Rapid LC-MS/MS quantification of the major benzodiazepines and their metabolites on dried blood spots using a simple and cost-effective sample pretreatment.

BACKGROUND: Dried blood spots (DBS) sampling has gained popularity in the bioanalytical community as an alternative to conventional plasma sampling, as it provides numerous benefits in terms of sample collection and logistics. The aim of this work was to show that these advantages can be coupled with a simple and cost-effective sample pretreatment, with subsequent rapid LC-MS/MS analysis for quantitation of 15 benzodiazepines, six metabolites and three Z-drugs. For this purpose, a simplified offline procedure was developed that consisted of letting a 5-µl DBS infuse directly into 100 µl of MeOH, in a conventional LC vial. RESULTS: The parameters related to the DBS pretreatment, such as extraction time or internal standard addition, were investigated and optimized, demonstrating that passive infusion in a regular LC vial was sufficient to quantitatively extract the analytes of interest. The method was validated according to international criteria in the therapeutic concentration ranges of the selected compounds. CONCLUSION: The presented strategy proved to be efficient for the rapid analysis of the selected drugs. Indeed, the offline sample preparation was reduced to a minimum, using a small amount of organic solvent and consumables, without affecting the accuracy of the method. Thus, this approach enables simple and rapid DBS analysis, even when using a non-DBS-dedicated autosampler, while lowering the costs and environmental impact.

Quantification of Myocardial Blood Flow in Absolute Terms Using (82)Rb PET Imaging: The RUBY-10 Study.

OBJECTIVES: The purpose of this study was to compare myocardial blood flow (MBF) and myocardial flow reserve (MFR) estimates from rubidium-82 positron emission tomography ((82)Rb PET) data using 10 software packages (SPs) based on 8 tracer kinetic models. BACKGROUND: It is unknown how MBF and MFR values from existing SPs agree for (82)Rb PET. METHODS: Rest and stress (82)Rb PET scans of 48 patients with suspected or known coronary artery disease were analyzed in 10 centers. Each center used 1 of 10 SPs to analyze global and regional MBF using the different kinetic models implemented. Values were considered to agree if they simultaneously had an intraclass correlation coefficient >0.75 and a difference <20% of the median across all programs. RESULTS: The most common model evaluated was the Ottawa Heart Institute 1-tissue compartment model (OHI-1-TCM). MBF values from 7 of 8 SPs implementing this model agreed best. Values from 2 other models (alternative 1-TCM and Axially distributed) also agreed well, with occasional differences. The MBF results from other models (e.g., 2-TCM and retention) were less in agreement with values from OHI-1-TCM. CONCLUSIONS: SPs using the most common kinetic model-OHI-1-TCM-provided consistent results in measuring global and regional MBF values, suggesting that they may be used interchangeably to process data acquired with a common imaging protocol.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

Lentiviral vectors efficiently transduce the EGFP gene into cardiomyocytes in vivo : immunohistology and Elisa quantification of the transgene

RESUME Les maladies cardio-vasculaires représentent la cause la plus importante de mortalité et de morbidité dans les pays occidentaux. La thérapie génique offre une nouvelle approche au traitement de ces maladies. L'expression de gènes protecteurs dans le myocarde par des technologies de transfert génique peut améliorer la fonction ventriculaire lors de l'insuffisance cardiaque ou stimuler la formation de nouveaux vaisseaux dans la maladie coronarienne. Etant donné qu'une majorité des maladies cardiaques sont des maladies chroniques, l'expression durable du gène thérapeutique introduit dans le coeur est souhaitable dans de nombreux cas. Malheureusement, l'utilité des vecteurs de transfert génique les plus utilisés en thérapie génique cardiovasculaire est limitée par une performance faible (ADN plasmidique) et une courte durée d'expression (adénovirus). Récemment, des vecteurs de transfert génique dérivés des lentivirus, une sous-famille des rétrovirus, ont retenu l'attention de la communauté scientifique en raison de leur capacité à exprimer des gènes à long terme. Contrairement aux vecteurs rétroviraux traditionnels, les vecteurs lentiviraux transduisent des gènes même dans des cellules qui ne se divisent pas, ce qui est le cas des cardiomyocytes adultes. Ces vecteurs présentent un profil de biosécurité comparable à celui des vecteurs rétroviraux traditionnels. Nous avons donc décidé de tester l'utilité des vecteurs lentiviraux pour le transfert génique dans des cardiomyocytes de rat adulte in vitro et in vivo. Plusieurs versions de vecteurs lentiviraux contenant différent promoteurs ont été construites. Ces vecteurs contenant le gène marqueur EGFP (enhanced green fluorescent protein) ont été testés dans des cardiomyocytes de rat in vitro, ainsi que dans des coeurs de rat in vivo. Le but de ces expériences était de déterminer la durée de l'expression du transgène après injection intramyocardique chez le rat. Pour ce faire, nous avons développé une technique ELISA pour détecter la protéine EGFP dans des extraits de tissu cardiaque. Les résultats ont montré que la protéine EGFP était encore présente à des niveaux significatifs jusqu'à dix semaines après l'injection de vecteurs lentiviraux, alors que l'expression transgénique obtenue avec un vecteur adénoviral traditionnel a été plus limitée dans le temps. Ces résultats démontrent la capacité des vecteurs lentiviraux à exprimer des gènes d'intérêt de manière performante et stable dans le cœur de rat adulte in vivo. SUMMARY Cardiovascular diseases are the first cause of morbidity and mortality in Western countries. Gene therapy offers a new approach to these diseases. Expression of therapeutic genes in the myocardium by gene transfer technologies can improve ventricular function in heart failure and stimulate neovascularization in coronary disease. Chronic heart diseases likely require sustained expression of the therapeutic gene within the heart itself. Unfortunately, the most commonly used vectors in cardiovascular gene therapy, i.e. plasmid DNA and recombinant adenovirus vectors, are limited by poor DNA uptake and transient transgene expression, respectively. Recently, lentivirus-derived vectors have attracted much interest because of their ability to achieve long-term transgene expression. In contrast to traditional retroviral vectors, lentiviral vectors are also able to transduce non- dividing cells, while presenting a comparable biosafety profile. Adult cardiomyocytes are terminally differentiated cells that do not divide under normal conditions. For these reasons, we have decided to evaluate the efficiency of lentiviral vectors for gene-transduction of adult cardiomyocytes both in vitro and in vivo. We constructed various types of lentiviral vectors containing various promoters. Vectors encoding EGFP as a reporter gene were tested in rat cardiomyocytes in vitro and in rat hearts in vivo. The aim of the experiments involved in this thesis work was to determine the duration of the expression of the transgene after rat intramyocardial injection using a quantitative assay. Therefore, an ELISA technique was set up to measure the EGFP protein in rat heart tissue extracts. Our results showed that the EGFP protein was still present at significant levels at ten weeks after lentiviral vector injection, whereas the duration of expression with adenoviral vectors was shorter. These results demonstrate that lentiviral vectors efficiently deliver genes and achieve sustained transgene expression in adult rat cardiomyocytes in vivo.

Quantification of the relation between surface morphodynamics and subsurface sedimentological product in sandy braided rivers

This study uses digital elevation models and ground-penetrating radar to quantify the relation between the surface morphodynamics and subsurface sedimentology in the sandy braided South Saskatchewan River, Canada. A unique aspect of the methodology is that both digital elevation model and ground-penetrating radar data were collected from the same locations in 2004, 2005, 2006 and 2007, thus enabling the surface morphodynamics to be tied explicitly to the associated evolving depositional product. The occurrence of a large flood in 2005 also allowed the influence of discharge to be assessed with respect to the processproduct relationship. The data demonstrate that the morphology of the study reach evolved even during modest discharges, but more extensive erosion was caused by the large flood. In addition, the study reach was dominated by compound bars before the flood, but switched to being dominated by unit bars during and after the flood. The extent to which the subsurface deposits (the product') were modified by the surface morphodynamics (the process') was quantified using the changes in radar-facies recorded in sequential ground-penetrating radar surveys. These surveys reveal that during the large flood there was an increase in the proportion of facies associated with bar margin accretion and larger dunes. In subsequent years, these facies became truncated and replaced with facies associated with smaller dune sets. This analysis shows that unit bars generally become truncated more laterally than vertically and, thus, they lose the high-angle bar margin deposits and smaller scale bar-top deposits. In general, the only fragments that remain of the unit bars are dune sets, thus making identification of the original unit barform problematic. This novel data set has implications for what may ultimately become preserved in the rock record.

Quantification of environment-driven changes in epiphytic macroinvertebrate communities associated to Phragmites australis

The epiphytic macroinvertebrate communities associated with the Common Reed, Phragmites australis (Cav.) Trin. ex Steudel, were examined seasonally from summer 2004 to spring 2005 in eleven coastal lagoons of the Llobregat Delta (NE Spain) following the method proposed by Kornijów & Kairesalo (1994). The aims of the study were to: 1) characterise and quantify changes in epiphytic macroinvertebrate communities along environmental gradients; 2) assess the contribution of elements of the epiphytic compartment to structuring the community; 3) define the optima and tolerances of selected epiphytic macroinvertebrate taxa for the most relevant ecological factors responsible for assemblage composition; and 4) identify possible epiphytic species assemblages that would allow a lagoon"s typology to be established, as well as their representative indicator species. Communities showed statistically significant seasonal variation, with two faunal peaks: one in summer, with high chironomid densities, and the other in winter, with high naidid densities. These peaks showed a clear response to the influence of environmental factors. Salinity explained the highest percentage of total variance (36%), while trophic variables (nutrients, phytoplanktonic chlorophyll-a, and total organic carbon) and epiphyton biomass (19.2 and 4% of total variance explained, respectively) were secondary. Three different epiphytic macroinvertebrate species assemblages could be defined. These assemblages were directly linked to conductivity conditions, which determined the rate of survival of certain taxa, and to the existence of a direct connection with the sea, which permitted the establishment of 'brackish-water' species. In spite of the existence of these species assemblages, the species composition and biomass of epiphytic macroinvertebrates and epiphyton differed substantially between lagoons; both elements were subject to changes in the environment, which finally determined the site-to-site variation in the density and composition of the macroinvertebrate population

Multicontrast MRI Quantification of Focal Inflammation and Degeneration in Multiple Sclerosis.

INTRODUCTION: Local microstructural pathology in multiple sclerosis patients might influence their clinical performance. This study applied multicontrast MRI to quantify inflammation and neurodegeneration in MS lesions. We explored the impact of MRI-based lesion pathology in cognition and disability. METHODS: 36 relapsing-remitting MS subjects and 18 healthy controls underwent neurological, cognitive, behavioural examinations and 3 T MRI including (i) fluid attenuated inversion recovery, double inversion recovery, and magnetization-prepared gradient echo for lesion count; (ii) T1, T2, and T2(*) relaxometry and magnetisation transfer imaging for lesion tissue characterization. Lesions were classified according to the extent of inflammation/neurodegeneration. A generalized linear model assessed the contribution of lesion groups to clinical performances. RESULTS: Four lesion groups were identified and characterized by (1) absence of significant alterations, (2) prevalent inflammation, (3) concomitant inflammation and microdegeneration, and (4) prevalent tissue loss. Groups 1, 3, 4 correlated with general disability (Adj-R (2) = 0.6; P = 0.0005), executive function (Adj-R (2) = 0.5; P = 0.004), verbal memory (Adj-R (2) = 0.4; P = 0.02), and attention (Adj-R (2) = 0.5; P = 0.002). CONCLUSION: Multicontrast MRI provides a new approach to infer in vivo histopathology of plaques. Our results support evidence that neurodegeneration is the major determinant of patients' disability and cognitive dysfunction.

Responding to Young People's Health Risks in Primary Care: A Cluster Randomised Trial of Training Clinicians in Screening and Motivational Interviewing.

OBJECTIVE: To evaluate the effectiveness of a complex intervention implementing best practice guidelines recommending clinicians screen and counsel young people across multiple psychosocial risk factors, on clinicians' detection of health risks and patients' risk taking behaviour, compared to a didactic seminar on young people's health. DESIGN: Pragmatic cluster randomised trial where volunteer general practices were stratified by postcode advantage or disadvantage score and billing type (private, free national health, community health centre), then randomised into either intervention or comparison arms using a computer generated random sequence. Three months post-intervention, patients were recruited from all practices post-consultation for a Computer Assisted Telephone Interview and followed up three and 12 months later. Researchers recruiting, consenting and interviewing patients and patients themselves were masked to allocation status; clinicians were not. SETTING: General practices in metropolitan and rural Victoria, Australia. PARTICIPANTS: General practices with at least one interested clinician (general practitioner or nurse) and their 14-24 year old patients. INTERVENTION: This complex intervention was designed using evidence based practice in learning and change in clinician behaviour and general practice systems, and included best practice approaches to motivating change in adolescent risk taking behaviours. The intervention involved training clinicians (nine hours) in health risk screening, use of a screening tool and motivational interviewing; training all practice staff (receptionists and clinicians) in engaging youth; provision of feedback to clinicians of patients' risk data; and two practice visits to support new screening and referral resources. Comparison clinicians received one didactic educational seminar (three hours) on engaging youth and health risk screening. OUTCOME MEASURES: Primary outcomes were patient report of (1) clinician detection of at least one of six health risk behaviours (tobacco, alcohol and illicit drug use, risks for sexually transmitted infection, STI, unplanned pregnancy, and road risks); and (2) change in one or more of the six health risk behaviours, at three months or at 12 months. Secondary outcomes were likelihood of future visits, trust in the clinician after exit interview, clinician detection of emotional distress and fear and abuse in relationships, and emotional distress at three and 12 months. Patient acceptability of the screening tool was also described for the intervention arm. Analyses were adjusted for practice location and billing type, patients' sex, age, and recruitment method, and past health risks, where appropriate. An intention to treat analysis approach was used, which included multilevel multiple imputation for missing outcome data. RESULTS: 42 practices were randomly allocated to intervention or comparison arms. Two intervention practices withdrew post allocation, prior to training, leaving 19 intervention (53 clinicians, 377 patients) and 21 comparison (79 clinicians, 524 patients) practices. 69% of patients in both intervention (260) and comparison (360) arms completed the 12 month follow-up. Intervention clinicians discussed more health risks per patient (59.7%) than comparison clinicians (52.7%) and thus were more likely to detect a higher proportion of young people with at least one of the six health risk behaviours (38.4% vs 26.7%, risk difference [RD] 11.6%, Confidence Interval [CI] 2.93% to 20.3%; adjusted odds ratio [OR] 1.7, CI 1.1 to 2.5). Patients reported less illicit drug use (RD -6.0, CI -11 to -1.2; OR 0·52, CI 0·28 to 0·96), and less risk for STI (RD -5.4, CI -11 to 0.2; OR 0·66, CI 0·46 to 0·96) at three months in the intervention relative to the comparison arm, and for unplanned pregnancy at 12 months (RD -4.4; CI -8.7 to -0.1; OR 0·40, CI 0·20 to 0·80). No differences were detected between arms on other health risks. There were no differences on secondary outcomes, apart from a greater detection of abuse (OR 13.8, CI 1.71 to 111). There were no reports of harmful events and intervention arm youth had high acceptance of the screening tool. CONCLUSIONS: A complex intervention, compared to a simple educational seminar for practices, improved detection of health risk behaviours in young people. Impact on health outcomes was inconclusive. Technology enabling more efficient, systematic health-risk screening may allow providers to target counselling toward higher risk individuals. Further trials require more power to confirm health benefits. TRIAL REGISTRATION: ISRCTN.com ISRCTN16059206.

Harmonization of training and assessment of medical specialists in physical and rehabilitation medicine across Europe - The contribution of the UEMS Board of Physical and Rehabilitation Medicine

Background.- The main goals of the European Board of Physical and Rehabili-tation Medicine (EBPRM), founded in 1991 as the third speciality board of theUnion of European Medical Specialists (UEMS), are to harmonize pre-graduate,post-graduate and continuous medical education in physical and rehabilitationmedicine (PRM) all over Europe. The harmonization of curricula of the medi-cal specialities and the assessment of medical specialists has become one of thepriorities of the UEMS and its working groups to which the EBPRM contributes.Action.- The EBPRM will continue to promote a specific minimal undergraduatecurriculum on PRM including issues like disability, participation and handicapto be taught all over Europe as a basis for general medical practice. The EBPRMwill also expand the existing EBPRM postgraduate curriculum into a detailedcatalogue of learning objectives. This catalogue will serve as a tool to boostharmonization of the national curricula across Europe as well as to structurethe content of the MCQ examination. It would be a big step forward towardsharmonization of European PRM specialist training if an important number ofcountries would use the certifying MCQ examination of the Board as a part ofthe national assessments for PRM specialists.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

Threshold Selection Criteria for Quantification of Lumbosacral Cerebrospinal Fluid and Root Volumes from MRI

BACKGROUND AND PURPOSE: The high variability of CSF volumes partly explains the inconsistency of anesthetic effects, but may also be due to image analysis itself. In this study, criteria for threshold selection are anatomically defined. METHODS: T2 MR images (n = 7 cases) were analyzed using 3-dimentional software. Maximal-minimal thresholds were selected in standardized blocks of 50 slices of the dural sac ending caudally at the L5-S1 intervertebral space (caudal blocks) and middle L3 (rostral blocks). Maximal CSF thresholds: threshold value was increased until at least one voxel in a CSF area appeared unlabeled and decreased until that voxel was labeled again: this final threshold was selected. Minimal root thresholds: thresholds values that selected cauda equina root area but not adjacent gray voxels in the CSF-root interface were chosen. RESULTS: Significant differences were found between caudal and rostral thresholds. No significant differences were found between expert and nonexpert observers. Average max/min thresholds were around 1.30 but max/min CSF volumes were around 1.15. Great interindividual CSF volume variability was detected (max/min volumes 1.6-2.7). CONCLUSIONS: The estimation of a close range of CSF volumes which probably contains the real CSF volume value can be standardized and calculated prior to certain intrathecal procedures

Characterization and quantification of polyradical character

The decomposition of 〈͈Ŝ²〉 into atomic and diatomic contributions (local spin analysis) is used to detect and quantify the polyradical character of molecular systems. A model triradical system is studied in detail, and the local spin analysis is used to distinguish several patterns of local spin distributions and spin-spin interactions that can be found for different electronic states. How close a real molecular system is to an ideal system of k perfectly localized spin centers is utilized to define a measure of its k-radical character. The spin properties and triradical character of the lowest-lying electronic states of a number of all σ, all π, and σ-π organic triradicals are discussed in detail. The local spin contributions exhibit good correlation with experimental triradical stabilization energies

Quantification of the bond-angle dispersion by Raman spectroscopy and the strain energy of amorphous silicon

A thorough critical analysis of the theoretical relationships between the bond-angle dispersion in a-Si, Δθ, and the width of the transverse optical Raman peak, Γ, is presented. It is shown that the discrepancies between them are drastically reduced when unified definitions for Δθ and Γ are used. This reduced dispersion in the predicted values of Δθ together with the broad agreement with the scarce direct determinations of Δθ is then used to analyze the strain energy in partially relaxed pure a-Si. It is concluded that defect annihilation does not contribute appreciably to the reduction of the a-Si energy during structural relaxation. In contrast, it can account for half of the crystallization energy, which can be as low as 7 kJ/mol in defect-free a-Si