PixFRET, an ImageJ plug-in for FRET calculation that can accommodate variations in spectral bleed-throughs.

Fluorescence resonance energy transfer (FRET) allows the user to investigate interactions between fluorescent partners. One crucial issue when calculating sensitized emission FRET is the correction for spectral bleed-throughs (SBTs), which requires to calculate the ratios between the intensities in the FRET and in the donor or acceptor settings, when only the donor or acceptor are present. Theoretically, SBT ratios should be constant. However, experimentally, these ratios can vary as a function of fluorophore intensity, and assuming constant values may hinder precise FRET calculation. One possible cause for such a variation is the use of a microscope set-up with different photomultipliers for the donor and FRET channels, a set-up allowing higher speed acquisitions on very dynamic fluorescent molecules in living cells. Herein, we show that the bias introduced by the differential response of the two PMTs can be circumvented by a simple modeling of the SBT ratios as a function of fluorophore intensity. Another important issue when performing FRET is the localization of FRET within the cell or a population of cells. We hence developed a freely available ImageJ plug-in, called PixFRET, that allows a simple and rapid determination of SBT parameters and the display of normalized FRET images. The usefulness of this modeling and of the plug-in are exemplified by the study of FRET in a system where two interacting nuclear receptors labeled with ECFP and EYFP are coexpressed in living cells.

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

Typing less common ovarian tumors: A training tool based on a pattern-based algorithm applied to a set of 20 virtual slides.

Context: Ovarian tumors (OT) typing is a competency expected from pathologists, with significant clinical implications. OT however come in numerous different types, some rather rare, with the consequence of few opportunities for practice in some departments. Aim: Our aim was to design a tool for pathologists to train in less common OT typing. Method and Results: Representative slides of 20 less common OT were scanned (Nano Zoomer Digital Hamamatsu®) and the diagnostic algorithm proposed by Young and Scully applied to each case (Young RH and Scully RE, Seminars in Diagnostic Pathology 2001, 18: 161-235) to include: recognition of morphological pattern(s); shortlisting of differential diagnosis; proposition of relevant immunohistochemical markers. The next steps of this project will be: evaluation of the tool in several post-graduate training centers in Europe and Québec; improvement of its design based on evaluation results; diffusion to a larger public. Discussion: In clinical medicine, solving many cases is recognized as of utmost importance for a novice to become an expert. This project relies on the virtual slides technology to provide pathologists with a learning tool aimed at increasing their skills in OT typing. After due evaluation, this model might be extended to other uncommon tumors.

Did tool-use evolve with enhanced physical cognitive abilities?

The use and manufacture of tools have been considered to be cognitively demanding and thus a possible driving factor in the evolution of intelligence. In this study, we tested the hypothesis that enhanced physical cognitive abilities evolved in conjunction with the use of tools, by comparing the performance of naturally tool-using and non-tool-using species in a suite of physical and general learning tasks. We predicted that the habitually tool-using species, New Caledonian crows and Galápagos woodpecker finches, should outperform their non-tool-using relatives, the small tree finches and the carrion crows in a physical problem but not in general learning tasks. We only found a divergence in the predicted direction for corvids. That only one of our comparisons supports the predictions under this hypothesis might be attributable to different complexities of tool-use in the two tool-using species. A critical evaluation is offered of the conceptual and methodological problems inherent in comparative studies on tool-related cognitive abilities.

Erythropoietin (EPO) immunoaffinity columns--a powerful tool for purifying EPO and its recombinant analogues.

The sample preparation method preceding the urinary erythropoietin (EPO) doping test is based on several concentration and ultrafiltration steps. In order to improve the quality of isoelectric focusing (IEF) gel results and therefore, the sensitivity of the EPO test, new sample preparation methods relying on affinity purification were recently proposed. This article focuses on the evaluation and validation of disposable immunoaffinity columns targeting both endogenous and recombinant EPO molecules in two World Anti-Doping Agency (WADA) accredited anti-doping laboratories. The use of the columns improved the resolution of the IEF profiles considerably when compared with the classical ultrafiltration method, and the columns' ability to ensure the isoform integrity of the endogenous and exogenous EPO molecules was confirmed. Immunoaffinity columns constitute therefore a potent and reliable tool for the preparation of urine samples and their use will significantly improve the sensitivity and specificity of the actual urinary EPO test.

V3 net charge: additional tool in HIV-1 tropism prediction.

Genotype-based algorithms are valuable tools for the identification of patients eligible for CCR5 inhibitors administration in clinical practice. Among the available methods, geno2pheno[coreceptor] (G2P) is the most used online tool for tropism prediction. This study was conceived to assess if the combination of G2P prediction with V3 peptide net charge (NC) value could improve the accuracy of tropism prediction. A total of 172 V3 bulk sequences from 143 patients were analyzed by G2P and NC values. A phenotypic assay was performed by cloning the complete env gene and tropism determination was assessed on U87_CCR5(+)/CXCR4(+) cells. Sequences were stratified according to the agreement between NC values and G2P results. Of sequences predicted as X4 by G2P, 61% showed NC values higher than 5; similarly, 76% of sequences predicted as R5 by G2P had NC values below 4. Sequences with NC values between 4 and 5 were associated with different G2P predictions: 65% of samples were predicted as R5-tropic and 35% of sequences as X4-tropic. Sequences identified as X4 by NC value had at least one positive residue at positions known to be involved in tropism prediction and positive residues in position 32. These data supported the hypothesis that NC values between 4 and 5 could be associated with the presence of dual/mixed-tropic (DM) variants. The phenotypic assay performed on a subset of sequences confirmed the tropism prediction for concordant sequences and showed that NC values between 4 and 5 are associated with DM tropism. These results suggest that the combination of G2P and NC could increase the accuracy of tropism prediction. A more reliable identification of X4 variants would be useful for better selecting candidates for Maraviroc (MVC) administration, but also as a predictive marker in coreceptor switching, strongly associated with the phase of infection.

Myocardial MIBG scintigraphy: a useful clinical tool? : A retrospective study in 50 parkinsonian patients.

Meta-iodbenzylguanidine scintigraphy (MIBG scintigraphy) shows reduced uptake in idiopathic Parkinson's disease (IPD), idiopathic REM sleep behavior disorder (IRBD) and Lewy body dementia (LBD), but not in other parkinsonian or dementia syndromes. We retrospectively reevaluated 50 patients. Concordance rate between last clinical diagnosis and scintigraphy diagnosis was only given in two-thirds of the patients. Confounding factors were: decreasing heart/mediastinum ratio (HMR) with progressive age, higher HMR in women and possibly interference with antihypertensive medication. Standardization of the methods and precise clinical guidelines are warranted for better clinical use.

Probabilistic evidential assessment of gunshot residue particle evidence (Part I): likelihood ratio calculation and case pre-assessment using Bayesian networks

Well developed experimental procedures currently exist for retrieving and analyzing particle evidence from hands of individuals suspected of being associated with the discharge of a firearm. Although analytical approaches (e.g. automated Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDS) microanalysis) allow the determination of the presence of elements typically found in gunshot residue (GSR) particles, such analyses provide no information about a given particle's actual source. Possible origins for which scientists may need to account for are a primary exposure to the discharge of a firearm or a secondary transfer due to a contaminated environment. In order to approach such sources of uncertainty in the context of evidential assessment, this paper studies the construction and practical implementation of graphical probability models (i.e. Bayesian networks). These can assist forensic scientists in making the issue tractable within a probabilistic perspective. The proposed models focus on likelihood ratio calculations at various levels of detail as well as case pre-assessment.

Micro-ordinateurs de poche : du gadget à l'irremplaçable [Handheld computers: the gadget that became an invaluable tool]

Les ordinateurs de poche ont le potentiel de devenir un outil de travail indispensable pour le médecin praticien. De part une facilité d'utilisation toujours plus grande, un format ultra-portable, une grande capacité de stockage de l'information et de synchronisation à de multiples bases de données, ils permettent l'accès en tout temps et littéralement au lit du malade à une gamme complète de ressources médicales. Cet article se veut une lllustration des différentes possibilités d'utilisation de ces nouveaux outils, au travers de situations cliniques banales rencontrées par tout praticien.

Cardiovascular Risk Prediction with Ultrasound

This paper addresses primary care physicians, cardiologists, internists, angiologists and doctors desirous of improving vascular risk prediction in primary care. Many cardiovascular risk factors act aggressively on the arterial wall and result in atherosclerosis and atherothrombosis. Cardiovascular prognosis derived from ultrasound imaging is, however, excellent in subjects without formation of intimal thickening or atheromas. Since ultrasound visualises the arterial wall directly, the information derived from the arterial wall may add independent incremental information to the knowledge of risk derived from global risk assessment. This paper provides an overview on plaque imaging for vascular risk prediction in two parts: Part 1: Carotid IMT is frequently used as a surrogate marker for outcome in intervention studies addressing rather large cohorts of subjects. Carotid IMT as a risk prediction tool for the prevention of acute myocardial infarction and stroke has been extensively studied in many patients since 1987, and has yielded incremental hazard ratios for these cardiovascular events independently of established cardiovascular risk factors. However, carotid IMT measurements are not used uniformly and therefore still lack widely accepted standardisation. Hence, at an individual, practicebased level, carotid IMT is not recommended as a risk assessment tool. The total plaque area of the carotid arteries (TPA) is a measure of the global plaque burden within both carotid arteries. It was recently shown in a large Norwegian cohort involving over 6000 subjects that TPA is a very good predictor for future myocardial infarction in women with an area under the curve (AUC) using a receiver operating curves (ROC) value of 0.73 (in men: 0.63). Further, the AUC for risk prediction is high both for vascular death in a vascular prevention clinic group (AUC 0.77) and fatal or nonfatal myocardial infarction in a true primary care group (AUC 0.79). Since TPA has acceptable reproducibility, allows calculation of posttest risk and is easily obtained at low cost, this risk assessment tool may come in for more widespread use in the future and also serve as a tool for atherosclerosis tracking and guidance for intensity of preventive therapy. However, more studies with TPA are needed. Part 2: Carotid and femoral plaque formation as detected by ultrasound offers a global view of the extent of atherosclerosis. Several prospective cohort studies have shown that cardiovascular risk prediction is greater for plaques than for carotid IMT. The number of arterial beds affected by significant atheromas may simply be added numerically to derive additional information on the risk of vascular events. A new atherosclerosis burden score (ABS) simply calculates the sum of carotid and femoral plaques encountered during ultrasound scanning. ABS correlates well and independently with the presence of coronary atherosclerosis and stenosis as measured by invasive coronary angiogram. However, the prognostic power of ABS as an independent marker of risk still needs to be elucidated in prospective studies. In summary, the large number of ways to measure atherosclerosis and related changes in human arteries by ultrasound indicates that this technology is not yet sufficiently perfected and needs more standardisation and workup on clearly defined outcome studies before it can be recommended as a practice-based additional risk modifier.

The Alcohol Use Disorders Identification Test (AUDIT) as a screening tool for excessive drinking in primary care: reliability and validity of a French version.

BACKGROUND: Excessive drinking is a major problem in Western countries. AUDIT (Alcohol Use Disorders Identification Test) is a 10-item questionnaire developed as a transcultural screening tool to detect excessive alcohol consumption and dependence in primary health care settings. OBJECTIVES: The aim of the study is to validate a French version of the Alcohol Use Disorders Identification Test (AUDIT). METHODS: We conducted a validation cross-sectional study in three French-speaking areas (Paris, Geneva and Lausanne). We examined psychometric properties of AUDIT as its internal consistency, and its capacity to correctly diagnose alcohol abuse or dependence as defined by DSM-IV and to detect hazardous drinking (defined as alcohol intake >30 g pure ethanol per day for men and >20 g of pure ethanol per day for women). We calculated sensitivity, specificity, positive and negative predictive values and Receiver Operator Characteristic curves. Finally, we compared the ability of AUDIT to accurately detect "alcohol abuse/dependence" with that of CAGE and MAST. RESULTS: 1207 patients presenting to outpatient clinics (Switzerland, n = 580) or general practitioners' (France, n = 627) successively completed CAGE, MAST and AUDIT self-administered questionnaires, and were independently interviewed by a trained addiction specialist. AUDIT showed a good capacity to discriminate dependent patients (with AUDIT > or =13 for males, sensitivity 70.1%, specificity 95.2%, PPV 85.7%, NPV 94.7% and for females sensitivity 94.7%, specificity 98.2%, PPV 100%, NPV 99.8%); and hazardous drinkers (with AUDIT > or =7, for males sensitivity 83.5%, specificity 79.9%, PPV 55.0%, NPV 82.7% and with AUDIT > or =6 for females, sensitivity 81.2%, specificity 93.7%, PPV 64.0%, NPV 72.0%). AUDIT gives better results than MAST and CAGE for detecting "Alcohol abuse/dependence" as showed on the comparative ROC curves. CONCLUSIONS: The AUDIT questionnaire remains a good screening instrument for French-speaking primary care.