Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling.

Continuous turnover of epithelia is ensured by the extensive self-renewal capacity of tissue-specific stem cells. Similarly, epithelial tumour maintenance relies on cancer stem cells (CSCs), which co-opt stem cell properties. For most tumours, the cellular origin of these CSCs and regulatory pathways essential for sustaining stemness have not been identified. In murine skin, follicular morphogenesis is driven by bulge stem cells that specifically express CD34. Here we identify a population of cells in early epidermal tumours characterized by phenotypic and functional similarities to normal bulge skin stem cells. This population contains CSCs, which are the only cells with tumour initiation properties. Transplants derived from these CSCs preserve the hierarchical organization of the primary tumour. We describe beta-catenin signalling as being essential in sustaining the CSC phenotype. Ablation of the beta-catenin gene results in the loss of CSCs and complete tumour regression. In addition, we provide evidence for the involvement of increased beta-catenin signalling in malignant human squamous cell carcinomas. Because Wnt/beta-catenin signalling is not essential for normal epidermal homeostasis, such a mechanistic difference may thus be targeted to eliminate CSCs and consequently eradicate squamous cell carcinomas.

Renal tubular NEDD4-2 deficiency causes NCC-mediated salt-dependent hypertension.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na+ channel (ENaC/SCNN1) to mediate Na+ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6-8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na+ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na+/K+ balance. Under a high-Na+ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K+ channel (ROMK), and total and phosphorylated thiazide-sensitive Na+Cl- cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na+/K+ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

Monitoring time-dependent degradation of phospholipids in sectioned tissues by MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) is useful for visualizing the localization of phospholipids on biological tissue surfaces creating great opportunities for IMS in lipidomic investigations. With advancements in IMS of lipids, there is a demand for large-scale tissue studies necessitating stable, efficient and well-defined sample handling procedures. Our work within this article shows the effects of different storage conditions on the phospholipid composition of sectioned tissues from mouse organs. We have taken serial sections from mouse brain, kidney and liver thaw mounted unto ITO-coated glass slides and stored them under various conditions later analyzing them at fixed time points. A global decrease in phospholipid signal intensity is shown to occur and to be a function of time and temperature. Contrary to the global decrease, oxidized phospholipid and lysophospholipid species are found to increase within 2 h and 24 h, respectively, when mounted sections are kept at ambient room conditions. Imaging experiments reveal that degradation products increase globally across the tissue. Degradation is shown to be inhibited by cold temperatures, with sample integrity maintained up to a week after storage in −80 °C freezer under N2 atmosphere. Overall, the results demonstrate a timeline of the effects of lipid degradation specific to sectioned tissues and provide several lipid species which can serve as markers of degradation. Importantly, the timeline demonstrates oxidative sample degradation begins appearing within the normal timescale of IMS sample preparation of lipids (i.e. 1-2 h) and that long-term degradation is global. Taken together, these results strengthen the notion that standardized procedures are required for phospholipid IMS of large sample sets, or in studies where many serial sections are prepared together but analyzed over time such as in 3-D IMS reconstruction experiments.

Prédiction des durées de séjour en réadaptation en fonction du statut fonctionnel. [Usefulness of functional status to predict length of stay in rehabilitation.]

Objectif : En Suisse, la réadaptation est financée en¦partie par l'assureur qui fixe préalablement à l'admission¦un nombre de jours (durée garantie) qu'il s'engage¦à rembourser. Lorsqu'une durée garantie est trop courte,¦une demande de prolongation est nécessaire, induisant¦des démarches administratives. Les objectifs de cette¦étude étaient a) d'étudier le lien entre durées garanties¦et caractéristiques du patient ; b) d'estimer les coûts¦liés aux demandes de prolongation ; c) d'évaluer¦l'impact de l'introduction d'un modèle d'attribution de¦durée garantie basé sur l'état fonctionnel du patient.¦Méthodes : Les corrélations entre état fonctionnel,¦durée effective et durée garantie ont été testées sur¦208 séjours représentatifs. Des durées garanties fictives¦ont été calculées à partir de la médiane de durée de¦séjour de 2 335 patients, groupés selon leur niveau¦fonctionnel (score des activités de base de la vie quotidienne¦(BAVQ) 0-1 vs 2-4 vs 5-6), puis comparées aux¦durées de séjour effectives et garanties.¦Résultats : L'état fonctionnel du patient n'est pas¦corrélé à la durée garantie, et 69 % des séjours nécessitent¦au moins une demande de prolongation, représentant¦2,6 équivalents temps plein en temps administratif¦projeté sur le canton. L'application du modèle proposé¦réduirait de 28 % les demandes de prolongation, et¦n'augmenterait que marginalement la proportion de¦jours garantis en surplus (11,2 % contre 6,5 % actuellement).¦Conclusion : L'utilisation systématique d'un modèle¦d'attribution de durées garanties basées sur l'état¦fonctionnel du patient permettrait de réduire sensiblement¦les coûts administratifs liés aux demandes de¦prolongation, sans entraîner de risque accru d'une augmentation¦de la durée de séjour.

Age-dependent gain of alternative splice forms and biased duplication explain the relation between splicing and duplication.

We analyze here the relation between alternative splicing and gene duplication in light of recent genomic data, with a focus on the human genome. We show that the previously reported negative correlation between level of alternative splicing and family size no longer holds true. We clarify this pattern and show that it is sufficiently explained by two factors. First, genes progressively gain new splice variants with time. The gain is consistent with a selectively relaxed regime, until purifying selection slows it down as aging genes accumulate a large number of variants. Second, we show that duplication does not lead to a loss of splice forms, but rather that genes with low levels of alternative splicing tend to duplicate more frequently. This leads us to reconsider the role of alternative splicing in duplicate retention.

Stage of change of cigarette smoking in drug dependent patients

Résumé En Suisse, les programmes de désaccoutumance au tabac se réfèrent généralement au modèle de préparation au changement de Prochaska et DiClemente (1983), Les patients atteints de maladies somatiques liées au tabagisme comme les pathologies cardiovasculaires ou pulmonaires accèdent facilement à ces programmes, contrairement aux patients présentant une dépendance à des drogues illicites. La prévalence de fumeurs dans cette population est pourtant élevée et les problèmes engendrés par le tabac sont importants, non seulement d'un point de vue individuel mais aussi en terme de santé publique. Il est par conséquent intéressant d'évaluer la motivation concernant la désaccoutumance au tabac de patients toxicomanes entreprenant un sevrage de drogues illicites. Dans cette étude, nous avons évalué les stades de préparation au changement concernant la dépendance au tabac chez 100 patients toxicomanes hospitalisés sur un mode volontaire dans le cadre d'un programme de sevrage à des drogues illégales. L'évaluation s'est faite à l'aide d'un auto-questionnaire dont les résultats indiquent qu'une minorité de patients sont décidés à interrompre la consommation de tabac. En effet, seul 15% des patients se trouvaient aux stades de contemplation ou de décision. De plus, 93% des sujets considéraient l'arrêt du tabac comme difficile ou très difficile. Ces données montrent qu'il existe un décalage important entre la motivation relative au sevrage de drogues illégales et la motivation liées à l'arrêt du tabac. En effet, malgré leur motivation élevée pour se sevrer de drogues illicites, la proportion de patients restant au stade de précontemplation concernant la désaccoutumance au tabac reste élevée. Diverses hypothèses permettent d'expliquer ces résultats, notamment la perception que la désaccoutumance au tabac est plus difficile à réaliser que le sevrage de substances illicites. Abstract Nicotine cessation programmes in Switzerland, which are commonly based on the stage of change model of Prochaska and DiClemente (1983), are rarely offered to patients with illicit drug dependence. This stands in contrast to the high smoking rates and the heavy burden of tobacco-related problems in these patients. The stage of change was therefore assessed by self-administered questionnaire in 100 inpatients attending an illegal drug withdrawal programme. Only 15% of the patients were in the contemplation or decision stage. 93% considered smoking cessation to be difficult or very difficult. These data show a discrepancy between the motivation to change illegal drug consumption habits and the motivation for smoking cessation. The high pro-portion of patients remaining in the precontemplation stage for smoking cessation, in spite of their motivation for illicit drug detoxification, may be due to the perception that cessation of smoking is more difficult than illicit drug abuse cessation.

Environment-dependent microevolution in a Mediterranean pine (Pinus pinaster Aiton).

BACKGROUND: A central question for understanding the evolutionary responses of plant species to rapidly changing environments is the assessment of their potential for short-term (in one or a few generations) genetic change. In our study, we consider the case of Pinus pinaster Aiton (maritime pine), a widespread Mediterranean tree, and (i) test, under different experimental conditions (growth chamber and semi-natural), whether higher recruitment in the wild from the most successful mothers is due to better performance of their offspring; and (ii) evaluate genetic change in quantitative traits across generations at two different life stages (mature trees and seedlings) that are known to be under strong selection pressure in forest trees. RESULTS: Genetic control was high for most traits (h2 = 0.137-0.876) under the milder conditions of the growth chamber, but only for ontogenetic change (0.276), total height (0.415) and survival (0.719) under the more stressful semi-natural conditions. Significant phenotypic selection gradients were found in mature trees for traits related to seed quality (germination rate and number of empty seeds). Moreover, female relative reproductive success was significantly correlated with offspring performance for specific leaf area (SLA) in the growth chamber experiment, and stem mass fraction (SMF) in the experiment under semi-natural conditions, two adaptive traits related to abiotic stress-response in pines. Selection gradients based on genetic covariance of seedling traits and responses to selection at this stage involved traits related to biomass allocation (SMF) and growth (as decomposed by a Gompertz model) or delayed ontogenetic change, depending also on the testing environment. CONCLUSIONS: Despite the evidence of microevolutionary change in adaptive traits in maritime pine, directional or disruptive changes are difficult to predict due to variable selection at different life stages and environments. At mature-tree stages, higher female effective reproductive success can be explained by differences in their production of offspring (due to seed quality) and, to a lesser extent, by seemingly better adapted seedlings. Selection gradients and responses to selection for seedlings also differed across experimental conditions. The distinct processes involved at the two life stages (mature trees or seedlings) together with environment-specific responses advice caution when predicting likely evolutionary responses to environmental change in Mediterranean forest trees.

Maturation-dependent neurotoxicity of lead acetate in vitro: implication of glial reactions.

Despite a wealth of data on the neurotoxic effects of lead at the cellular and molecular levels, the reasons for its development-dependent neurotoxicity are still unclear. Here, the maturation-dependent effects of lead acetate were analyzed in immature and differentiated brain cells cultured in aggregates. Markers of general cytotoxicity as well as cell-type-specific markers of glial and neuronal cells showed that immature brain cells were more sensitive to lead than the differentiated counterparts, demonstrating that the development-dependent neurotoxicity of lead can be reproduced in aggregating brain cell cultures. After 10 days of treatment, astrocytes were found to be more affected by lead acetate than neurons in immature cultures, and microglial cells were strongly activated. Eleven days after cessation of the treatment, lead acetate caused a partial loss of astrocytes and an intense reactivity of the remaining ones. Furthermore, microglial cells expressed a macrophagic phenotype, and the loss of activity of neuron-specific enzymes was aggravated. In differentiated cultures, no reactive gliosis was found. It is hypothetized that the intense glial reactions (microgliosis and astrogliosis) observed in immature cultures contribute to the development-dependent neurotoxicity of lead.

Does history of falling predict length of stay in post-acute geriatric rehabilitation?

Objectives:To investigate the associations between falls before hospital¦admission, falls during hospitalization, and length of stay in elderly¦people admitted to post-acute geriatric rehabilitation. Method: History¦of falling in the previous 12 months before admission was recorded¦among 249 older persons (mean age 82.3±7.4 years, 69.1% women)¦consecutively admitted to post-acute rehabilitation. Data on medical,¦functional and cognitive status were collected upon admission. Falls¦during hospitalization and length of stay were recorded at discharge.¦Results: Overall, 92 (40.4%) patients reported no fall in the 12 months¦before admission; 63(27.6%) reported 1 fall, and 73(32.0%) reported¦multiple falls. Previous falls occurrence (one or more falls) was significantly¦associated with in-stay falls (19.9% of previous fallers fell¦during the stay vs 7.6% in patients without history of falling, P=.01),¦and with a longer length of stay (22.4 ± 10.1 days vs 27.1 ± 14.3 days,¦P=.01). In multivariate robust regression controlling for gender, age,¦functional and cognitive status, history of falling remained significantly¦associated with longer rehabilitation stay (2.8 days more in single fallers,¦p=.05, and 3.3 days more in multiple fallers, p=.0.1, compared to¦non-fallers). Conclusion: History of falling in the 12 months prior to¦post acute geriatric rehabilitation is independently associated with a¦longer rehabilitation length of stay. Previous fallers have also an¦increased risk of falling during rehabilitation stay. This suggests that¦hospital fall prevention measures should particularly target these high¦riskpatients.

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

Vertebrate conserved non coding DNA regions have a high persistence length and a short persistence time.

BACKGROUND: The comparison of complete genomes has revealed surprisingly large numbers of conserved non-protein-coding (CNC) DNA regions. However, the biological function of CNC remains elusive. CNC differ in two aspects from conserved protein-coding regions. They are not conserved across phylum boundaries, and they do not contain readily detectable sub-domains. Here we characterize the persistence length and time of CNC and conserved protein-coding regions in the vertebrate and insect lineages. RESULTS: The persistence length is the length of a genome region over which a certain level of sequence identity is consistently maintained. The persistence time is the evolutionary period during which a conserved region evolves under the same selective constraints.Our main findings are: (i) Insect genomes contain 1.60 times less conserved information than vertebrates; (ii) Vertebrate CNC have a higher persistence length than conserved coding regions or insect CNC; (iii) CNC have shorter persistence times as compared to conserved coding regions in both lineages. CONCLUSION: Higher persistence length of vertebrate CNC indicates that the conserved information in vertebrates and insects is organized in functional elements of different lengths. These findings might be related to the higher morphological complexity of vertebrates and give clues about the structure of active CNC elements.Shorter persistence time might explain the previously puzzling observations of highly conserved CNC within each phylum, and of a lack of conservation between phyla. It suggests that CNC divergence might be a key factor in vertebrate evolution. Further evolutionary studies will help to relate individual CNC to specific developmental processes.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.