Evidence for different expression profiles for c-Met, EGFR, PTEN and the mTOR pathway in low and high grade endometrial carcinomas in a cohort of consecutive women. Occurrence of PIK3CA and K-Ras mutations and microsatellite instability.

Molecular and genetic investigations in endometrial carcinogenesis may have prognostic and therapeutic implications. We studied the expression of EGFR, c-Met, PTEN and the mTOR signalling pathway (phospho-AKT/phospho-mTOR/phospho-RPS6) in 69 consecutive tumours and 16 tissue microarrays. We also analysed PIK3CA, K-Ras mutations and microsatellite instability (MSI). We distinguished two groups: group 1 (grade 1 and 2 endometrioid cancers) and group 2 (grade 3 endometrioid and type II clear and serous cell cancers). We hypothesised that these histological groups might have different features. We found that a) survival was higher in group 1 with less aggressive tumours (P⟨0.03); b) EGFR (P=0.01), PTEN and the AKT/mTOR/RPS6 signalling pathway were increased in group 1 versus group 2 (P=0.05 for phospho-mTOR); c) conversely, c-Met was higher (P⟨0.03) in group 2 than in group 1; d) In group 1, EGFR was correlated with c-Met, phospho-mTOR, phospho-RPS6 and the global activity of the phospho-AKT/phospho-mTOR/phospho-RPS6 pathway. In group 2, EGFR was correlated only with the phospho-AKT/phospho-mTOR/phospho-RPS6 pathway, whereas c-Met was correlated with PTEN; e) survival was higher for tumours with more than 50% PTEN-positive cells; f) K-RAS and PIK3CA mutations occurred in 10-12% of the available tumours and MSI in 40.4%, with a loss of MLH1 and PMS2 expression. Our results for endometrial cancers provide the first evidence for a difference in status between groups 1 and 2. The patients may benefit from different targeted treatments, anti-EGFR agents and rapamycin derivatives (anti-mTOR) for group 1 and an anti c-MET/ligand complex for group 2.

Linear random knots and their scaling behavior

We present here a nonbiased probabilistic method that allows us to consistently analyze knottedness of linear random walks with up to several hundred noncorrelated steps. The method consists of analyzing the spectrum of knots formed by multiple closures of the same open walk through random points on a sphere enclosing the walk. Knottedness of individual "frozen" configurations of linear chains is therefore defined by a characteristic spectrum of realizable knots. We show that in the great majority of cases this method clearly defines the dominant knot type of a walk, i.e., the strongest component of the spectrum. In such cases, direct end-to-end closure creates a knot that usually coincides with the knot type that dominates the random closure spectrum. Interestingly, in a very small proportion of linear random walks, the knot type is not clearly defined. Such walks can be considered as residing in a border zone of the configuration space of two or more knot types. We also characterize the scaling behavior of linear random knots.

Comparison between a linear ion trap and a triple quadruple MS in the sensitive detection of large peptides at femtomole amounts on column.

In addition to the importance of sample preparation and extract separation, MS detection is a key factor in the sensitive quantification of large undigested peptides. In this article, a linear ion trap MS (LIT-MS) and a triple quadrupole MS (TQ-MS) have been compared in the detection of large peptides at subnanomolar concentrations. Natural brain natriuretic peptide, C-peptide, substance P and D-Junk-inhibitor peptide, a full D-amino acid therapeutic peptide, were chosen. They were detected by ESI and simultaneous MS(1) and MS(2) acquisitions. With direct peptide infusion, MS(2) spectra revealed that fragmentation was peptide dependent, milder on the LIT-MS and required high collision energies on the TQ-MS to obtain high-intensity product ions. Peptide adsorption on surfaces was overcome and peptide dilutions ranging from 0.1 to 25 nM were injected onto an ultra high-pressure LC system with a 1 mm id analytical column and coupled with the MS instruments. No difference was observed between the two instruments when recording in LC-MS(1) acquisitions. However, in LC-MS(2) acquisitions, a better sensitivity in the detection of large peptides was observed with the LIT-MS. Indeed, with the three longer peptides, the typical fragmentation in the TQ-MS resulted in a dramatic loss of sensitivity (> or = 10x).

A general linear relaxometry model of R1 using imaging data.

PURPOSE: The longitudinal relaxation rate (R1 ) measured in vivo depends on the local microstructural properties of the tissue, such as macromolecular, iron, and water content. Here, we use whole brain multiparametric in vivo data and a general linear relaxometry model to describe the dependence of R1 on these components. We explore a) the validity of having a single fixed set of model coefficients for the whole brain and b) the stability of the model coefficients in a large cohort. METHODS: Maps of magnetization transfer (MT) and effective transverse relaxation rate (R2 *) were used as surrogates for macromolecular and iron content, respectively. Spatial variations in these parameters reflected variations in underlying tissue microstructure. A linear model was applied to the whole brain, including gray/white matter and deep brain structures, to determine the global model coefficients. Synthetic R1 values were then calculated using these coefficients and compared with the measured R1 maps. RESULTS: The model's validity was demonstrated by correspondence between the synthetic and measured R1 values and by high stability of the model coefficients across a large cohort. CONCLUSION: A single set of global coefficients can be used to relate R1 , MT, and R2 * across the whole brain. Our population study demonstrates the robustness and stability of the model. Magn Reson Med, 2014. © 2014 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. Magn Reson Med 73:1309-1314, 2015. © 2014 Wiley Periodicals, Inc.

The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H(2) receptor.

In previous studies we showed that the wild-type histamine H(2) receptor stably expressed in Chinese hamster ovary cells is constitutively active. Because constitutive activity of the H(2) receptor is already found at low expression levels (300 fmol/mg protein) this receptor is a relatively unique member of the G-protein-coupled receptor (GPCR) family and a useful tool for studying GPCR activation. In this study the role of the highly conserved DRY motif in activation of the H(2) receptor was investigated. Mutation of the aspartate 115 residue in this motif resulted in H(2) receptors with high constitutive activity, increased agonist affinity, and increased signaling properties. In addition, the mutant receptors were shown to be highly structurally instable. Mutation of the arginine 116 residue in the DRY motif resulted also in a highly structurally instable receptor; expression of the receptor could only be detected after stabilization with either an agonist or inverse agonist. Moreover, the agonist affinity at the Arg-116 mutant receptors was increased, whereas the signal transduction properties of these receptors were decreased. We conclude that the Arg-116 mutant receptors can adopt an active conformation but have a decreased ability to couple to or activate the G(s)-protein. This study examines the pivotal role of the aspartate and arginine residues of the DRY motif in GPCR function. Disruption of receptor stabilizing constraints by mutation in the DRY motif leads to the formation of active GPCR conformations, but concomitantly to GPCR instability.

Direct numerical simulations of interface dynamics to link capillary pressure and total surface energy

The flow of two immiscible fluids through a porous medium depends on the complex interplay between gravity, capillarity, and viscous forces. The interaction between these forces and the geometry of the medium gives rise to a variety of complex flow regimes that are difficult to describe using continuum models. Although a number of pore-scale models have been employed, a careful investigation of the macroscopic effects of pore-scale processes requires methods based on conservation principles in order to reduce the number of modeling assumptions. In this work we perform direct numerical simulations of drainage by solving Navier-Stokes equations in the pore space and employing the Volume Of Fluid (VOF) method to track the evolution of the fluid-fluid interface. After demonstrating that the method is able to deal with large viscosity contrasts and model the transition from stable flow to viscous fingering, we focus on the macroscopic capillary pressure and we compare different definitions of this quantity under quasi-static and dynamic conditions. We show that the difference between the intrinsic phase-average pressures, which is commonly used as definition of Darcy-scale capillary pressure, is subject to several limitations and it is not accurate in presence of viscous effects or trapping. In contrast, a definition based on the variation of the total surface energy provides an accurate estimate of the macroscopic capillary pressure. This definition, which links the capillary pressure to its physical origin, allows a better separation of viscous effects and does not depend on the presence of trapped fluid clusters.

Gravity-driven slug motion in capillary tubes

The velocity of a liquid slug falling in a capillary tube is lower than predicted for Poiseuille flow due to presence of menisci, whose shapes are determined by the complex interplay of capillary, viscous, and gravitational forces. Due to the presence of menisci, a capillary pressure proportional to surface curvature acts on the slug and streamlines are bent close to the interface, resulting in enhanced viscous dissipation at the wedges. To determine the origin of drag-force increase relative to Poiseuille flow, we compute the force resultant acting on the slug by integrating Navier-Stokes equations over the liquid volume. Invoking relationships from differential geometry we demonstrate that the additional drag is due to viscous forces only and that no capillary drag of hydrodynamic origin exists (i.e., due to hydrodynamic deformation of the interface). Requiring that the force resultant is zero, we derive scaling laws for the steady velocity in the limit of small capillary numbers by estimating the leading order viscous dissipation in the different regions of the slug (i.e., the unperturbed Poiseuille-like bulk, the static menisci close to the tube axis and the dynamic regions close to the contact lines). Considering both partial and complete wetting, we find that the relationship between dimensionless velocity and weight is, in general, nonlinear. Whereas the relationship obtained for complete-wetting conditions is found in agreement with the experimental data of Bico and Quere [J. Bico and D. Quere, J. Colloid Interface Sci. 243, 262 (2001)], the scaling law under partial-wetting conditions is validated by numerical simulations performed with the Volume of Fluid method. The simulated steady velocities agree with the behavior predicted by the theoretical scaling laws in presence and in absence of static contact angle hysteresis. The numerical simulations suggest that wedge-flow dissipation alone cannot account for the entire additional drag and that the non-Poiseuille dissipation in the static menisci (not considered in previous studies) has to be considered for large contact angles.

A non-linear Bayesian approach for upscaling local-scale hydraulic conductivity measurements based on local- and regional-scale geophysical data

Significant progress has been made with regard to the quantitative integration of geophysical and hydrological data at the local scale. However, extending the corresponding approaches to the regional scale represents a major, and as-of-yet largely unresolved, challenge. To address this problem, we have developed an upscaling procedure based on a Bayesian sequential simulation approach. This method is then applied to the stochastic integration of low-resolution, regional-scale electrical resistivity tomography (ERT) data in combination with high-resolution, local-scale downhole measurements of the hydraulic and electrical conductivities. Finally, the overall viability of this upscaling approach is tested and verified by performing and comparing flow and transport simulation through the original and the upscaled hydraulic conductivity fields. Our results indicate that the proposed procedure does indeed allow for obtaining remarkably faithful estimates of the regional-scale hydraulic conductivity structure and correspondingly reliable predictions of the transport characteristics over relatively long distances.

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry.

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.

Analysis of Markers for Recent Alcohol Consumption by Capillary Zone Electrophoresis and an Immunochemical Method

Le but de ce travail doctoral était le développement de méthodes analytiques pour la détermination dethyl glucuronide et dethyl sulfate. Ces deux substances sont des métabolites directs de lethanol qui peuvent être détectées pendant des heures jusqu'à des jours dans des fluides corporels, après que léthanol ait été complètement éliminé du corps humain. Ce sont donc des marqueurs de consommation récente d'alcool.La majorité des expériences ont été effectuées en utilisant l'électrophorèse capillaire. Il était envisagé de fournir des méthodes utilisables dans des laboratoires de routine. Des méthodes électrophorétiques ont été développées et optimisées pour la détermination dethyl sulfate dans le sérum et l'urine ainsi que pour lethyl glucuronide dans le sérum. Lethyl glucuronide urinaire a pu être déterminé par un immunoassay commerciale qui a en plus été adapté avec succès pour des échantillons de sérum. Avec toutes ces méthodes d'analyse il était possible d'observer les deux marqueurs de consommation d'alcool récente, même une consommation aussi basse qu'un verre de boissons alcooliques.Finalement, une étude englobant plus de 100 échantillons aété effectuée avec l'ambition de déterminer les valeurs de référence pour lethyl glucuronide dans le sérum et l'urine. De plus, la nécessité de normaliser les échantillons d'urine par rapport à la dilution a été investiguée. Grâce à cette étude des valeurs de cut-off et une base statistique pour l'interprétation probabiliste ont pu être proposées.

Outcome of a Modified Broström-Gould Procedure for Lateral Ankle Instability

Introduction: Ankle sprains affect 200'000 persons/year in Switzerland. Most incidences are successfully treated by conservative measures but 20% require reconstruction for symptomatic chronic lateral ankle instability. This study evaluates the functional outcome after a modified Broström-Gould technique as measured by different clinical scores and compares the functional outcome of this technique with other surgical treatments of ankle instability. Methods: This retrospective cohort study evaluates 47 patients who underwent a modified Broström-Gould procedure using suture anchors to refix the lateral ankle capsuloligamentary structures at our institution from 2005 to 2009 with a minimum follow-up of one year (13-72 Mo). All patients were operated by one single surgeon and evaluated by an independent examiner. The function was assessed using 4 scores including: the AOFAS (American Orthopaedic Foot and Ankle Society's Score) hindfoot score; the FAAM (Foot and Ankle Ability Measurement); the CAIT (Cumberland Ankle Instability Tool); the CAIS (Chronic Ankle Instability Scale). Results: Six patients were excluded leaving 41 patients for examination. 34 patients (83%) thought that their ankle was more stable after the surgery, 7 (17%) did not feel any difference. 27 patients were very satisfied, 11 satisfied and 3 not satisfied. Reasons for non satisfaction included persistent instability and pain. Ankle mobility returned to normal in 93% of patients. Five patients had transcient hypoesthesy in the area of the superficial peroneal nerve. One patient suffered from a superficial infection treated successfully by local measures. 80% had the perception of a normal ankle, 20% thought to be below normal. At follow-up the AOFAS was 89/100 (37-100), the FAAM 85/100% (35-100%), the CAIT 20/30 (5-30), and the CAIS 74/100% (27-100%). Conclusions: The modified Broström-Gould procedure, which belongs to the anatomic ankle stabilizations is relatively simple and offers good outcome that satisfied 93% of the patients in the present study. No active stabilisator is sacrificed. Preservation of the ankle mobility is better and the complication rate is lower than after non-anatomical procedures described in the literature. The CAIT appeared as the most severe score compared to the other scales used in our study.

Comparison of microsatellite instability and chromosomal instability in predicting survival of patients with T3N0 colorectal cancer.

BACKGROUND: At least 2 apparently independent mechanisms, microsatellite instability (MSI) and chromosomal instability, are implicated in colorectal tumorigenesis. Their respective roles in predicting clinical outcomes of patients with T3N0 colorectal cancer remain unknown. METHODS: Eighty-eight patients with a sporadic T3N0 colon or rectal adenocarcinoma were followed up for a median of 67 months. For chromosomal instability analysis, Ki-ras mutations were determined by single-strand polymerase chain reaction, and p53 protein staining was studied by immunohistochemistry. For MSI analysis, DNA was amplified by polymerase chain reaction at 7 microsatellite targets (BAT25, BAT26, D17S250, D2S123, D5S346, transforming growth factor receptor II, and BAX). RESULTS: Overall 5-year survival rate was 72%. p53 protein nuclear staining was detected in 39 patients (44%), and MSI was detected in 21 patients (24%). MSI correlated with proximal location (P <.001) and mucinous content (P <.001). In a multivariate analysis, p53 protein expression carried a significant risk of death (relative risk = 4.0, 95% CI = 1.6 to 10.1, P =.004). By comparison, MSI was not a statistically significant prognostic factor for survival in this group (relative risk = 2.2, 95% CI = 0.6 to 7.3, P =.21). CONCLUSIONS: p53 protein overexpression provides better prognostic discrimination than MSI in predicting survival of patients with T3N0 colorectal cancer. Although MSI is associated with specific clinicopathologic parameters, it did not predict overall survival in this group. Assessment of p53 protein expression by immunocytochemistry provides a simple means to identify a subset of T3N0 patients with a 4-times increased risk for death.