Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry I. Screening analysis.

The general strategy to perform anti-doping analyses of urine samples starts with the screening for a wide range of compounds. This step should be fast, generic and able to detect any sample that may contain a prohibited substance while avoiding false negatives and reducing false positive results. The experiments presented in this work were based on ultra-high-pressure liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry. Thanks to the high sensitivity of the method, urine samples could be diluted 2-fold prior to injection. One hundred and three forbidden substances from various classes (such as stimulants, diuretics, narcotics, anti-estrogens) were analysed on a C(18) reversed-phase column in two gradients of 9min (including two 3min equilibration periods) for positive and negative electrospray ionisation and detected in the MS full scan mode. The automatic identification of analytes was based on retention time and mass accuracy, with an automated tool for peak picking. The method was validated according to the International Standard for Laboratories described in the World Anti-Doping Code and was selective enough to comply with the World Anti-Doping Agency recommendations. In addition, the matrix effect on MS response was measured on all investigated analytes spiked in urine samples. The limits of detection ranged from 1 to 500ng/mL, allowing the identification of all tested compounds in urine. When a sample was reported positive during the screening, a fast additional pre-confirmatory step was performed to reduce the number of confirmatory analyses.

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.

Platforms for high-throughput screening of Wnt/Frizzled antagonists.

Signaling cascades initiated by Wnt lipoglycoproteins and their receptors of the Frizzled family regulate many aspects of animal development and physiology. Improper activation of this signaling promotes carcinogenic transformation and metastasis. Development of agents blocking the Wnt-Frizzled signaling is of prime interest for drug discovery. Despite certain progress no such agents are as yet brought to the market or even to clinical trials. One reason for these delays might be the use of suboptimal readout assays. In this article we overview existing and developing assay platforms to screen for Wnt-Frizzled antagonists. Among those, G protein-activating assays built on the emerging GPCR properties of Frizzleds are highlighted.

A simple and reliable method for the screening of transgenic tobacco plants

Even though much improvement has been made in plant transformation methods, the screening of transgenic plants is often a laborious work. Most approaches for detecting the transgene in transformed plants are still timeconsuming, and can be quite expensive. The objective of this study was to search for a simpler method to screen for transgenic plants. The infiltration of kanamycin (100 mg/mL) into tobacco leaves resulted in conspicuous chlorotic spots on the non-transgenic plant leaves, while no spots were seen on the leaves of transformed plants. This reaction occurred regardless of age of the tested plants, and the method has proven to be simple, fast, non-destructive, relatively cheap, and reliable. These results were comparable to those obtained by the polymerase chain reaction (PCR) amplification of the transgene using specific primers.

Subplate subpopulations in the hypoxic-ischemic neonatal rat brain

Subplate neurons are among the earliest born cells of the neocortex and play a fundamental role in cortical development, in particular in the formation of thalamocortical connections. Subplate abnormalities have been described in several neuropathological disorders including schizophrenia, autism and periventricular eukomalacia (Eastwood and Harrison, Schizophr Res, 79, 2005; McQuillen and Ferriero, Brain Pathol, 15, 2005). We have identified and confirmed a range of specific markers for murine subplate using a microarray based approach and found that different subplate subpopulations are characterized by distinct expression patterns of these genes (Hoerder-Suabedissen et al., Cereb Cortex, 19, 2009). In this current study, we are making use of these markers to investigate neuropathological changes of the subplate after cerebral hypoxia-ischemia (HI) in the neonatal rat. First, we characterized the expression of a number of murine subplate markers in the postnatal rat using immunohistochemistry and in situ hybridization. While several genes (Nurr1, Cplx3, Ctgf and Tmem163) presented very similar expression patterns as in the mouse, others (Ddc, MoxD1 and TRH) were completely absent in the rat cortex. This finding suggests important differences in the subplate populations of these two rodent species. In a neonatal rat model of HI, selective vulnerability of subplate has been suggested using BrdU birthdating methods (McQuillen et al., J Neurosci, 15, 2003). We hypothesized that certain subplate subpopulations could be more susceptible than others and analyzed the above subplate markers in a similar yet slightly milder HI model. Two-day old male rat pups underwent permanent occlusion of the right common carotid artery followed by a period of hypoxia (6% O2, 1.5h or 2h) and were analyzed six days later. Preliminary counts on three subplate subpopulations (Nurr1+, Cplx3+ and Ctgf+ cells, respectively) showed similar reductions in cell numbers for all three groups. In addition, we found that the majority of cases which show changes in the subplate also exhibit lesions in the deep cortical layers VI (identified by FoxP2 expression) and sometimes even layer V (revealed by Er81 immunoreactivity), which questions the selective susceptibility of subplate over other cortical layers under the conditions we used in our model. Supported by MRC, FMO holds a Berrow Scholarship, Lincoln College, Oxford.

Detailed analysis and follow-up studies of a high-throughput screening for indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulator of immune responses and therefore an important therapeutic target for the treatment of diseases that involve pathological immune escape, such as cancer. Here, we describe a robust and sensitive high-throughput screen (HTS) for IDO1 inhibitors using the Prestwick Chemical Library of 1200 FDA-approved drugs and the Maybridge HitFinder Collection of 14,000 small molecules. Of the 60 hits selected for follow-up studies, 14 displayed IC50 values below 20 μM under the secondary assay conditions, and 4 showed an activity in cellular tests. In view of the high attrition rate we used both experimental and computational techniques to identify and to characterize compounds inhibiting IDO1 through unspecific inhibition mechanisms such as chemical reactivity, redox cycling, or aggregation. One specific IDO1 inhibitor scaffold, the imidazole antifungal agents, was chosen for rational structure-based lead optimization, which led to more soluble and smaller compounds with micromolar activity.

Screening of Bacillus thuringiensis strains effective against mosquitoes

The objective of this work was to evaluate 210 Bacillus thuringiensis strains against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective. These strains were isolated from different regions of Brazil and are stored in a Bacillus spp. collection at Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brazil. The selected strains were characterized by morphological (microscopy), biochemical (SDS-PAGE 10%) and molecular (PCR) methods. Six B. thuringiensis strains were identified as mosquito-toxic after the selective bioassays. None of the strains produced the expected PCR products for detection of cry4, cry11 and cyt1A genes. These results indicate that the activity of mosquitocidal Brazilian strains are not related with Cry4, Cry11 or Cyt proteins, so they could be used as an alternative bioinsecticide against mosquitoes.

Multicenter study of a new fully automated HBsAg screening assay with enhanced sensitivity for the detection of HBV mutants.

In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.

An efficient algorithm to perform multiple testing in epistasis screening

Background: Research in epistasis or gene-gene interaction detection for human complex traits has grown over the last few years. It has been marked by promising methodological developments, improved translation efforts of statistical epistasis to biological epistasis and attempts to integrate different omics information sources into the epistasis screening to enhance power. The quest for gene-gene interactions poses severe multiple-testing problems. In this context, the maxT algorithm is one technique to control the false-positive rate. However, the memory needed by this algorithm rises linearly with the amount of hypothesis tests. Gene-gene interaction studies will require a memory proportional to the squared number of SNPs. A genome-wide epistasis search would therefore require terabytes of memory. Hence, cache problems are likely to occur, increasing the computation time. In this work we present a new version of maxT, requiring an amount of memory independent from the number of genetic effects to be investigated. This algorithm was implemented in C++ in our epistasis screening software MBMDR-3.0.3. We evaluate the new implementation in terms of memory efficiency and speed using simulated data. The software is illustrated on real-life data for Crohn’s disease. Results: In the case of a binary (affected/unaffected) trait, the parallel workflow of MBMDR-3.0.3 analyzes all gene-gene interactions with a dataset of 100,000 SNPs typed on 1000 individuals within 4 days and 9 hours, using 999 permutations of the trait to assess statistical significance, on a cluster composed of 10 blades, containing each four Quad-Core AMD Opteron(tm) Processor 2352 2.1 GHz. In the case of a continuous trait, a similar run takes 9 days. Our program found 14 SNP-SNP interactions with a multiple-testing corrected p-value of less than 0.05 on real-life Crohn’s disease (CD) data. Conclusions: Our software is the first implementation of the MB-MDR methodology able to solve large-scale SNP-SNP interactions problems within a few days, without using much memory, while adequately controlling the type I error rates. A new implementation to reach genome-wide epistasis screening is under construction. In the context of Crohn’s disease, MBMDR-3.0.3 could identify epistasis involving regions that are well known in the field and could be explained from a biological point of view. This demonstrates the power of our software to find relevant phenotype-genotype higher-order associations.

Association of moderate alcohol use and binge drinking during pregnancy with neonatal health.

Background:  Heavy drinking and smoking during pregnancy are known to have a negative impact on the unborn child. However, the impact of low-to-moderate alcohol consumption and binge drinking has been debated recently. The aim of this study was to examine the relationship of moderate prenatal drinking and binge drinking with birthweight, being small for gestational age (SGA) at birth, preterm birth, and neonatal asphyxia. Methods:  Moderate alcohol drinking, binge drinking, and several possible confounders were assessed in 1,258 pregnant women; information on neonatal health was obtained at birth. Results:  Results indicate that 30.8% of the women drank at low levels (<2 glasses/wk), 7.9% drank moderately (2 to 4 glasses/wk), and 0.9% showed higher levels of drinking (≥5 glasses/wk); 4.7% reported binge drinking (defined as ≥3 glasses/occasion). 6.4% of the children were SGA (<10th percentile of birthweight adjusted for gestational age), 4.6% were preterm (<37th week of gestation), and 13.0% showed asphyxia (arterial cord pH <7.10 and/or arterial cord lactate >6.35 mmol and/or Apgar score <7 at 5 minutes). When controlling for maternal age, citizenship, occupational status, parity, smoking, use of prescription/over-the-counter drugs, illicit drug use, and child gender moderate drinking was related to lower birthweight (p < 0.01), and moderate drinking and binge drinking were associated with neonatal asphyxia at trend level (p = 0.06 and p = 0.09). Moderate drinking and binge drinking were not related to length of gestation. Conclusions:  In contrast to recent reviews in the field, our results assume that moderate drinking and binge drinking are risk factors for neonatal health.

Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease.

OBJECTIVES: Reactivation of latent tuberculosis (TB) in inflammatory bowel disease (IBD) patients treated with antitumor necrosis factor-alpha medication is a serious problem. Currently, TB screening includes chest x-rays and a tuberculin skin test (TST). The interferon-gamma release assay (IGRA) QuantiFERON-TB Gold In-Tube (QFT-G-IT) shows better specificity for diagnosing TB than the skin test. This study evaluates the two test methods among IBD patients. METHODS: Both TST and IGRA were performed on 212 subjects (114 Crohn's disease, 44 ulcerative colitis, 10 indeterminate colitis, 44 controls). RESULTS: Eighty-one percent of IBD patients were under immunosuppressive therapy; 71% of all subjects were vaccinated with Bacille Calmette Guérin; 18% of IBD patients and 43% of controls tested positive with the skin test (P < 0.0001). Vaccinated controls tested positive more often with the skin test (52%) than did vaccinated IBD patients (23%) (P = 0.011). Significantly fewer immunosuppressed patients tested positive with the skin test than did patients not receiving therapy (P = 0.007); 8% of patients tested positive with the QFT-G-IT test (14/168) compared to 9% (4/44) of controls. Test agreement was significantly higher in the controls (P = 0.044) compared to the IBD group. CONCLUSIONS: Agreement between the two test methods is poor in IBD patients. In contrast to the QFT-G-IT test, the TST is negatively influenced by immunosuppressive medication and vaccination status, and should thus be replaced by the IGRA for TB screening in immunosuppressed patients having IBD.