Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo.

PURPOSE: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or "wet" Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. METHODS: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with "wet" AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8(+/-) mice expressing ß-galactosidase. Aged Mfge8(+/-) and Mfge8(-/-) mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. RESULTS: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8(-/-) mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8(-/-) mice as compared to controls. CONCLUSIONS: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8(-/-) mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.

Data-based priors for vector autoregressions with drifting coefficients

This paper proposes full-Bayes priors for time-varying parameter vector autoregressions (TVP-VARs) which are more robust and objective than existing choices proposed in the literature. We formulate the priors in a way that they allow for straightforward posterior computation, they require minimal input by the user, and they result in shrinkage posterior representations, thus, making them appropriate for models of large dimensions. A comprehensive forecasting exercise involving TVP-VARs of different dimensions establishes the usefulness of the proposed approach.

Eumelanin-based coloration and fitness parameters in birds: a meta-analysis

Although melanin is the most common pigment in animal integuments, the adaptive function of variation in melanin-based coloration remains poorly understood. The individual fitness returns associated with melanin pigments can be variable across species as these pigments can have physical and biological protective properties and genes involved in melanogenesis may vary in the intensity of pleiotropic effects. Moreover, dark and pale coloration can also enhance camouflage in alternative habitats and melanin-based coloration can be involved in social interactions. We investigated whether darker or paler individuals achieve a higher fitness in birds, a taxon wherein associations between melanin-based coloration and fitness parameters have been studied in a large number of species. A meta-analysis showed that the degree of melanin-based coloration was not significantly associated with laying date, clutch size, brood size, and survival across 26 species. Similar results were found when restricting the analyses to non-sexually dimorphic birds, colour polymorphic and monomorphic species, in passerines and non-passerines and in species for which inter-individual variation in melanism is due to colour intensity. However, eumelanic coloration was positively associated with clutch and brood size in sexually dimorphic species and those that vary in the size of black patches, respectively. Given that greater extent of melanin-based coloration was positively associated with reproductive parameters and survival in some species but negatively in other species, we conclude that in birds the sign and magnitude of selection exerted on melanin-based coloration is species- or trait-specific.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Sustained improvement in left ventricular function after bone marrow derived cell therapy in patients with acute ST elevation myocardial infarction. A 5-year follow-up from the Stem Cell Transplantation in Ischaemic Myocardium Study.

BACKGROUND: Intracoronary injection of autologous bone marrow-derived mononucleated cells (BM-MNC) may improve LV function shortly after acute ST elevation myocardial infarction (STEMI), but little is known about the long-term durability of the treatment effect. METHODS: In a single-centre trial a total of 60 patients with acute anterior STEMI, successful reperfusion therapy and a left ventricular ejection fraction (LVEF) of <50% were screened for the study. 23 patients were actively treated with intracoronary infusion of BM-MNC within a median of 3 days. The open-label control group consisted of 19 patients who did not consent to undergo BM-MNC treatment but agreed to undergo regular clinical and echocardiographic follow-up for up to 5 years after AMI. RESULTS: Whereas at 4 months there was no significant difference between the increase in LVEF in the BM-MNC group and the control group (+7.0%, 95%CI 3.6; 10.4) vs. +3.9%, 95%CI -2.1; 10), the absolute increase at 5 years remained stable in the BM-MNC but not in the control group (+7.95%, 95%CI 3.5; 12.4 vs. -0.5%, 95%CI -5.4; 4.4; p for interaction between groups = 0.035). DISCUSSION: In this single-centre, open-labelled study, intracoronary administration of BM-MNC is feasible and safe in the short term. It is also associated with sustained improvement of left ventricular function in patients with acute myocardial infarction, encouraging phase III studies to examine the potential BM-MNC effect on clinical outcome.

Genomik und personalisierte Medizin [Genomics and personalized medicine].

Personalized medicine has a substantial potential to transform the way diseases will be predicted, prevented and treated. The field will greatly benefit from novel DNA sequencing technologies, in particular commoditization of individual whole genome sequencing. This evolution cannot be stopped, and the medical and scientific community, as well as the society at large, have the responsibility to anticipate the expected benefits from this revolution, but also the potential risks associated with it. Massive investments will be needed for the potential of personalized medicine to be realized, and for the field to come to maturity. In particular, a paradigm change in the way clinical research is done is needed. Switzerland and its Western part pro-actively anticipate these changes.

Frequent users of emergency departments present very high prevalence of mental health and substance use disorders, which are largely underdiagnosed by clinicians

Background: The objective of this study was to determine if mental health and substance use diagnoses were equally detected in frequent users (FUs) compared to infrequent users (IUs) of emergency departments (EDs). Methods: In a sample of 399 adult patients (>= 18 years old) admitted to a teaching hospital ED, we compared the mental health and substance use disorders diagnoses established clinically and consigned in the medical files by the ED physicians to data obtained in face-to-face research interviews using the Primary Care Evaluation of Mental Disorders (PRIME-MD) and the Alcohol, Smoking and Involvement Screening Test (ASSIST). Between November 2009 and June 2010, 226 FUs (>4 visits within a year) who attended the ED were included, and 173 IUs (<= 4 visits within a year) were randomly selected from a pool of identified patients to comprise the comparison group. Results: For mental health disorders identified by the PRIME-MD, FUs were more likely than IUs to have an anxiety (34 vs. 16%, Chi2(1) = 16.74, p <0.001), depressive (47 vs. 25%, Chi2(1) = 19.11, p <0.001) or posttraumatic stress (PTSD) disorder (11 vs. 5%, Chi2(1) = 4.87, p = 0.027). Only 3/76 FUs (4%) with an anxiety disorder, 16/104 FUs (15%) with a depressive disorder and none of the 24 FUs with PTSD were detected by the ED medical staff. None of the 27 IUs with an anxiety disorder, 6/43 IUs (14%) with a depressive disorder and none of the 8 IUs with PTSD were detected. For substance use disorders identified by the ASSIST, FUs were more at risk than IUs for alcohol (24 vs. 7%, Chi2(1) = 21.12, p <0.001) and drug abuse/dependence (36 vs. 25%, Chi2(1) = 5.52, p = 0.019). Of the FUs, 14/54 (26%) using alcohol and 8/81 (10%) using drugs were detected by the ED physicians. Of the IUs, 5/12 (41%) using alcohol and none of the 43 using drugs were detected. Overall, there was no significant difference in the rate of detection of mental health and substance use disorders between FUs and IUs (Fisher's Exact Test: anxiety, p = 0.567; depression, p = 1.000; PTSD, p = 1.000; alcohol, p = 0.517; and drugs, p = 0.053). Conclusions: While the prevalence of mental health and substance use disorders was higher among FUs, the rates of detection were not significantly different for FUs vs. IUs. However, it may be that drug disorders among FUs were more likely to be detected.

Morphological study of adult male worms of Schistosoma mansoni Sambon, 1907 by confocal laser scanning microscopy

Aiming to detail data obtained through brightfield microscopy (BM) on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice, stained with Langeron's carmine and analyzed under a confocal laser scanning microscope CLSM 410 (Carl Zeiss). The reproductive system presented a single and lobate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphous and isolated from the normal ones. Collecting tubules (excretory ducts), followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.

Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

Purely Endoscopic Keyhole Supraorbital Approaches for Anterior and Middle Skull Base Tumors

Anterior and middle skull base tumors, mainly meningiomas, are usually operated on using a sub-frontal route with a microscope. With modern radiotherapy, the goal of skull base surgery moves from a radical surgery with high rate of side effect to a functional concept that aims to remove as much as possible of the tumor without compromising the neurological status of patients. Minimally skull base surgery benefits from keyhole and endoscopy techniques. For 3 2 decades, the development of endoscopy helps to imagine innovative approaches for skull base tumors such as the endonasal route. Nonetheless, CSF leak issue and the absence of direct control of the tumor margins may limit the interest of such a route. Keyhole craniotomies have been developed with microscope but vision issue limits their use. Combining advantages of both techniques appears therefore natural and gave birth to intracranial assisted and more recently to fully endoscopic keyhole surgery. For anterior or middle skull base tumors, Keyhole supraorbital approaches can be done either by a trans-eyebrow or trans-eyelid routes. A step-by-step description of these fully endoscopic alternative routes summarizing advantages and drawbacks compared to others (traditional sub-frontal or more recent endonasal approaches) is reported in this chapter by the authors.