Phase I and phase II xenobiotic reactions and metabolism of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in aggregating liver cell cultures.

Aggregating fetal liver cell cultures were tested for their ability to metabolize xenobiotics using ethoxycoumarin-O-deethylase (ECOD), as marker of phase I metabolism, and glutathione S-transferase (GST), as marker for phase II reactions. Significant basal activities, stable over 14 days in culture were measured for both ECOD and GST activities. The prototype cytochrome P450 inducers, 3-methylcholanthrene (3-MC) and phenobarbital (PB), increased ECOD and GST activities reaching an optimum 7 days after culturing, followed by a decline in activity. This decline was partially prevented by 1% dimethyl sulfoxide (DMSO) added chronically to the culture medium. DMSO was also found to induce ECOD activity and to a lesser extent GST activity. Furthermore, it potentiated in a dose-dependent manner the induction of ECOD by PB. The food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is metabolically transformed through a number of pathways in vivo. It was therefore used to examine the metabolic capacity in fetal and adult liver cell aggregates. Metabolism of MeIQx was mainly through N2-conjugation, resulting in formation of the N2-glucuronide and sulfamate conjugates for non-induced fetal liver cells. These metabolites were also found in large amounts in non-induced adult liver cells. Low levels of cytochrome P450-mediated ring-hydroxylated metabolites were detected in both non-induced fetal and adult liver cells. After induction with arochlor (PCB) or 3-MC, the major pathway was ring-hydroxylation (cytochrome P450 dependent), followed by conjugation to beta-glucuronic or sulfuric acid. The presence of the glucuronide conjugate of N-hydroxy-MeIQx, a mutagenic metabolite, suggested an induction of P450 CYP1A2. The metabolism of MeIQx by liver cell aggregates is very similar to that observed in vivo and suggests that aggregating liver cell cultures are a useful model for in vitro metabolic studies in toxicology.

Mitochondrial DNA sequence variation within the remnant populations of the endangered numbat (Marsupialia: Myrmecobiidae: Myrmecobius fasciatus).

The numbat has been reduced to two populations in Western Australia. To better understand the effects of range reduction on gene flow and genetic variation, and to address questions crucial for the species' management, we analysed mitochondrial DNA (mtDNA) sequences of free-ranging individuals and museum specimens. The results suggest recent connectivity between the remnant populations, although one of those may have lost significant amounts of genetic diversity during the recent population size reduction. We propose that for management purposes the remnant populations should be treated as a single historical lineage and that, subject to certain caveats, consideration should be given to population augmentation by translocation.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

Docetaxel, cisplatin, and fluorouracil; docetaxel and cisplatin; and epirubicin, cisplatin, and fluorouracil as systemic treatment for advanced gastric carcinoma: a randomized phase II trial of the Swiss Group for Clinical Cancer Research.

PURPOSE: This randomized phase II trial evaluated two docetaxel-based regimens to see which would be most promising according to overall response rate (ORR) for comparison in a phase III trial with epirubicin-cisplatin-fluorouracil (ECF) as first-line advanced gastric cancer therapy. PATIENTS AND METHODS: Chemotherapy-naïve patients with measurable unresectable and/or metastatic gastric carcinoma, a performance status <or= 1, and adequate hematologic, hepatic, and renal function randomly received <or= eight 3-weekly cycles of ECF (epirubicin 50 mg/m(2) on day 1, cisplatin 60 mg/m(2) on day 1, and fluorouracil [FU] 200 mg/m(2)/d on days 1 to 21), TC (docetaxel initially 85 mg/m(2) on day 1 [later reduced to 75 mg/m(2) as a result of toxicity] and cisplatin 75 mg/m(2) on day 1), or TCF (TC plus FU 300 mg/m(2)/d on days 1 to 14). Study objectives included response (primary), survival, toxicity, and quality of life (QOL). RESULTS: ORR was 25.0% (95% CI, 13% to 41%) for ECF, 18.5% (95% CI, 9% to 34%) for TC, and 36.6% (95% CI, 23% to 53%) for TCF (n = 119). Median overall survival times were 8.3, 11.0, and 10.4 months for ECF, TC, and TCF, respectively. Toxicity was acceptable, with one toxic death (TC arm). Grade 3 or 4 neutropenia occurred in more treatment cycles with docetaxel (TC, 49%; TCF, 57%; ECF, 34%). Global health status/QOL substantially improved with ECF and remained similar to baseline with both docetaxel regimens. CONCLUSION: Time to response and ORR favor TCF over TC for further evaluation, particularly in the neoadjuvant setting. A trend towards increased myelosuppression and infectious complications with TCF versus TC or ECF was observed.

Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study.

BACKGROUND: Hypertension can be controlled adequately with existing drugs such as angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Nevertheless, treatment success is often restricted by patients not adhering to treatment. Immunisation against angiotensin II could solve this problem. We investigated the safety and efficacy of CYT006-AngQb-a vaccine based on a virus-like particle-that targets angiotensin II to reduce ambulatory blood pressure. METHODS: In this multicentre, double-blind, randomised, placebo-controlled phase IIa trial, 72 patients with mild-to-moderate hypertension were randomly assigned with a computer-generated randomisation list to receive subcutaneous injections of either 100 mug CYT006-AngQb (n=24), 300 mug CYT006-AngQb (24), or placebo (24), at weeks 0, 4, and 12. 24-h ambulatory blood pressure was measured before treatment and at week 14. The primary outcomes were safety and tolerability. Analyses were done by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00500786. FINDINGS: Two patients in the 100 mug group, three in the 300 mug group, and none in the placebo group discontinued study treatment. All patients were included in safety analyses; efficacy analyses did not include the five dropouts, for whom no data were available at week 14. Five serious adverse events were reported (two in the 100 mug group, two in the 300 mug group, and one in the placebo group); none were deemed to be treatment related. Most side-effects were mild, transient reactions at the injection site. Mild, transient influenza-like symptoms were seen in three patients in the 100 mug group, seven in the 300 mug group, and none in the placebo group. In the 300 mug group, there was a reduction from baseline in mean ambulatory daytime blood pressure at week 14 by -9.0/-4.0 mm Hg compared with placebo (p=0.015 for systolic and 0.064 for diastolic). The 300 mug dose reduced the early morning blood-pressure surge compared with placebo (change at 0800 h -25/-13 mm Hg; p<0.0001 for systolic, p=0.0035 for diastolic). INTERPRETATION: Immunisation with CYT006-AngQb was associated with no serious adverse events; most observed adverse events were consistent with local or systemic responses similar to those seen with other vaccines. The 300 mug dose reduced blood pressure in patients with mild-to-moderate hypertension during the daytime, especially in the early morning. FUNDING: Cytos Biotechnology AG.

Narcolepsy and familial advanced sleep-phase syndrome: molecular genetics of sleep disorders.

Sleep disorders are very prevalent and represent an emerging worldwide epidemic. However, research into the molecular genetics of sleep disorders remains surprisingly one of the least active fields. Nevertheless, rapid progress is being made in several prototypical disorders, leading recently to the identification of the molecular pathways underlying narcolepsy and familial advanced sleep-phase syndrome. Since the first reports of spontaneous and induced loss-of-function mutations leading to hypocretin deficiency in human and animal models of narcolepsy, the role of this novel neurotransmission pathway in sleep and several other behaviors has gained extensive interest. Also, very recent studies using an animal model of familial advanced sleep-phase syndrome shed new light on the regulation of circadian rhythms.

Hybrid Multiscale Finite Volume method for two-phase flow in porous media

We present a novel hybrid (or multiphysics) algorithm, which couples pore-scale and Darcy descriptions of two-phase flow in porous media. The flow at the pore-scale is described by the Navier?Stokes equations, and the Volume of Fluid (VOF) method is used to model the evolution of the fluid?fluid interface. An extension of the Multiscale Finite Volume (MsFV) method is employed to construct the Darcy-scale problem. First, a set of local interpolators for pressure and velocity is constructed by solving the Navier?Stokes equations; then, a coarse mass-conservation problem is constructed by averaging the pore-scale velocity over the cells of a coarse grid, which act as control volumes; finally, a conservative pore-scale velocity field is reconstructed and used to advect the fluid?fluid interface. The method relies on the localization assumptions used to compute the interpolators (which are quite straightforward extensions of the standard MsFV) and on the postulate that the coarse-scale fluxes are proportional to the coarse-pressure differences. By numerical simulations of two-phase problems, we demonstrate that these assumptions provide hybrid solutions that are in good agreement with reference pore-scale solutions and are able to model the transition from stable to unstable flow regimes. Our hybrid method can naturally take advantage of several adaptive strategies and allows considering pore-scale fluxes only in some regions, while Darcy fluxes are used in the rest of the domain. Moreover, since the method relies on the assumption that the relationship between coarse-scale fluxes and pressure differences is local, it can be used as a numerical tool to investigate the limits of validity of Darcy's law and to understand the link between pore-scale quantities and their corresponding Darcy-scale variables.

The candidate tuberculosis vaccine Mtb72F/AS02 in PPD positive adults: A randomized controlled phase I/II study.

Prevention of tuberculosis (TB) through vaccination would substantially reduce the global TB burden. Mtb72F/AS02 is a candidate TB vaccine shown to be immunogenic and well tolerated in PPD-negative adults. We evaluated the safety and immunogenicity of Mtb72F/AS02 in Mycobacterium-primed adults (BCG-vaccinated, or infected adults who had received post-exposure chemoprophylaxis or treatment for pulmonary TB disease). In this observer-blind controlled trial, 20 BCG-vaccinated adults and 18 adults previously infected with Mycobacterium tuberculosis (Mtb), were randomized 3:1 to receive three doses of Mtb72F/AS02 or AS02 at one-month intervals, and followed for 6 months post third vaccination. Mtb72F/AS02 was well tolerated in BCG-vaccinated adults, and tended to be more reactogenic in Mtb-infected adults. Adverse events were mainly self-limiting, resolving without sequelae. No serious adverse events were reported. The adverse events in Mtb72F/AS02 vaccinees were not clearly associated with vaccine-induced responses (as assessed by proinflammatory cytokines, total IgE and C-reactive protein levels). No Th2 T-cell responses, or vaccine-induced T-cell responses to Mtb antigens (CFP-10/PPD/ESAT-6) were detected by ICS. In both cohorts, Mtb72F/AS02 induced persistent polyfunctional Mtb72F-specific CD4(+) T-cell responses and anti-Mtb72F humoral responses. IFN-γ was detectable in serum one day post each vaccination. Further evaluation of the candidate vaccine, Mtb72F/AS02, is warranted. Trial registration: ClinicalTrials.gov identifier: NCT00146744.

Genetic and environmental components of phenotypic variation in immune response and body size of a colonial bird, Delichon urbica (the house martin).

Directional selection for parasite resistance is often intense in highly social host species. Using a partial cross-fostering experiment we studied environmental and genetic variation in immune response and morphology in a highly colonial bird species, the house martin (Delichon urbica). We manipulated intensity of infestation of house martin nests by the haematophagous parasitic house martin bug Oeciacus hirundinis either by spraying nests with a weak pesticide or by inoculating them with 50 bugs. Parasitism significantly affected tarsus length, T cell response, immunoglobulin and leucocyte concentrations. We found evidence of strong environmental effects on nestling body mass, body condition, wing length and tarsus length, and evidence of significant additive genetic variance for wing length and haematocrit. We found significant environmental variance, but no significant additive genetic variance in immune response parameters such as T cell response to the antigenic phytohemagglutinin, immunoglobulins, and relative and absolute numbers of leucocytes. Environmental variances were generally greater than additive genetic variances, and the low heritabilities of phenotypic traits were mainly a consequence of large environmental variances and small additive genetic variances. Hence, highly social bird species such as the house martin, which are subject to intense selection by parasites, have a limited scope for immediate microevolutionary response to selection because of low heritabilities, but also a limited scope for long-term response to selection because evolvability as indicated by small additive genetic coefficients of variation is weak.

Solid phase microextraction as a short-term sampling technique for BTEX occupational exposure

Solid phase microextraction (SPME) has been widely used for many years in various applications, such as environmental and water samples, food and fragrance analysis, or biological fluids. The aim of this study was to suggest the SPME method as an alternative to conventional techniques used in the evaluation of worker exposure to benzene, toluene, ethylbenzene, and xylene (BTEX). Polymethylsiloxane-carboxen (PDMS/CAR) showed as the most effective stationary phase material for sorbing BTEX among other materials (polyacrylate, PDMS, PDMS/divinylbenzene, Carbowax/divinylbenzene). Various experimental conditions were studied to apply SPME to BTEX quantitation in field situations. The uptake rate of the selected fiber (75 microm PDMS/CAR) was determined for each analyte at various concentrations, relative humidities, and airflow velocities from static (calm air) to dynamic (> 200 cm/s) conditions. The SPME method also was compared with the National Institute of Occupational Safety and Health method 1501. Unlike the latter, the SPME approach fulfills the new requirement for the threshold limit value-short term exposure limit (TLV-STEL) of 2.5 ppm for benzene (8 mg/m(3))

Phase-I/II radio-immunotherapy study with Iodine-131-labeled anti-CEA monoclonal antibody F6 F(ab')2 in patients with non-resectable liver metastases from colorectal cancer.

Experimental studies in nude mice with human colon-carcinoma grafts demonstrated the therapeutic efficiency of F(ab')2 fragments to carcinoembryonic antigen (CEA) labeled with a high dose of 131Iodine. A phase I/II study was designed to determine the maximum tolerated dose of 131I-labeled F(ab')2 fragments (131I-F(ab')2) from anti-CEA monoclonal antibody F6, its limiting organ toxicity and tumor uptake. Ten patients with non-resectable liver metastases from colorectal cancer (9 detected by CT scan and 1 by laparotomy) were treated with 131I-F(ab')2, doses ranging from 87 mCi to 300 mCi for the first 5 patients, with a constant 300-mCi dose for the last 5 patients. For all the patients, autologous bone marrow was harvested and stored before treatment. Circulating CEA ranged from 2 to 126 ng/ml. No severe adverse events were observed during or immediately following infusion of therapeutic doses. The 9 patients with radiologic evidence of liver metastases showed uptake of 131I-F(ab')2 in the metastases, as observed by single-photon-emission tomography. The only toxicity was hematologic, and no severe aplasia was observed when up to 250 mCi was infused. At the 300-mCi dose, 5 out of 6 patients presented grade-3 or -4 hematologic toxicity, with a nadir for neutrophils and thrombocytes ranging from 25 to 35 days after infusion. In these 5 cases, bone marrow was re-infused. No clinical complications were observed during aplasia. The tumor response could be evaluated in 9 out of 10 patients. One patient showed a partial response of one small liver metastasis (2 cm in diameter) and a stable evolution of the other metastases, 2 patients had stable disease, and 6 showed tumor progression at the time of evaluation (2 or 3 months after injection) by CT scan. This phase-I/II study demonstrated that a dose of 300 mCi of 131I-F(ab')2 from the anti-CEA Mab F6 is well tolerated with bone-marrow rescue, whereas a dose of 200 mCi can be infused without severe bone-marrow toxicity.

Concomitant cisplatin and hyperfractionated radiotherapy in locally advanced head and neck cancer: 10-year follow-up of a randomized phase III trial (SAKK 10/94).

Purpose: To compare the long-term outcome of treatment with concomitant cisplatin and hyperfractionated radiotherapy versus treatment with hyperfractionated radiotherapy alone in patients with locally advanced head and neck cancer.Methods and Materials: From July 1994 to July 2000, a total of 224 patients with squamous cell carcinoma of the head and neck were randomized to receive either hyperfractionated radiotherapy alone (median total dose, 74.4 Gy; 1.2 Gy twice daily; 5 days per week) or the same radiotherapy combined with two cycles of cisplatin (20 mg/m(2) for 5 consecutive days during weeks 1 and 5). The primary endpoint was the time to any treatment failure; secondary endpoints were locoregional failure, metastatic failure, overall survival, and late toxicity assessed according to Radiation Therapy Oncology Group criteria.Results: Median follow-up was 9.5 years (range, 0.1-15.4 years). Median time to any treatment failure was not significantly different between treatment arms (hazard ratio [HR], 1.2 [95% confidence interval [CM 0.9-1.7; p = 0.17]). Rates of locoregional failure-free survival (HR, 1.5 [95% CI, 1.1-2.1;p = 0.021), distant metastasis-free survival (HR, 1.6 [95% CI, 1.1-2.5; p = 0.021), and cancer-specific survival (HR, 1.6 [95% CI, 1.0-2.5;p = 0.03]) were significantly improved in the combined-treatment arm, with no difference in major late toxicity between treatment arms. However, overall survival was not significantly different (HR, 1.3 [95% CI, 0.9-1.8; p = 0.11]).Conclusions: After long-term follow-up, combined-treatment with cisplatin and hyperfractionated radiotherapy maintained improved rates of locoregional control, distant metastasis-free survival, and cancer-specific survival compared to that of hyperfractionated radiotherapy alone, with no difference in major late toxicity. (C) 2012 Elsevier Inc.