Free-living energy expenditure assessed by two different methods in rural Gambian men.

OBJECTIVE: To assess total free-living energy expenditure (EE) in Gambian farmers with two independent methods, and to determine the most realistic free-living EE and physical activity in order to establish energy requirements for rural populations in developing countries. DESIGN: In this cross-sectional study two methods were applied at the same time. SETTING: Three rural villages and Dunn Nutrition Centre Keneba, MRC, The Gambia. SUBJECTS: Eight healthy, male subjects were recruited from three rural Gambian villages in the sub-Sahelian area (age: 25 +/- 4y; weight: 61.2 +/- 10.1 kg; height: 169.5 +/- 6.5 cm, body mass index: 21.2 +/- 2.5 kg/m2). INTERVENTION: We assessed free-living EE with two inconspicuous and independent methods: the first one used doubly labeled water (DLW) (2H2 18O) over a period of 12 days, whereas the second one was based on continuous heart rate (HR) measurements on two to three days using individual regression lines (HR vs EE) established by indirect calorimetry in a respiration chamber. Isotopic dilution of deuterium (2H2O) was also used to assess total body water and hence fat-free mass (FFM). RESULTS: EE assessed by DLW was found to be 3880 +/- 994 kcal/day (16.2 +/- 4.2 MJ/day). Expressed per unit body weight the EE averaged 64.2 +/- 9.3 kcal/kg/d (269 +/- 38 kJ/kg/d). These results were consistent with the EE results assessed by HR: 3847 +/- 605 kcal/d (16.1 +/- 2.5 MJ/d) or 63.4 +/- 8.2 kcal/kg/d (265 +/- 34kJ/kg/d). Physical activity index, expressed as a multiple of basal metabolic rate (BMR), averaged 2.40 +/- 0.41 (DLW) or 2.40 +/- 0.28 (HR). CONCLUSIONS: These findings suggest an extremely high level of physical activity in Gambian men during intense agricultural work (wet season). This contrasts with the relative food shortage, previously reported during the harvesting period. We conclude that the assessment of EE during the agricultural season in non-industrialized countries needs further investigations in order to obtain information on the energy requirement of these populations. For this purpose the use of the DLW and HR methods have been shown to be useful and complementary.

Evaluation of free libraries for visual recognition of art imagery on mobile devices

This paper describes a systematic research about free software solutions and techniques for art imagery computer recognition problem.

Distribution of free and conjugated cannabinoids in human bile samples.

The metabolism of Δ(9)-tetrahydrocannabinol (THC) is relatively complex, and over 80 metabolites have been identified. However, much less is known about the formation and fate of cannabinoid conjugates. Bile excretion is known to be an important route for the elimination of phase II metabolites. A liquid chromatography-tandem mass spectrometry LC-MS/MS procedure for measuring cannabinoids in oral fluid was adapted, validated and applied to 10 bile samples. THC, 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), Δ(9)-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol glucuronide (THCCOOH-gluc) and Δ(9)-tetrahydrocannabinol glucuronide (THC-gluc) were determined following solid-phase extraction and LC-MS/MS. High concentrations of THCCOOH-gluc were found in bile samples (range: 139-21,275 ng/mL). Relatively high levels of THCCOOH (7.7-1548 ng/mL) and THC-gluc (38-1366 ng/mL) were also measured. THC-A, the plant precursor of THC, was the only cannabinoid that was not detected. These results show that biliary excretion is an important route of elimination for cannabinoids conjugates and that their enterohepatic recirculation is a significant factor to consider when analyzing blood elimination profiles of cannabinoids. Furthermore, we suggest that the bile is the matrix of choice for the screening of phase II cannabinoid metabolites.

Effect of weight reduction in moderately overweight patients on recorded ambulatory blood pressure and free cytosolic platelet calcium.

Although platelet cytosolic calcium has been shown to decrease during pharmacological treatment of hypertension, there is no evidence that cytosolic calcium also falls during a nonpharmacological reduction in blood pressure. To provide such evidence, we examined prospectively the relation between platelet cytosolic calcium and ambulatory blood pressure during weight reduction in moderately overweight (body mass index [BMI] greater than 25), mildly hypertensive individuals. The experimental group (responders: BMI reduction greater than 5%) consisted of 19 patients who lost 8.5 +/- 2.9 kg (mean +/- SD, p less than 0.05) during a 10-week hypocaloric diet, whereas the control group (nonresponders: BMI reduction less than 5%) consisted of 12 patients who showed no relevant change in body weight (-2.0 +/- 1.3 kg) during the same period of time. The moderate weight loss of the responders decreased blood pressure by 14/5 mm Hg (p less than 0.05), as measured by ambulatory monitoring, which renders a placebo effect unlikely. This nonpharmacological reduction in blood pressure was accompanied by a proportional 11% decrease (p less than 0.05) in platelet cytosolic calcium and also by significant (p less than 0.05) decreases in plasma catecholamines and serum cholesterol. These findings establish the concept of a nonpharmacological reduction in free cytosolic platelet calcium in humans and add further evidence suggesting a link between intracellular calcium homeostasis and blood pressure regulation.

Relationship of imatinib-free plasma levels and target genotype with efficacy and tolerability

Imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). Using a nonlinear mixed effects population model, individual estimates of pharmacokinetic parameters were derived and used to estimate imatinib exposure (area under the curve, AUC) in 58 patients. Plasma-free concentration was deduced from a model incorporating plasma levels of alpha(1)-acid glycoprotein. Associations between AUC (or clearance) and response or incidence of side effects were explored by logistic regression analysis. Influence of KIT genotype was also assessed in GIST patients. Both total (in GIST) and free drug exposure (in CML and GIST) correlated with the occurrence and number of side effects (e.g. odds ratio 2.7+/-0.6 for a two-fold free AUC increase in GIST; P<0.001). Higher free AUC also predicted a higher probability of therapeutic response in GIST (odds ratio 2.6+/-1.1; P=0.026) when taking into account tumour KIT genotype (strongest association in patients harbouring exon 9 mutation or wild-type KIT, known to decrease tumour sensitivity towards imatinib). In CML, no straightforward concentration-response relationships were obtained. Our findings represent additional arguments to further evaluate the usefulness of individualizing imatinib prescription based on a therapeutic drug monitoring programme, possibly associated with target genotype profiling of patients.

Use of new biochemical plasmatic markers, plasma free and total metanephrines, for the diagnosis and follow-up of neuroblastoma in children with clinical correlation

Background: Neuroblastoma is a paediatrictumour derived from the neural crest. Biochemical diagnosis and follow up rely on quantitation of urinary catecholamines (dopamine and noradrenaline) and their metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) (gold-standard). When combined, these analyses have a sensitivity of 95%. However, they are clearly limited by inaccuracy of urine collection in young children and normalisation of catecholamine concentrations by creatininuria. Recent development in biochemical diagnosis of pheochromocytoma, another neural crest tumour found in adults, shows that plasmatic measurement of methoxylated catecholamines called metanephrines are more sensitive and specific than other biomarkers. Moreover, a study to determine the reference intervals for metanephrines in a pediatric population has recently been completed. The aim of this work is to describe the role of metanephrines monitoring in the follow up of neuroblastoma. Method: This retrospective study included patients with neuroblastoma in whom the following parameters were determined: plasma free and total metanephrines, plasma catecholamines, 24h urinary catecholamines and metanephrines in absolute value and corrected by creatinine, VMA and HVA at the diagnosis and during treatment at the University Hospital of Lausanne (Switzerland). Eleven patients aged between the first day of life and 7 years old were followed between 2005 and 2012. Clinical outcome and biochemical concentrations of the analytes were correlated. Results: At diagnosis, plasma free and total normetanephrines and methoxytyramine have a sensitivity of 100% compared to 85% for the actual gold standard. Metanephrine remain below the upper reference limit as expected since these tumours do not produce adrenaline. The relationship between biochemical markers and clinical outcome is illustrated graphically. Plasma or urinary normetanephrine and methoxytyramine correlate better with the history of the patient than VMA and HVA, as evaluated by ordinal logistic regression. Concentrations of analytes in urine show a better correlation with clinical events when the results are corrected by creatininuria. Conclusion: Normetanephrine and methoxytyramine reflect disease history in neuroblastoma patients and could play a significant role in the follow up of this type of tumour. Formal studies in a sufficient number of patients are needed to confirm this preliminary observation.

Rituximab treatment in Myasthenia gravis: an open-label trial

Myasthenia gravis (MG), an antibody (AB)-mediated autoimmune disorder, responds to treatments targeting the humoral response such as intravenous immunoglobulines (IVIG) and plasma exchange treatments (PEX). Rituximab (RTX), a monoclonal anti-CD20 AB that depletes the specific B lymphocyte population should be efficient and is being used for resistant MG patients in small cohorts. Objectives: This is an observational prospective study that aims to determine the efficacy of RTX in MG, the duration of the clinical effect after treatment and the possible sparing effect on other immunosuppressive drugs.Methods: Between January 2009 and December 2010, 8 MG (2 with anti-MUSK AB) patients (62.5% female) with mean age of 41 years (range 24-79 yo), were treated by RTX. The patients treated were those who experienced serious side-effects and/or treatment failure. In three cases the criteria for treatment was the need to spare frequent recurrent plasmapheresis or IGIV treatment. We compared the functional tests before and prospectively after the treatment (schema used for one cure: 2 9 1gr within 15 days interval), the duration of the efficiency (follow-up of 4-24 months) and we repeated the cures based on clinical criteria.Results: Two patients (25%) underwent 3 RTX cures, 2 (25%) underwent 2 cures and the others (50%) one cure. No adverse events were observed. Six patients (75%) showed a clinical response with improvement of the functional scores and reduction of the concomitant immunosuppressive treatments (75% for prednisone, 35% for other immunosuppressive drugs) that persists over a period of 4-9 months. Follow-up of clinical state and lymphocyte count showed an inverse correlation between the CD 19 count and the clinical state of the patients.Conclusion: In this small series of patients RTX treatment shows significant improvement of clinical state of MG refractory to conventional treatment patients, without side-effects reported, even in patients that were retreated. Larger studies should be held to determine if RTX could be an alternative to plasmapheresis and IVIG as second-line treatment in MG.

Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression.

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2Kd cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse beta 2-microglobulina (beta 2-ma). When infected with the heavy chain-encoding virus alone, H-2Kd was produced in a beta 2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, approximately 10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2Kd-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro, in cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 microM) of an H-2Kd-restricted octapeptide epitope from Plasmodium berghei. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse beta 2-m but not to cells expressing free heavy chain only.

Intermittent atrial tachycardia promotes repolarization alternans and conduction slowing during rapid rates, and increases susceptibility to atrial fibrillation in a free-behaving sheep model.

INTRODUCTION: Paroxysmal atrial fibrillation (AF) may be triggered by intermittent atrial tachycardia, and ultimately lead to persistent AF. However, the mechanisms by which intermittent atrial tachycardia promotes sustained AF are not well understood. METHODS AND RESULTS: Eight sheep were chronically implanted with 2 pacemakers for the recording of broadband right atrial unipolar electrograms, and for the delivery of electrophysiological stimulation protocols and intermittent right atrial tachycardia. Right atrial kinetics of activation recovery interval (ARI) as a surrogate for action potential duration, of conduction time and velocity, and of repolarization alternans were analyzed at incremental pacing rates during the remodeling process induced by weeks of intermittent atrial tachycardia until the development of sustained AF. Intermittent atrial tachycardia decreased ARI and blunted its rate adaptation, facilitated atrial capture, and slowed conduction at high rates, and increased susceptibility to pacing-induced AF. In spite of blunted ARI rate adaptation, right atrial repolarization alternans was maintained during remodeling, and further increased in magnitude just before rapid pacing-induced AF. CONCLUSION: This study suggests that weeks of intermittent right atrial tachycardia result in a gradual electrical remodeling favorable for wavebreaks and reentry that may facilitate fibrillation.

Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures

Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types. Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols. However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally. Defining optimal culture parameters for ex vivo expansion has been a major challenge. We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions. Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved. Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion. Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture. With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.

Multi-scale label interaction to improve object detection and segmentation

The project aims at advancing the state of the art in the use of context information for classification of image and video data. The use of context in the classification of images has been showed of great importance to improve the performance of actual object recognition systems. In our project we proposed the concept of Multi-scale Feature Labels as a general and compact method to exploit the local and global context. The feature extraction from the discriminative probability or classification confidence label field is of great novelty. Moreover the use of a multi-scale representation of the feature labels lead to a compact and efficient description of the context. The goal of the project has been also to provide a general-purpose method and prove its suitability in different image/video analysis problem. The two-year project generated 5 journal publications (plus 2 under submission), 10 conference publications (plus 2 under submission) and one patent (plus 1 pending). Of these publications, a relevant number make use of the main result of this project to improve the results in detection and/or segmentation of objects.

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.