I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Untargeted profiling of urinary steroid metabolites after testosterone ingestion: opening new perspectives for antidoping testing.

AIM: Antidoping procedures are expected to greatly benefit from untargeted metabolomic approaches through the discovery of new biomarkers of prohibited substances abuse. RESULTS: Endogenous steroid metabolites were monitored in urine samples from a controlled elimination study of testosterone undecanoate after ingestion. A platform coupling ultra-high pressure LC with high-resolution quadrupole TOF MS was used and high between-subject metabolic variability was successfully handled using a multiblock data analysis strategy. Links between specific subsets of metabolites and influential genetic polymorphisms of the UGT2B17 enzyme were highlighted. CONCLUSION: This exploratory metabolomic strategy constitutes a first step toward a better understanding of the underlying patterns driving the high interindividual variability of steroid metabolism. Promising biomarkers were selected for further targeted study.

The use of methylamphetamine chemical profiling in an intelligence-led perspective and the observation of inhomogeneity within seizures

This study focuses on methylamphetamine (MA) seizures made by the Australian Federal Police (AFP) to investigate the use of chemical profiling in an intelligence perspective. Correlation coefficients were used to obtain a similarity degree between a population of linked samples and a population of unlinked samples. Although it was demonstrated that a general framework can be followed for the use of any forensic case data in an intelligence-led perspective, threshold values have to be re-evaluated for each type of illicit drug investigated. Unlike the results obtained in a previous study on 3,4-methylenedioxymethylamphetamine (MDMA) seizures, chemical profiles of MA samples coming from the same seizure showed relative inhomogeneity, limiting their ability to link seizures. Different hypotheses were investigated to obtain a better understanding of this inhomogeneity although no trend was observed. These findings raise an interesting discussion in regards to the homogeneity and representativeness of illicit drug seizures (for intelligence purposes). Further, it also provides some grounds to discuss the initial hypotheses and assumptions that most forensic science studies are based on.

Impact of normalization on miRNA microarray expression profiling.

Profiling miRNA levels in cells with miRNA microarrays is becoming a widely used technique. Although normalization methods for mRNA gene expression arrays are well established, miRNA array normalization has so far not been investigated in detail. In this study we investigate the impact of normalization on data generated with the Agilent miRNA array platform. We have developed a method to select nonchanging miRNAs (invariants) and use them to compute linear regression normalization coefficients or variance stabilizing normalization (VSN) parameters. We compared the invariants normalization to normalization by scaling, quantile, and VSN with default parameters as well as to no normalization using samples with strong differential expression of miRNAs (heart-brain comparison) and samples where only a few miRNAs are affected (by p53 overexpression in squamous carcinoma cells versus control). All normalization methods performed better than no normalization. Normalization procedures based on the set of invariants and quantile were the most robust over all experimental conditions tested. Our method of invariant selection and normalization is not limited to Agilent miRNA arrays and can be applied to other data sets including those from one color miRNA microarray platforms, focused gene expression arrays, and gene expression analysis using quantitative PCR.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue.

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_ 5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_ 5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Interest of qualitative and quantitative profiling of endocannabinoids for evaluation of their implication in drug addiction process

Introduction: In the middle of the 90's, the discovery of endogenous ligands for cannabinoid receptors opened a new era in this research field. Amides and esters of arachidonic acid have been identified as these endogenous ligands. Arachidonoylethanolamide (anandamide or AEA) and 2-Arachidonoylglycerol (2-AG) seem to be the most important of these lipid messengers. In addition, virodhamine (VA), noladin ether (2-AGE), and N-arachidonoyl dopamine (NADA) have been shown to bind to CB receptors with varying affinities. During recent years, it has become more evident that the EC system is part of fundamental regulatory mechanisms in many physiological processes such as stress and anxiety responses, depression, anorexia and bulimia, schizophrenia disorders, neuroprotection, Parkinson disease, anti-proliferative effects on cancer cells, drug addiction, and atherosclerosis. Aims: This work presents the problematic of EC analysis and the input of Information Dependant Acquisition based on hybrid triple quadrupole linear ion trap (QqQLIT) system for the profiling of these lipid mediators. Methods: The method was developed on a LC Ultimate 3000 series (Dionex, Sunnyvale, CA, USA) coupled to a QTrap 4000 system (Applied biosystems, Concord, ON, Canada). The ECs were separated on an XTerra C18 MS column (50 × 3.0 mm i.d., 3.5 μm) with a 5 min gradient elution. For confirmatory analysis, an information-dependant acquisition experiment was performed with selected reaction monitoring (SRM) as survey scan and enhanced produced ion (EPI) as dependant scan. Results: The assay was found to be linear in the concentration range of 0.1-5 ng/mL for AEA, 0.3-5 ng/mL for VA, 2-AGE, and NADA and 1-20 ng/mL for 2-AG using 0.5 mL of plasma. Repeatability and intermediate precision were found less than 15% over the tested concentration ranges. Under non-pathophysiological conditions, only AEA and 2-AG were actually detected in plasma with concentration ranges going from 104 to 537 pg/mL and from 2160 to 3990 pg/mL respectively. We have particularly focused our scopes on the evaluation of EC level changes in biological matrices through drug addiction and atherosclerosis processes. We will present preliminary data obtained during pilot study after administration of cannabis on human patients. Conclusion: ECs have been shown to play a key role in regulation of many pathophysiological processes. Medical research in these different fields continues to growth in order to understand and to highlight the predominant role of EC in the CNS and peripheral tissues signalisation. The profiling of these lipids needs to develop rapid, highly sensitive and selective analytical methods.

The fou2 gain-of-function allele and the wild-type allele of Two Pore Channel 1 contribute to different extents or by different mechanisms to defense gene expression in Arabidopsis.

The fatty acid oxygenation up-regulated 2 (fou2) mutant in Arabidopsis thaliana creates a gain-of-function allele in a non-selective cation channel encoded by the Two Pore Channel 1 (TPC1) gene. This mutant genetically implicates cation fluxes in the control of the positive feedback loop whereby jasmonic acid (JA) stimulates its own synthesis. In this study we observed extensive transcriptome reprogramming in healthy fou2 leaves closely resembling that induced by treatment with methyl jasmonate, biotic stresses and the potassium starvation response. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA-inducible proteins. In agreement with these analyses, epistasis studies performed by crossing fou2 with aos indicated that elevated levels of JA in fou2 are the major determinant of the mutant phenotype. In addition, generation of fou2 aba1-5, fou2 etr1-1 and fou2 npr1-1 double mutants showed that the fou2 phenotype was only weakly affected by ABA levels and unaffected by mutations in NPR1 and ETR1. The results now suggest possible mechanisms whereby fou2 could induce JA synthesis/signaling early in the wound response. In contrast to fou2, transcriptome analysis of a loss-of-function allele of TPC1, tpc1-2, revealed no differential expression of JA biosynthesis genes in resting leaves. However, the analysis disclosed reduced mRNA levels of the pathogenesis-related genes PDF1.2a and THI2.1 in healthy and diseased tpc1-2 leaves. The results suggest that wild-type TPC1 contributes to their expression by mechanisms somewhat different from those affecting their expression in fou2.

Proteomic analysis of the shistosome tegument and its surface membranes

The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS), and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

Analysis of prion strains by PrPSc profiling in sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Prion diseases are a group of invariably fatal neurodegenerative disorders affecting humans and a wide range of mammals. An essential part of the infectious agent, termed the prion, is composed of an abnormal isoform (PrPSc) of a host-encoded normal cellular protein (PrPC). The conversion of PrPC to PrPSc is thought to play a crucial role in the development of prion diseases and leads to PrPSc deposition, mainly in the central nervous system. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form of human prion disease, presents with a marked clinical heterogeneity. This diversity is accompanied by a molecular signature which can be defined by histological, biochemical, and genetic means. The molecular classification of sCJD is an important tool to aid in the understanding of underlying disease mechanisms and the development of therapy protocols. Comparability of classifications is hampered by disparity of applied methods and inter-observer variability. METHODS AND FINDINGS: To overcome these difficulties, we developed a new quantification protocol for PrPSc by using internal standards on each Western blot, which allows for generation and direct comparison of individual PrPSc profiles. By studying PrPSc profiles and PrPSc type expression within nine defined central nervous system areas of 50 patients with sCJD, we were able to show distinct PrPSc distribution patterns in diverse subtypes of sCJD. Furthermore, we were able to demonstrate the co-existence of more than one PrPSc type in individuals with sCJD in about 20% of all patients and in more than 50% of patients heterozygous for a polymorphism on codon 129 of the gene encoding the prion protein (PRNP). CONCLUSION: PrPSc profiling represents a valuable tool for the molecular classification of human prion diseases and has important implications for their diagnosis by brain biopsy. Our results show that the co-existence of more than one PrPSc type might be influenced by genetic and brain region-specific determinants. These findings provide valuable insights into the generation of distinct PrPSc types.

The profiling of MDMA tablets: a study of the combination of physical characteristics and organic impurities as sources of information

The profiling of MDMA tablets can be carried out using different sets of characteristics. The first type of measurements performed on MDMA tablets are physical characteristics (i.e. post-tabletting characteristics). They yield preliminary profiling data that may be valuable in a first stage for investigation purposes. However organic impurities (i.e. pre-tabletting characteristics) are generally considered to bring more reliable information, particularly for presentation of evidence in court. This work aimed therefore at evaluating the added value of combining pre-tabletting characteristics and post-tabletting characteristics of seized MDMA tablets. In approximately half of the investigated cases, the post-tabletting links were confirmed with organic impurities analyses. In the remaining cases, post-tabletting batches (post-TBs) were divided in several pre-tabletting batches (pre-TBs), thus supporting the hypothesis that several production batches of MDMA powder (pre-TBs) were used to produce one single post-TB (i.e. tablets having the same shape, diameter, thickness, weight and score; but different organic impurities composition). In view of the obtained results, the hypotheses were discussed through illustrating examples. In conclusion, both sets of characteristics were found relevant alone and combined together. They actually provide distinct information about MDMA illicit production and trafficking.

Small nucleolar RNA expression profiling identifies potential prognostic markers in peripheral T-cell lymphoma.

Peripheral T-cell lymphoma (PTCL) is a rare, heterogeneous type of non-Hodgkin lymphoma (NHL) that, in general, is associated with a poor clinical outcome. Therefore, a current major challenge is the discovery of new prognostic tools for this disease. In the present study, a cohort of 122 patients with PTCL was collected from a multicentric T-cell lymphoma consortium (TENOMIC). We analyzed the expression of 80 small nucleolar RNAs (snoRNAs) using high-throughput quantitative PCR. We demonstrate that snoRNA expression analysis may be useful in both the diagnosis of some subtypes of PTCL and the prognostication of both PTCL-not otherwise specified (PTCL-NOS; n = 26) and angio-immunoblastic T-cell lymphoma (AITL; n = 46) patients treated with chemotherapy. Like miRNAs, snoRNAs are globally down-regulated in tumor cells compared with their normal counterparts. In the present study, the snoRNA signature was robust enough to differentiate anaplastic large cell lymphoma (n = 32) from other PTCLs. For PTCL-NOS and AITL, we obtained 2 distinct prognostic signatures with a reduced set of 3 genes. Of particular interest was the prognostic value of HBII-239 snoRNA, which was significantly over-expressed in cases of AITL and PTCL-NOS that had favorable outcomes. Our results suggest that snoRNA expression profiles may have a diagnostic and prognostic significance for PTCL, offering new tools for patient care and follow-up.

Overview of coded light projection techniques for automatic 3D profiling

Obtaining automatic 3D profile of objects is one of the most important issues in computer vision. With this information, a large number of applications become feasible: from visual inspection of industrial parts to 3D reconstruction of the environment for mobile robots. In order to achieve 3D data, range finders can be used. Coded structured light approach is one of the most widely used techniques to retrieve 3D information of an unknown surface. An overview of the existing techniques as well as a new classification of patterns for structured light sensors is presented. This kind of systems belong to the group of active triangulation method, which are based on projecting a light pattern and imaging the illuminated scene from one or more points of view. Since the patterns are coded, correspondences between points of the image(s) and points of the projected pattern can be easily found. Once correspondences are found, a classical triangulation strategy between camera(s) and projector device leads to the reconstruction of the surface. Advantages and constraints of the different patterns are discussed

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach.

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.

Genomic and proteomic approaches towards an understanding of sleep.

The basic functions of sleep are still unclear, however, recent advances in genomics and proteomics have begun to contribute to our understanding of both normal and pathological sleep. In this review, we focus primarily on normal sleep and wake that have been studied in model organisms such as mice. Mice have been especially valuable since many different inbred strains exist that differ in sleep-related traits, and genes can be altered by either mutagenesis or targeted approaches. Advances in QTL (Quantitative Trait Loci) analysis have also helped to identify important sleep related genes, and several other QTLs have been mapped as a first step toward finding the genes that underlie basic sleep traits. In addition to more traditional genetic approaches, the abundance of different mRNAs across sleep and wake can now be studied and compared in different brain regions much more thoroughly using microarray methods. Progress at the protein level has been more difficult, but a few studies have begun to investigate changes in proteins during sleep and wake, and we present some of our own preliminary data in this area. A knowledge of which genes and proteins control or respond to changes in sleep will not only help answer fundamental questions, but may also suggest novel drug targets for improving multiple aspects of sleep and wake.