102 research outputs found
Human colon organoids reveal distinct physiologic and oncogenic Wnt responses.
No full textConstitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC
Development of ecotoxicity testing in the standard organism Folsomia candida (Collembola)
No full textChemical analyses of the environment can document contamination by various xenobiotics but it is also important to understand the effect of pollutants on living organisms. Thus, in the present work the author investigated the effect of chemicals (imidacloprid and silver nanoparticles) at the molecular and cellular biological organization levels of a standard test organism, Folsomia candida (Collembola).In the study with the insecticide imidacloprid, the effect of the chemical was focused on the detoxifying enzyme glutathione S-transferase (GST) from F. candida. Test animals were treated with different concentrations of imidacloprid for 48 hours. Changes in steady state levels of GST mRNA and GST enzyme activity were investigated. Furthermore, enzyme extracts were separated according to their sizes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the resolved protein bands were detected by silver staining. Additionally, the size of the glutathione (GSH) pool in Collembola was also determined. Moreover, a predicted protein sequence of putative GSTs was identified with animals from the control group.A 3 fold up-regulation of GST steady state mRNA levels was detected in the samples treated with 5.0 mg L-1 imidacloprid compared to the control, while a 2.5 and a 2 fold up-regulation was found in organisms treated with 2.5 and 7.5 mg L-1 imidacloprid, respectively. GST activity increased with increasing imidacloprid amounts from an initial activity of 0.11 μmol min-1 mg-1 protein in the control group up to 0.25 μmol min-1 mg-1 protein in the sample treated with the 5.0 mg L-1 of pesticide. In contrast, the total amount of GSH decreased with increasing imidacloprid concentration.The results suggest that the alteration of the steady state level of GST mRNA, GST activity as well as GSH level may be involved in the response of F. candida upon the exposure to imidacloprid and can be used as biomarkers to monitor the toxic effects of imidacloprid and other environmental pollutants on Collembola.In case of the study with silver nanoparticles (AgNPs), chemical effects were studied (in comparison to silver nitrate) focusing on molecular and cellular alterations as ecotoxicological endpoints. At the molecular level, an up-regulation of metallothionein-containing protein (MTC) mRNA in AgNPs treated groups indicated toxic heavy metal stress effects caused by the release of silver ions from AgNPs, which was similar to animal groups treated with silver nitrate. Alteration of the steady state level of GST mRNA was also detected in animals treated with AgNPs and AgNO3. At the cellular level, the relation between GST activity and the size of the GSH pool was examined and it was found that the change of GST activity from different animal groups was not significant, whereas the GSH pool (reduced and oxidized forms) decreased with increasing concentration of AgNPs.In order to obtain direct evidence whether AgNPs causes oxidative stress, treated animals were incubated with non-fluorescent probe, 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA). A fluorescence signal was observed in both the AgNPs- and AgNO3 treated groups pointing to the production of reactive oxygen species (RS). Since ROS formation in F. candida is difficult to quantify, yeast strain BY4742 and mutants lacking oxidative-stress related protective enzymes were exploited as a further eukaryote model organism. As results, AgNPs and AgNO3 were found to also affect growth of yeast and induced oxidative stress.The effect of AgNPs on Collembola and yeast strains was similar to the one from AgNO3. However, AgNPs is less toxic due to the slow release of silver ions. In summary, the toxic effect of AgNPs on F. candida is caused by a combination of the release of silver ions from the AgNPs and formation of reactive species
Plasticity within stem cell hierarchies in mammalian epithelia
No full textTissue homeostasis and regeneration are fueled by resident stem cells that have the capacity to self-renew, and to generate all the differentiated cell types that characterize a particular tissue. Classical models of such cellular hierarchies propose that commitment and differentiation occur unidirectionally, with the arrows 'pointing away' from the stem cell. Recent studies, all based on genetic lineage tracing, describe various strategies employed by epithelial stem cell hierarchies to replace damaged or lost cells. While transdifferentiation from one tissue type into another ('metaplasia') appears to be generally forbidden in nonpathological contexts, plasticity within an individual tissue stem cell hierarchy may be much more common than previously appreciated. In this review, we discuss recent examples of such plasticity in selected mammalian epithelia, highlighting the different modes of regeneration and their implications for our understanding of cellular hierarchy and tissue self-renewal
Un modèle analytique des transferts de technologie
No full textThis study deals with international technology transfers. A theoretical and analytical model is presented as well as applications to real international economic problems. In the first part the author reminds the Ricardian model with a continuous of goods. In the second part, the model is applied to the study of technology transfers and an attempt is made to deal with endogeneous technological change using Pugel's approach. In the last part, the model is applied to the following questions: free technology transfer, the choice of an appropriate technology, the relation between wage rate and technology transfers.
Un modèle analytique des transferts de technologie
No full textThis study deals with international technology transfers. A theoretical and analytical model is presented as well as applications to real international economic problems. In the first part the author reminds the Ricardian model with a continuous of goods. In the second part, the model is applied to the study of technology transfers and an attempt is made to deal with endogeneous technological change using Pugel's approach. In the last part, the model is applied to the following questions: free technology transfer, the choice of an appropriate technology, the relation between wage rate and technology transfers
The transcriptional regulator FUBP1 influences disease outcome in murine and human myeloid leukemia
No full textDynamic formation of microvillus inclusions during enterocyte differentiation in Munc18-2–deficient intestinal organoids
No full textBackground & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID.
Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions.
Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin–positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane.
Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border
Transcriptional repression by the T-box proteins tbx18 and tbx15 depends on groucho corepressors
No full text10.1074/jbc.M703724200JOURNAL OF BIOLOGICAL CHEMISTRY2823525748-2575
Colorectal cancer organoid–stroma biobank allows subtype-specific assessment of individualized therapy responses
No full textIn colorectal cancers, the tumor microenvironment plays a key role in prognosis and therapy efficacy. Patient-derived tumor organoids (PDTO) show enormous potential for preclinical testing; however, cultured tumor cells lose important characteristics, including the consensus molecular subtypes (CMS). To better reflect the cellular heterogeneity, we established the colorectal cancer organoid–stroma biobank of matched PDTOs and cancer-associated fibroblasts (CAF) from 30 patients. Context-specific phenotyping showed that xenotransplantation or coculture with CAFs improves the transcriptomic fidelity and instructs subtype-specific stromal gene expression. Furthermore, functional profiling in coculture exposed CMS4-specific therapeutic resistance to gefitinib and SN-38 and prognostic expression signatures. Chemogenomic library screening identified patient- and therapy-dependent mechanisms of stromal resistance including MET as a common target. Our results demonstrate that colorectal cancer phenotypes are encrypted in the cancer epithelium in a plastic fashion that strongly depends on the context. Consequently, CAFs are essential for a faithful representation of molecular subtypes and therapy responses ex vivo
Immune escape of colorectal tumors via local LRH-1/Cyp11b1-mediated synthesis of immunosuppressive glucocorticoids.
No full textControl of tumor development and growth by the immune system critically defines patient fate and survival. What regulates the escape of colorectal tumors from destruction by the immune system is currently unclear. Here, we investigated the role of intestinal synthesis of glucocorticoids in the tumor development during inflammation-induced mouse model of colorectal cancer. We demonstrate that the local synthesis of immunoregulatory glucocorticoids has dual roles in the regulation of intestinal inflammation and tumor development. In the inflammation phase LRH-1/Nr5A2-regulated and Cyp11b1-mediated intestinal glucocorticoid synthesis prevents tumor development and growth. In established tumors, however, tumor-autonomous Cyp11b1-mediated glucocorticoid synthesis suppresses anti-tumor immune responses and promotes immune escape. Transplantation of glucocorticoid synthesis-proficient colorectal tumor organoids into immunocompetent recipient mice resulted in rapid tumor growth, whereas transplantation of Cyp11b1-deleted and glucocorticoid synthesis-deficient tumor organoids was characterized by reduced tumor growth and increased immune cell infiltration. In human colorectal tumors, high expression of steroidogenic enzymes correlated with the expression of other immune checkpoints and suppressive cytokines, and negatively correlated with overall patients' survival. Thus, LRH-1-regulated tumor-specific glucocorticoid synthesis contributes to tumor immune escape and represents a novel potential therapeutic target
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