Application of direct-infusion ESI-MS/MS for toxicological screening.

BACKGROUND: Direct-infusion ESI-MS/MS is a powerful approach for the identification of substances in complex mixtures. The aim of this work was to investigate its applicability to the toxicological screening of blood samples. A simple protein precipitation was used, followed by a 15 min infusion of the extract in the mass spectrometer. RESULTS: The application of the procedure to commercial quality controls and authentic post-mortem blood samples demonstrated that the direct-infusion ESI-MS/MS approach enables the simultaneous identification of substances that require different chromatographic conditions. However, poor sensitivity was observed for benzodiazepine, amphetamines and opiate compounds. CONCLUSION: Considering the facile implementation and positive performance of direct-infusion ESI-MS/MS, this approach may to be a valuable complementary technique for systematic toxicological analysis procedures.

COLOX: a new blood-based test for colorectal cancer (CRC)screening

BACKGROUND: The objective is to develop a cost-effective, reliable and non invasive screening test able to detect early CRCs and adenomas. This is done on a nucleic acids multigene assay performed on peripheral blood mononuclear cells (PBMCs). METHODS: A colonoscopy-controlled study was conducted on 179 subjects. 92 subjects (21 CRC, 30 adenoma >1 cm and 41 controls) were used as training set to generate a signature. Other 48 subjects kept blinded (controls, CRC and polyps) were used as a test set. To determine organ and disease specificity 38 subjects were used: 24 with inflammatory bowel disease (IBD),14 with other cancers (OC). Blood samples were taken and PBMCs were purified. After the RNA extraction, multiplex RT-qPCR was applied on 92 different candidate biomarkers. After different univariate and multivariate analysis 60 biomarkers with significant p-values (<0.01) were selected. 2 distinct biomarker signatures are used to separate patients without lesion from those with CRC or with adenoma, named COLOX CRC and COLOX POL. COLOX performances were validated using random resampling method, bootstrap. RESULTS: COLOX CRC and POL tests successfully separate patients without lesions from those with CRC (Se 67%, Sp 93%, AUC 0.87), and from those with adenoma > 1cm (Se 63%, Sp 83%, AUC 0.77). 6/24 patients in the IBD group and 1/14 patients in the OC group have a positive COLOX CRC. CONCLUSION: The two COLOX tests demonstrated a high Se and Sp to detect the presence of CRCs and adenomas > 1 cm. A prospective, multicenter, pivotal study is underway in order to confirm these promising results in a larger cohort.

Development of a two-step screening ESI-TOF-MS method for rapid determination of significant stress-induced metabolome modifications in plant leaf extracts: the wound response in Arabidopsis thaliana as a case study.

To study the stress-induced effects caused by wounding under a new perspective, a metabolomic strategy based on HPLC-MS has been devised for the model plant Arabidopsis thaliana. To detect induced metabolites and precisely localise these compounds among the numerous constitutive metabolites, HPLC-MS analyses were performed in a two-step strategy. In a first step, rapid direct TOF-MS measurements of the crude leaf extract were performed with a ballistic gradient on a short LC-column. The HPLC-MS data were investigated by multivariate analysis as total mass spectra (TMS). Principal components analysis (PCA) and hierarchical cluster analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrated a clear clustering of plant specimens selecting the highest discriminating ions given by the complete data analysis, leading to the specific detection of discrete-induced ions (m/z values). Furthermore, pool constitution with plants of homogeneous behaviour was achieved for confirmatory analysis. In this second step, long high-resolution LC profilings on an UPLC-TOF-MS system were used on pooled samples. This allowed to precisely localise the putative biological marker induced by wounding and by specific extraction of accurate m/z values detected in the screening procedure with the TMS spectra.

Etude de la variation interlaboratoire du test de dépistage prénatal des défauts de fermeture du tube neural [Interlaboratory variation in the prenatal screening test for neural tube closing defects].

Early detection of neural-tude defects is possible by determining Alpha-fetoprotein (AFP) in maternal serum. 16'685 pregnant women were observed. Three methods for the determination of the "normal" range are compared. The first one, already used in similar studies, makes use of a constant multiple of the median. The other two ones make use of robust estimates of location and scale. Their comparison shows the interest of the robust methods to reduce the interlaboratory variability.

Dépistage néonatal systématique de la mucoviscidose en Suisse: dès janvier 2011 [Implementation of the neonatal cystic fibrosis screening program in Switzerland: beginning January 2011].

The diagnosis of cystic fibrosis (CF) is often delayed because of the nonspecificity of a wide variety of clinical symptoms at disease onset. Newborn screening for CF has been advocated to reduce delays in diagnosis, facilitating preventive care for early respiratory and nutritional involvement. According to American and European consensus and experience of existing programs, a Swiss Nationwide Cystic Fibrosis Newborn Screening Program started in January 2011. Screening strategy combines two steps: an immunoreactive trypsinogen assay and DNA mutation analysis in dried blood samples at day 4 (Guthrie cards).

Screening for gestational diabetes: variation in guidelines.

OBJECTIVE: To compare published guidelines concerning screening for gestational diabetes. STUDY DESIGN: Systematic search and comparative analysis of published guidelines. Appraisal of guidelines quality. Simulation analysis. RESULTS: Ten published guidelines proposed either universal screening (5), selective screening (3) or screening when clinically indicated (2). Variations of testing schedules and blood glucose thresholds were observed. The quality of the published guidelines was low, on average 22 (8-51) percentage points on the assessment scale. These differences would have led to large variations in the number of patients to be screened. CONCLUSIONS: Large variations between guidelines have been observed which would translate in large practice variations, if the guidelines were systematically applied. These variations are partially explained by the absence of definite evidence that universal or selective screening for gestational diabetes do more good than harm on infant and maternal health. The methodology of developing guidelines should be more evidence based, systematic and explicit.

Two Signatures For A New Blood-Based Test For Colorectal Cancer Screening

Background: Detection rates for adenoma and early colorectal cancer (CRC) are unsatisfactory due to low compliance towards invasive screening procedures such as colonoscopy. There is a large unmet screening need calling for an accurate, non-invasive and cost-effective test to screen for early neoplastic and pre-neoplastic lesions. Our goal is to identify effective biomarker combinations to develop a screening test aimed at detecting precancerous lesions and early CRC stages, based on a multigene assay performed on peripheral blood mononuclear cells (PBMC).Methods: A pilot study was conducted on 92 subjects. Colonoscopy revealed 21 CRC, 30 adenomas larger than 1 cm and 41 healthy controls. A panel of 103 biomarkers was selected by two approaches: a candidate gene approach based on literature review and whole transcriptome analysis of a subset of this cohort by Illumina TAG profiling. Blood samples were taken from each patient and PBMC purified. Total RNA was extracted and the 103 biomarkers were tested by multiplex RT-qPCR on the cohort. Different univariate and multivariate statistical methods were applied on the PCR data and 60 biomarkers, with significant p-value (< 0.01) for most of the methods, were selected.Results: The 60 biomarkers are involved in several different biological functions, such as cell adhesion, cell motility, cell signaling, cell proliferation, development and cancer. Two distinct molecular signatures derived from the biomarker combinations were established based on penalized logistic regression to separate patients without lesion from those with CRC or adenoma. These signatures were validated using bootstrapping method, leading to a separation of patients without lesion from those with CRC (Se 67%, Sp 93%, AUC 0.87) and from those with adenoma larger than 1cm (Se 63%, Sp 83%, AUC 0.77). In addition, the organ and disease specificity of these signatures was confirmed by means of patients with other cancer types and inflammatory bowel diseases.Conclusions: The two defined biomarker combinations effectively detect the presence of CRC and adenomas larger than 1 cm with high sensitivity and specificity. A prospective, multicentric, pivotal study is underway in order to validate these results in a larger cohort.

Rapid sample pre-treatment prior to GC-MS and GC-MS/MS urinary toxicological screening.

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC-MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200 μL of urine in less than 30 min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5 min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC-MS run to 10 min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC-MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC-COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

Genotype-phenotype analysis of Jervell and Lange-Nielsen syndrome in six families from Saudi Arabia.

We sought to explore the genotype-phenotype of Jervell and Lange-Nielsen syndrome (JLNS) patients in Saudi Arabia. We have also assessed the plausible effect of consanguinity into the pathology of JLNS. Six families with at least one JLNS-affected member attended our clinic between 2011 and 2013. Retrospective and prospective clinical data were collected and genetic investigation was performed. Pathogenic mutations in the KCNQ1 gene were detected in all JLNS patients. The homozygous mutations detected were Leu273Phe, Asp202Asn, Ile567Thr, and c.1486_1487delCT and compound heterozygous mutations were c.820_ 830del and c.1251+1G>T. All living JLNS patients except one had a QTc of >500 ms and a history of recurrent syncope. β-Blockers abolished the cardiac-related events in all patients except two siblings with homozygous Ile567Thr mutation. Four of the six mutations were originally reported in autosomal dominant long QT syndrome (LQTS) patients. Eighty percent of the heterozygote mutation carriers showed prolongation of QTc, but majority of these reported no symptoms attributable to arrhythmias. Mutations detected in this study will be advantageous in tribe and region-specific cascade screening of LQTS in Saudi Arabia.

Prevalence and risk of Down syndrome in monozygotic and dizygotic multiple pregnancies in Europe: implications for prenatal screening.

OBJECTIVE: To determine risk of Down syndrome (DS) in multiple relative to singleton pregnancies, and compare prenatal diagnosis rates and pregnancy outcome. DESIGN: Population-based prevalence study based on EUROCAT congenital anomaly registries. SETTING: Eight European countries. POPULATION: 14.8 million births 1990-2009; 2.89% multiple births. METHODS: DS cases included livebirths, fetal deaths from 20 weeks, and terminations of pregnancy for fetal anomaly (TOPFA). Zygosity is inferred from like/unlike sex for birth denominators, and from concordance for DS cases. MAIN OUTCOME MEASURES: Relative risk (RR) of DS per fetus/baby from multiple versus singleton pregnancies and per pregnancy in monozygotic/dizygotic versus singleton pregnancies. Proportion of prenatally diagnosed and pregnancy outcome. STATISTICAL ANALYSIS: Poisson and logistic regression stratified for maternal age, country and time. RESULTS: Overall, the adjusted (adj) RR of DS for fetus/babies from multiple versus singleton pregnancies was 0.58 (95% CI 0.53-0.62), similar for all maternal ages except for mothers over 44, for whom it was considerably lower. In 8.7% of twin pairs affected by DS, both co-twins were diagnosed with the condition. The adjRR of DS for monozygotic versus singleton pregnancies was 0.34 (95% CI 0.25-0.44) and for dizygotic versus singleton pregnancies 1.34 (95% CI 1.23-1.46). DS fetuses from multiple births were less likely to be prenatally diagnosed than singletons (adjOR 0.62 [95% CI 0.50-0.78]) and following diagnosis less likely to be TOPFA (adjOR 0.40 [95% CI 0.27-0.59]). CONCLUSIONS: The risk of DS per fetus/baby is lower in multiple than singleton pregnancies. These estimates can be used for genetic counselling and prenatal screening.

Optimization of HS-SPME/GC-MS analysis and its use in the profiling of illicit ecstasy tablets (Part 1)

A headspace solid-phase microextraction procedure (HS-SPME) was developed for the profiling of traces present in 3,4-methylenedioxymethylampethamine (MDMA). Traces were first extracted using HS-SPME and then analyzed by gas chromatography-mass spectroscopy (GC-MS). The HS-SPME conditions were optimized using varying conditions. Optimal results were obtained when 40 mg of crushed MDMA sample was heated at 80 °C for 15 min, followed by extraction at 80 °C for 15 min with a polydimethylsiloxane/divinylbenzene coated fibre. A total of 31 compounds were identified as traces related to MDMA synthesis, namely precursors, intermediates or by-products. In addition some fatty acids used as tabletting materials and caffeine used as adulterant, were also detected. The use of a restricted set of 10 target compounds was also proposed for developing a screening tool for clustering samples having close profile. 114 seizures were analyzed using an SPME auto-sampler (MultiPurpose Samples MPS2), purchased from Gerstel GMBH & Co. (Germany), and coupled to GC-MS. The data was handled using various pre-treatment methods, followed by the study of similarities between sample pairs based on the Pearson correlation. The results show that HS-SPME, coupled with the suitable statistical method is a powerful tool for distinguishing specimens coming from the same seizure and specimens coming from different seizures. This information can be used by law enforcement personnel to visualize the ecstasy distribution network as well as the clandestine tablet manufacturing.

Venture capital financing : screening and contracting under assymetric information

Structure of the Thesis This thesis consists of 5 sections. Section 1 starts with the problem definition and the presentation of the objectives of this thesis. Section 2 introduces a presentation of the theoretical foundations of Venture financing and a review of the main theories developed on Venture investing. It includes a taxonomy of contracting clauses relevant in venture contracting, the conflicts they address, and presents some general observations on contractual clauses. Section 3 presents the research findings on the analysis of a European VC's deal flow and investment screening linked to the prevailing market conditions. Section 4 focuses an empirical study of a European VC's investment process, the criteria it uses to make its investments. It presents empirical findings on the investment criteria over time, business cycles, and investment types. It also links these criteria to the VC's subsequent performance. Finally, section 5 presents an empirical research on the comparison of the legal contracts signed between European and United States Venture Capitalists and the companies they finance. This research highlights some of the contracting practices in Europe and the United States.

Multiway Array Decomposition Analysis of EEGs in Alzheimer’s Disease

Methods for the extraction of features from physiological datasets are growing needs as clinical investigations of Alzheimer’s disease (AD) in large and heterogeneous population increase. General tools allowing diagnostic regardless of recording sites, such as different hospitals, are essential and if combined to inexpensive non-invasive methods could critically improve mass screening of subjects with AD. In this study, we applied three state of the art multiway array decomposition (MAD) methods to extract features from electroencephalograms (EEGs) of AD patients obtained from multiple sites. In comparison to MAD, spectral-spatial average filter (SSFs) of control and AD subjects were used as well as a common blind source separation method, algorithm for multiple unknown signal extraction (AMUSE). We trained a feed-forward multilayer perceptron (MLP) to validate and optimize AD classification from two independent databases. Using a third EEG dataset, we demonstrated that features extracted from MAD outperformed features obtained from SSFs AMUSE in terms of root mean squared error (RMSE) and reaching up to 100% of accuracy in test condition. We propose that MAD maybe a useful tool to extract features for AD diagnosis offering great generalization across multi-site databases and opening doors to the discovery of new characterization of the disease.

Population based screening - the difficulty of how to do more good than harm and how to achieve it.

Screening people without symptoms of disease is an attractive idea. Screening allows early detection of disease or elevated risk of disease, and has the potential for improved treatment and reduction of mortality. The list of future screening opportunities is set to grow because of the refinement of screening techniques, the increasing frequency of degenerative and chronic diseases, and the steadily growing body of evidence on genetic predispositions for various diseases. But how should we decide on the diseases for which screening should be done and on recommendations for how it should be implemented? We use the examples of prostate cancer and genetic screening to show the importance of considering screening as an ongoing population-based intervention with beneficial and harmful effects, and not simply the use of a test. Assessing whether screening should be recommended and implemented for any named disease is therefore a multi-dimensional task in health technology assessment. There are several countries that already use established processes and criteria to assess the appropriateness of screening. We argue that the Swiss healthcare system needs a nationwide screening commission mandated to conduct appropriate evidence-based evaluation of the impact of proposed screening interventions, to issue evidence-based recommendations, and to monitor the performance of screening programmes introduced. Without explicit processes there is a danger that beneficial screening programmes could be neglected and that ineffective, and potentially harmful, screening procedures could be introduced.

Depistage mutationnel des genes de la peripherine/RDS, rhodopsine et ROM-1 dans 69 cas index de retinite pigmentaire et autres dystrophies retiniennes [Mutational screening of peripherin/RDS genes, rhodopsin and ROM-1 in 69 index cases with retinitis pigmentosa and other retinal dystrophies]

PURPOSE: Phenotypic, genetic and molecular characterization of 69 index patients with retinitis pigmentosa (RP) and various inherited retinal diseases. PATIENTS AND METHOD: patients went through complete ocular examination and blood samples were drawn for mutational screening of three candidate genes: rhodopsin (RHO), peripherin/RDS, and ROM-1. RESULTS: the most frequent type of RP among our population was the autosomal dominant (43.6%). Three RHO mutations were found among the RP patients. A RDS mutation was detected in three unrelated families segregating dominant macular dystrophy. DISCUSSION AND CONCLUSIONS: 18% of the autosomal dominant RP patients presented a RHO mutation; RDS R172W mutation was present in 25% of the dominant macular dystrophies.