation are discussed.

Free-living amoebae: Biological by-passes in water treatment.

Free-living amoebae constitute reservoirs for many bacteria including not only well-known pathogens but also emerging pathogens responsible for respiratory diseases, and contribute to the protection, survival and dissemination of these bacteria in water systems, despite the application of disinfection or thermal treatments. In this article we review the available information on the presence of free-living amoebae and amoebae-resisting bacteria in drinking water systems, on the factors that contribute to their presence in the water and/or the biofilms, on the possible control measures and their effectiveness, and we identify some gaps in current knowledge needing further research.

The Impact of CAROtid plaque Screening on Smoking (CAROSS) cessation and control of other cardiovascular risk factors: rationale and design of a randomized controlled trial

BACKGROUND: Screening tests for subclinical cardiovascular disease, such as markers of atherosclerosis, are increasingly used in clinical prevention to identify individuals at high cardiovascular risk. Being aware of these test results might also enhance patient motivation to change unhealthy behaviors but the effectiveness of such a screening strategy has been poorly studied. METHODS: The CAROtid plaque Screening trial on Smoking cessation (CAROSS) is a randomized controlled trial in 530 regular smokers aged 40-70 years to test the hypothesis that carotid plaque screening will influence smokers' behavior with an increased rate of smoking cessation (primary outcome) and an improved control of other cardiovascular risk factors (secondary outcomes) after 1-year follow-up. All smokers will receive a brief advice for smoking cessation,and will subsequently be randomly assigned to either the intervention group (with plaques screening) or the control group (without plaque screening). Carotid ultrasound will be conducted with a standard protocol. Smokers with at least one carotid plaque will receive pictures of their own plaques with a structured explanation on the general significance of plaques. To ensure equal contact conditions, smokers not undergoing ultrasound and those without plaque will receive a relevant explanation on the risks associated with tobacco smoking. Study outcomes will be compared between smokers randomized to plaque screening and smokers not submitted to plaque screening. SUMMARY: This will be the first trial to assess the impact of carotid plaque screening on 1-year smoking cessation rates and levels of control of other cardiovascular risk factors.

Small and long non-coding RNAs in cardiac homeostasis and regeneration

Cardiovascular diseases and in particular heart failure are major causes of morbidity and mortality in the Western world. Recently, the notion of promoting cardiac regeneration as a means to replace lost cardiomyocytes in the damaged heart has engendered considerable research interest. These studies envisage the utilization of both endogenous and exogenous cellular populations, which undergo highly specialized cell fate transitions to promote cardiomyocyte replenishment. Such transitions are under the control of regenerative gene regulatory networks, which are enacted by the integrated execution of specific transcriptional programs. In this context, it is emerging that the non-coding portion of the genome is dynamically transcribed generating thousands of regulatory small and long non-coding RNAs, which are central orchestrators of these networks. In this review, we discuss more particularly the biological roles of two classes of regulatory non-coding RNAs, i.e. microRNAs and long non-coding RNAs, with a particular emphasis on their known and putative roles in cardiac homeostasis and regeneration. Indeed, manipulating non-coding RNA-mediated regulatory networks could provide keys to unlock the dormant potential of the mammalian heart to regenerate. This should ultimately improve the effectiveness of current regenerative strategies and discover new avenues for repair. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Genetic Variations and Diseases in UniProtKB/Swiss-Prot: The Ins and Outs of Expert Manual Curation.

During the last few years, next-generation sequencing (NGS) technologies have accelerated the detection of genetic variants resulting in the rapid discovery of new disease-associated genes. However, the wealth of variation data made available by NGS alone is not sufficient to understand the mechanisms underlying disease pathogenesis and manifestation. Multidisciplinary approaches combining sequence and clinical data with prior biological knowledge are needed to unravel the role of genetic variants in human health and disease. In this context, it is crucial that these data are linked, organized, and made readily available through reliable online resources. The Swiss-Prot section of the Universal Protein Knowledgebase (UniProtKB/Swiss-Prot) provides the scientific community with a collection of information on protein functions, interactions, biological pathways, as well as human genetic diseases and variants, all manually reviewed by experts. In this article, we present an overview of the information content of UniProtKB/Swiss-Prot to show how this knowledgebase can support researchers in the elucidation of the mechanisms leading from a molecular defect to a disease phenotype.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase.

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37-1.73]; combined P=2.58 · 10(-13)). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25-1.44; P=1.032 · 10(-14)). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16-3.66) for systolic and 1.40 (95% CI: 0.25-2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression.

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

Safety and accuracy of robot-assisted versus fluoroscopy-guided pedicle screw insertion for degenerative diseases of the lumbar spine: a matched cohort comparison.

Object Recent years have been marked by efforts to improve the quality and safety of pedicle screw placement in spinal instrumentation. The aim of the present study is to compare the accuracy of the SpineAssist robot system with conventional fluoroscopy-guided pedicle screw placement. Methods Ninety-five patients suffering from degenerative disease and requiring elective lumbar instrumentation were included in the study. The robot cohort (Group I; 55 patients, 244 screws) consisted of an initial open robot-assisted subgroup (Subgroup IA; 17 patients, 83 screws) and a percutaneous cohort (Subgroup IB, 38 patients, 161 screws). In these groups, pedicle screws were placed under robotic guidance and lateral fluoroscopic control. In the fluoroscopy-guided cohort (Group II; 40 patients, 163 screws) screws were inserted using anatomical landmarks and lateral fluoroscopic guidance. The primary outcome measure was accuracy of screw placement on the Gertzbein-Robbins scale (Grade A to E and R [revised]). Secondary parameters were duration of surgery, blood loss, cumulative morphine, and length of stay. Results In the robot group (Group I), a perfect trajectory (A) was observed in 204 screws (83.6%). The remaining screws were graded B (n = 19 [7.8%]), C (n = 9 [3.7%]), D (n = 4 [1.6%]), E (n = 2 [0.8%]), and R (n = 6 [2.5%]). In the fluoroscopy-guided group (Group II), a completely intrapedicular course graded A was found in 79.8% (n = 130). The remaining screws were graded B (n = 12 [7.4%]), C (n = 10 [6.1%]), D (n = 6 [3.7%]), and E (n = 5 [3.1%]). The comparison of "clinically acceptable" (that is, A and B screws) was neither different between groups (I vs II [p = 0.19]) nor subgroups (Subgroup IA vs IB [p = 0.81]; Subgroup IA vs Group II [p = 0.53]; Subgroup IB vs Group II [p = 0.20]). Blood loss was lower in the robot-assisted group than in the fluoroscopy-guided group, while duration of surgery, length of stay, and cumulative morphine dose were not statistically different. Conclusions Robot-guided pedicle screw placement is a safe and useful tool for assisting spine surgeons in degenerative spine cases. Nonetheless, technical difficulties remain and fluoroscopy backup is advocated.