Importance of influx and efflux systems and xenobiotic metabolizing enzymes in intratumoral disposition of anticancer agents.

In this review, intratumoral drug disposition will be integrated into the wide range of resistance mechanisms to anticancer agents with particular emphasis on targeted protein kinase inhibitors. Six rules will be established: 1. There is a high variability of extracellular/intracellular drug level ratios; 2. There are three main systems involved in intratumoral drug disposition that are composed of SLC, ABC and XME enzymes; 3. There is a synergistic interplay between these three systems; 4. In cancer subclones, there is a strong genomic instability that leads to a highly variable expression of SLC, ABC or XME enzymes; 5. Tumor-expressed metabolizing enzymes play a role in tumor-specific ADME and cell survival and 6. These three systems are involved in the appearance of resistance (transient event) or in the resistance itself. In addition, this article will investigate whether the overexpression of some ABC and XME systems in cancer cells is just a random consequence of DNA/chromosomal instability, hypo- or hypermethylation and microRNA deregulation, or a more organized modification induced by transposable elements. Experiments will also have to establish if these tumor-expressed enzymes participate in cell metabolism or in tumor-specific ADME or if they are only markers of clonal evolution and genomic deregulation. Eventually, the review will underline that the fate of anticancer agents in cancer cells should be more thoroughly investigated from drug discovery to clinical studies. Indeed, inhibition of tumor expressed metabolizing enzymes could strongly increase drug disposition, specifically in the target cells resulting in more efficient therapies.

Orthogonal organic and aqueous-based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous-based buffer for tissue section preparation before matrix-assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water-acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14-day mouse fetus whole-body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on-tissue MALDI analysis compared with solely conventional organic rinsing.

Evaluation of a mixed approach combining stationary and wearable systems to monitor gait over long distance.

Thanks to decades of research, gait analysis has become an efficient tool. However, mainly due to the price of the motion capture systems, standard gait laboratories have the capability to measure only a few consecutive steps of ground walking. Recently, wearable systems were proposed to measure human motion without volume limitation. Although accurate, these systems are incompatible with most of existing calibration procedures and several years of research will be necessary for their validation. A new approach consisting of using a stationary system with a small capture volume for the calibration procedure and then to measure gait using a wearable system could be very advantageous. It could benefit from the knowledge related to stationary systems, allow long distance monitoring and provide new descriptive parameters. The aim of this study was to demonstrate the potential of this approach. Thus, a combined system was proposed to measure the 3D lower body joints angles and segmental angular velocities. It was then assessed in terms of reliability towards the calibration procedure, repeatability and concurrent validity. The dispersion of the joint angles across calibrations was comparable to those of stationary systems and good reliability was obtained for the angular velocities. The repeatability results confirmed that mean cycle kinematics of long distance walks could be used for subjects' comparison and pointed out an interest for the variability between cycles. Finally, kinematics differences were observed between participants with different ankle conditions. In conclusion, this study demonstrated the potential of a mixed approach for human movement analysis.

δ 15N measurement of organic and inorganic substances by EA-IRMS: a speciation-dependent procedure

Little attention has been paid so far to the influence of the chemical nature of the substance when measuring δ 15N by elemental analysis (EA)-isotope ratio mass spectrometry (IRMS). Although the bulk nitrogen isotope analysis of organic material is not to be questioned, literature from different disciplines using IRMS provides hints that the quantitative conversion of nitrate into nitrogen presents difficulties. We observed abnormal series of δ 15N values of laboratory standards and nitrates. These unexpected results were shown to be related to the tailing of the nitrogen peak of nitrate-containing compounds. A series of experiments were set up to investigate the cause of this phenomenon, using ammonium nitrate (NH4NO3) and potassium nitrate (KNO3) samples, two organic laboratory standards as well as the international secondary reference materials IAEA-N1, IAEA-N2-two ammonium sulphates [(NH4)2SO4]-and IAEA-NO-3, a potassium nitrate. In experiment 1, we used graphite and vanadium pentoxide (V2O5) as additives to observe if they could enhance the decomposition (combustion) of nitrates. In experiment 2, we tested another elemental analyser configuration including an additional section of reduced copper in order to see whether or not the tailing could originate from an incomplete reduction process. Finally, we modified several parameters of the method and observed their influence on the peak shape, δ 15N value and nitrogen content in weight percent of nitrogen of the target substances. We found the best results using mere thermal decomposition in helium, under exclusion of any oxygen. We show that the analytical procedure used for organic samples should not be used for nitrates because of their different chemical nature. We present the best performance given one set of sample introduction parameters for the analysis of nitrates, as well as for the ammonium sulphate IAEA-N1 and IAEA-N2 reference materials. We discuss these results considering the thermochemistry of the substances and the analytical technique itself. The results emphasise the difference in chemical nature of inorganic and organic samples, which necessarily involves distinct thermochemistry when analysed by EA-IRMS. Therefore, they should not be processed using the same analytical procedure. This clearly impacts on the way international secondary reference materials should be used for the calibration of organic laboratory standards.

Organic and inorganic geochemistry of Ljubija siderite deposits, NW Bosnia and Herzegovina

The Ljubija siderite deposits, hosted by a Carboniferous sedimentary complex within the Inner Dinarides, occur as stratabound replacement-type ore bodies in limestone blocks and as siderite-sulfides veins in shale. Three principal types of ore textures have been recognized including massive dark siderite and ankerite, siderite with zebra texture, and siderite veins. The ore and host rocks have been investigated by a combination of inorganic (major, trace, and rare earth element concentrations), organic (characterization of hydrocarbons including biomarkers), and stable isotope geochemical methods (isotope ratios of carbonates, sulfides, sulfates, kerogen, and individual hydrocarbons). New results indicate a marine origin of the host carbonates and a hydrothermal-metasomatic origin of the Fe mineralization. The differences in ore textures (e.g., massive siderite, zebra siderite) are attributed to physicochemical variations (e.g., changes in acidity, temperature, and/or salinity) of the mineralizing fluids and to the succession and intensity of replacement of host limestone. Vein siderite was formed by precipitation from hydrothermal fluids in the late stage of mineralization. The equilibrium fractionation of stable isotopes reveals higher formation temperatures for zebra siderites (around 245A degrees C) then for siderite vein (around 185A degrees C). Sulfur isotope ratios suggest Permian seawater or Permian evaporites as the main sulfur source. Fluid inclusion composition confirms a contribution of the Permian seawater to the mineralizing fluids and accord with a Permian mineralization age. Organic geochemistry data reflect mixing of hydrocarbons at the ore site and support the hydrothermal-metasomatic origin of the Ljubija iron deposits.

Heat capacity measurements on the equiatomic compounds in Ni-X (X = Al, In, Si, Ge and Bi) and M-Sb (with M = Ni, Co and Fe) systems

Molar heat capacities of the binary compounds NiAl, NiIn, NiSi, NiGe, NiBi, NiSb, CoSb and FeSb were determined every 10 K by differential scanning calorimetry in the temperature range 310-1080 K. The experimental results have been fitted versus temperature according to C-p = a + b . T + c . T-2 + d . T-2. Results are given, discussed and compared to estimations found in the literature. Two compounds, NiBi and FeSb, are subject to transformations between 460 and 500 K. (C) 1999 Elsevier Science Ltd. All rights reserved.

Targeted inactivation of hypocretin receptors in dopaminergic and noradrenergic systems alters brain activity in wakefulness

Since the discovery of hypocretins/orexins (Hcrt/Ox) in 1998, several narcoleptic mouse models, such as Hcrt-KO, Hcrtrl-KO, Hcrtr2-KO and double receptors KO mice, and orexin-ataxin transgenic mice were generated. The available Hcrt mouse models do not allow the dissection of the specific role of Hcrt in each target region. Dr. Anne Vassalli generated loxP-flanked alleles for each Hcrt receptor, which are manipulated by Cre recombinase to generate mouse lines with disrupted Hcrtrl or Hcrtr2 (or both) in cell type-specific manner. The role of noradrenaline (NA) and dopamine (OA) in ttie regulation of vigilance states is well documented. The purpose of this thesis is to explore the role of the Hcrt input into these two monoaminergic systems. Chronic loss of Hcrtrl in NA neurons consolidated paradoxical sleep (PS), and altered wakefulness brain activity in baseline, during the sleep deprivation (SD), and when mice were challenged by a novel environment, or exposed to nest-building material. The analysis of alterations in the sleep EEG delta power showed a consistent correlation with the changes in the preceding waking quality in these mice. Targeted inactivation of Hcrt input into DA neurons showed that Hcrtr2 inactivation present the strongest phenotype. The loss of Hcrtr2 in DA neurons caused modified brain activities in spontaneous wakefulness, during SD, and in novel environmental conditions. In addition to alteration of wakefulness quality and quantity, conditional inactivation of Hcrtr2 in DA neurons caused an increased in time spent in PS in baseline and a delayed and less complete PS recovery after SD. In the first 30 min of sleep recovery, single (i.e. for Hcrtrl or Hcrtr2) conditional knockout receptor mice had opposite changes in delta activity, including an increased power density in the fast delta range with specific inactivation of Hcrtr2, but a decreased power density in the same range with specific inactivation of Hcrtrl in DA cells. These studies demonstrate a complex impact of Hcrt receptors signaling in both NA and DA system, not only on quantity and quality of wakefulness, but also on PS amount regulation as well as on SWS delta power expression. -- Depuis la découverte des hypocrétines/orexines (Hcrt/Ox) en 1998, plusieurs modèles de souris, narcoleptiques telles que Hcrt-KO, Hcrtr2-KO et récepteurs doubles KO et les souris transgéniques orexine-ataxine ont été générés. Les modèles de souris Hcrt disponibles ne permettaient pas la dissection du rôle spécifique de l'Hcrt dans chaque noyau neuronal cible. Notre laboratoire a généré des allèles loxP pour chacun des 2 gènes codant pour les récepteurs Hcrtr, qui sont manipulés par recombinase Cre pour générer des lignées de souris avec Hcrtrl inactivé, ou Hcrtr2 inactivé, (ou les deux), spécifiquement dans un type cellulaire particulier. Le rôle de la noradrénaline (NA) et la dopamine (DA) dans la régulation des états de vigilance est bien documentée. Le but de cette thèse est d'étudier le rôle de l'afférence Hcrt dans ces deux systèmes monoaminergiques au niveau de l'activité cérébrale telle qu'elle apparaît dans l'électroencéphalogramme (EEG). Mon travail montre que la perte chronique de Hcrtrl dans les neurones NA consolide le sommeil paradoxal (PS), et l'activité cérébrale de l'éveil est modifiée en condition spontanée, au cours d'une experience de privation de sommeil (SD), et lorsque les souris sont présentées à un nouvel environnement, ou exposées à des matériaux de construction du nid. Ces modifications de l'éveil sont corrélées à des modifications de puissance de l'activité delta du sommeil lent qui le suit. L'inactivation ciblée des Hcrtrs dans les neurones DA a montré que l'inactivation Hcrtr2 conduit au phénotype le plus marqué. La perte de Hcrtr2 dans les neurones DA mène à des modification d'activité cérébrale en éveil spontané, pendant SD, ainsi que dans des conditions environnementales nouvelles. En plus de l'altération de la qualité de l'éveil et de la quantité, l'inactivation conditionnelle de Hcrtr2 dans les neurones DA a provoqué une augmentation du temps passé en sommeil paradoxal (PS) en condition de base, et une reprise retardée et moins complète du PS après SD. Dans les 30 premières minutes de la récupération de sommeil, les modèles inactivés pour un seul des récepteurs (ie pour Hcrtrl ou Hcrtr2 seulement) montrent des changements opposés en activité delta, en particulier une densité de puissance accrue dans le delta rapide avec l'inactivation spécifique de Hcrtr2, mais une densité de puissance diminuée dans cette même gamme chez les souris inactivées spécifiquement en Hcrtrl dans les neurones DA. Ces études démontrent un impact complexe de l'inactivation de la neurotransmission au niveau des récepteurs d'Hcrt dans les deux compartiments NA et DA, non seulement sur la quantité et la qualité de l'éveil, mais aussi sur la régulation de quantité de sommeil paradoxal, ainsi que sur l'expression de la puissance delta pendant le sommeil lent.

Complexité, médecine générale et réformes des systèmes de santé [Complexity, general practice and reforms of health systems].

The family doctor facing complexity must decide in situations of low certainty and low agreement. Complexity is in part subjective but can also be measured. Changes in the health systems aim to reduce health costs. They tend to give priority to simple situations and to neglect complexity. One role of an academic institute of family medicine is to present and promote the results of scientific research supporting the principles of family medicine, taking into account both the local context and health systems reforms. In Switzerland the new challenge is the introduction of managed care.

Mutism and Amnesia following High-Voltage Electrical Injury: Psychogenic Symptomatology Triggered by Organic Dysfunction?

Background: Mutism and dense retrograde amnesia are found both in organic and dissociative contexts. Moreover, dissociative symptoms may be modulated by right prefrontal activity. A single case, M.R., developed left hemiparesis, mutism and retrograde amnesia after a high-voltage electric shock without evidence of lasting brain lesions. M.R. suddenly recovered from his mutism following a mild brain trauma 2 years later. Methods: M.R.'s neuropsychological pattern and anatomoclinical correlations were studied through (i) language and memory assessment to characterize his deficits, (ii) functional neuroimaging during a standard language paradigm, and (iii) assessment of frontal and left insular connectivity through diffusion tractography imaging and transcranial magnetic stimulation. A control evaluation was repeated after recovery. Findings: M.R. recovered from the left hemiparesis within 90 days of the accident, which indicated a transient right brain impairment. One year later, neurobehavioral, language and memory evaluations strongly suggested a dissociative component in the mutism and retrograde amnesia. Investigations (including MRI, fMRI, diffusion tensor imaging, EEG and r-TMS) were normal. Twenty-seven months after the electrical injury, M.R. had a very mild head injury which was followed by a rapid recovery of speech. However, the retrograde amnesia persisted. Discussion: This case indicates an interaction of both organic and dissociative mechanisms in order to explain the patient's symptoms. The study also illustrates dissociation in the time course of the two different dissociative symptoms in the same patient.

Method to assess and optimise dependability of complex macro-systems: application to a railway signalling system

Achieving a high degree of dependability in complex macro-systems is challenging. Because of the large number of components and numerous independent teams involved, an overview of the global system performance is usually lacking to support both design and operation adequately. A functional failure mode, effects and criticality analysis (FMECA) approach is proposed to address the dependability optimisation of large and complex systems. The basic inductive model FMECA has been enriched to include considerations such as operational procedures, alarm systems. environmental and human factors, as well as operation in degraded mode. Its implementation on a commercial software tool allows an active linking between the functional layers of the system and facilitates data processing and retrieval, which enables to contribute actively to the system optimisation. The proposed methodology has been applied to optimise dependability in a railway signalling system. Signalling systems are typical example of large complex systems made of multiple hierarchical layers. The proposed approach appears appropriate to assess the global risk- and availability-level of the system as well as to identify its vulnerabilities. This enriched-FMECA approach enables to overcome some of the limitations and pitfalls previously reported with classical FMECA approaches.

Cycle length of spermatogenesis in shrews (mammalia: soricidae) with high and low metabolic rates and different mating systems.

The aim of the present study was to establish and compare the durations of the seminiferous epithelium cycles of the common shrew Sorex araneus, which is characterized by a high metabolic rate and multiple paternity, and the greater white-toothed shrew Crocidura russula, which is characterized by a low metabolic rate and a monogamous mating system. Twelve S. araneus males and fifteen C. russula males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determinations, we applied the classical method of estimation and linear regression as a new method. With regard to variance, and even with a relatively small sample size, the new method seems to be more precise. In addition, the regression method allows the inference of information for every animal tested, enabling comparisons of different factors with cycle lengths. Our results show that not only increased testis size leads to increased sperm production, but it also reduces the duration of spermatogenesis. The calculated cycle lengths were 8.35 days for S. araneus and 12.12 days for C. russula. The data obtained in the present study provide the basis for future investigations into the effects of metabolic rate and mating systems on the speed of spermatogenesis.