A cancer rainbow mouse for visualizing the functional genomics of oncogenic clonal expansion.

Field cancerization is a premalignant process marked by clones of oncogenic mutations spreading through the epithelium. The timescales of intestinal field cancerization can be variable and the mechanisms driving the rapid spread of oncogenic clones are unknown. Here we use a Cancer rainbow (Crainbow) modelling system for fluorescently barcoding somatic mutations and directly visualizing the clonal expansion and spread of oncogenes. Crainbow shows that mutations of ß-catenin (Ctnnb1) within the intestinal stem cell results in widespread expansion of oncogenes during perinatal development but not in adults. In contrast, mutations that extrinsically disrupt the stem cell microenvironment can spread in adult intestine without delay. We observe the rapid spread of premalignant clones in Crainbow mice expressing oncogenic Rspondin-3 (RSPO3), which occurs by increasing crypt fission and inhibiting crypt fixation. Crainbow modelling provides insight into how somatic mutations rapidly spread and a plausible mechanism for predetermining the intratumor heterogeneity found in colon cancers

Mouse Background Strain Profoundly Influences Paneth Cell Function and Intestinal Microbial Composition

Increasing evidence supports the central role of Paneth cells in maintaining intestinal host-microbial homeostasis. However, the direct impact of host genotype on Paneth cell function remains unclear. Here, we characterize key differences in Paneth cell function and intestinal microbial composition in two widely utilized, genetically distinct mouse strains (C57BL/6 and 129/SvEv). In doing so, we demonstrate critical influences of host genotype on Paneth cell activity and the enteric microbiota.Paneth cell numbers were determined by flow cytometry. Antimicrobial peptide (AMP) expression was evaluated using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), acid urea-polyacrylamide gel electrophoresis, and mass spectrometry. Effects of mouse background on microbial composition were assessed by reciprocal colonization of germ-free mice from both background strains, followed by compositional analysis of resultant gut bacterial communities using terminal restriction fragment length polymorphism analysis and 16 S qPCR. Our results revealed that 129/SvEv mice possessed fewer Paneth cells and a divergent AMP profile relative to C57BL/6 counterparts. Novel 129/SvEv á-defensin peptides were identified, including Defa2/18v, Defa11, Defa16, and Defa18. Host genotype profoundly affected the global profile of the intestinal microbiota, while both source and host factors were found to influence specific bacterial groups. Interestingly, ileal α-defensins from 129/SvEv mice displayed attenuated antimicrobial activity against pro-inflammatory E. coli strains, a bacterial species found to be expanded in these animals.This work establishes the important impact of host genotype on Paneth cell function and the composition of the intestinal microbiota. It further identifies specific AMP and microbial alterations in two commonly used inbred mouse strains that have varying susceptibilities to a variety of disorders, ranging from obesity to intestinal inflammation. This will be critical for future studies utilizing these murine backgrounds to study the effects of Paneth cells and the intestinal microbiota on host health and disease

The use of opioids at the end of life: knowledge level of pharmacists and cooperation with physicians

Contains fulltext : 96464.pdf (publisher's version ) (Open Access)PURPOSE: What is the level of knowledge of pharmacists concerning pain management and the use of opioids at the end of life, and how do they cooperate with physicians? METHODS: A written questionnaire was sent to a sample of community and hospital pharmacists in the Netherlands. The questionnaire was completed by 182 pharmacists (response rate 45%). RESULTS: Pharmacists were aware of the most basic knowledge about opioids. Among the respondents, 29% erroneously thought that life-threatening respiratory depression was a danger with pain control, and 38% erroneously believed that opioids were the preferred drug for palliative sedation. One in three responding pharmacists did not think his/her theoretical knowledge was sufficient to provide advice on pain control. Most pharmacists had working agreements with physicians on euthanasia (81%), but fewer had working agreements on palliative sedation (46%) or opioid therapy (25%). Based on the experience of most of responding pharmacists (93%), physicians were open to unsolicited advice on opioid prescriptions. The majority of community pharmacists (94%) checked opioid prescriptions most often only after dispensing, while it was not a common practice among the majority of hospital pharmacists (68%) to check prescriptions at all. CONCLUSIONS: Although the basic knowledge of most pharmacists was adequate, based on the responses to the questionnaire, there seems to be a lack of knowledge in several areas, which may hamper pharmacists in improving the quality of care when giving advice to physicians and preventing or correcting mistakes if necessary. If education is improved, a more active role of the pharmacist may improve the quality of end-of-life pharmacotherapy

The CSF-1 receptor fashions the intestinal stem cell niche

AbstractGastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5+ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r−/− defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche

Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal

The canonical Wnt/β-catenin signaling pathway governs diverse developmental, homeostatic and pathologic processes. Palmitoylated Wnt ligands engage cell surface Frizzled (Fzd) receptors and Lrp5/6 co-receptors enabling β-catenin nuclear translocation and Tcf/Lef-dependent gene transactivation1–3. Mutations in Wnt downstream signaling components have revealed diverse functions presumptively attributed to Wnt ligands themselves, although direct attribution remains elusive, as complicated by redundancy between 19 mammalian Wnts and 10 Fzds1 and Wnt hydrophobicity2,3. For example, individual Wnt ligand mutations have not revealed homeostatic phenotypes in the intestinal epithelium4, an archetypal canonical Wnt pathway-dependent rapidly self-renewing tissue whose regeneration is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs)5–9. R-spondin ligands (Rspo1–4) engage distinct Lgr4-6 and Rnf43/Znrf3 receptor classes10–13, markedly potentiate canonical Wnt/β-catenin signaling and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo8,14–17. However, the interchangeability, functional cooperation and relative contributions of Wnt versus Rspo ligands to in vivo canonical Wnt signaling and ISC biology remain unknown. Here, we deconstructed functional roles of Wnt versus Rspo ligands in the intestinal crypt stem cell niche. We demonstrate that the default fate of Lgr5+ ISCs is lineage commitment, escape from which requires both Rspo and Wnt ligands. However, gain-of-function studies using Rspo versus a novel non-lipidated Wnt analog reveal qualitatively distinct, non-interchangeable roles for these ligands in ISCs. Wnts are insufficient to induce Lgr5+ ISC self-renewal, but rather confer a basal competency by maintaining Rspo receptor expression that enables Rspo to actively drive and specify the extent of stem cell expansion. This functionally non-equivalent yet cooperative interplay between Wnt and Rspo ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precision control of tissue regeneration

Real and Monetary Policy Convergence: EMU Crisis to the CFA Zone

A major lesson of the EMU crisis is that serious disequilibria result from regional monetary arrangements not designed to be robust to a variety of shocks. The purpose of this paper is to assess these disequilibria within the CEMAC, UEMOA and CFA zones. In the assessments, monetary policy targets inflation and financial dynamics of depth, efficiency, activity and size while real sector policy targets economic performance in terms of GDP growth. We also provide the speed of convergence and time required to achieve a 100% convergence. But for financial intermediary size within the CFA zone, findings for the most part support only unconditional convergence. There is no form of convergence within the CEMAC zone. The broad insignificance of conditional convergence results have substantial policy implications. Monetary and real policies which are often homogenous for member states are thwarted by heterogeneous structural and institutional characteristics which give rise to different levels and patterns of financial intermediary development. Therefore member states should work towards harmonizing cross-country differences in structural and institutional characteristics that hamper the effectiveness of monetary policies