Simultaneous Visualization of Both Signaling Cascade Activity and End-Point Gene Expression in Single Cells

We have developed an approach for simultaneous detection of individual endogenous protein modifications and mRNA molecules in single cells in situ. For this purpose we combined two methods previously developed in our lab: in situ proximity ligation assay for the detection of individual protein interactions and -modifications and in situ detection of single mRNA molecules using padlock probes. As proof-of-principle, we demonstrated the utility of the method for simultaneous detection of phosphorylated PDGFRβ and DUSP6/MKP-3 mRNA molecules in individual human fibroblasts upon PDGF-BB stimulation. Further we applied drugs disrupting the PDGFRβ signaling pathway at various sites to show that this combined method can concurrently monitor the molecular effect of the drugs, i.e. inhibition of downstream signaling from the targeted node in the signaling pathway. Due to its ability to detect different types of molecules in single cells in situ the method presented here can contribute to a deeper understanding of cell-to-cell variations and can be applied to e.g. pinpoint effector sites of drugs in a signaling pathway

Franskundervisning og det virtuelle læringsrum

Undervisning i fransk er under stadig udvikling, og da det for mig har været tæt for- bundet med såvel den teknologiske udvikling af computere som udbredelsen af Internet, vil jeg berette om nogle af de erfaringer, jeg har gjort mig, krydret med konkrete eksempler, og om den fremtid, jeg forestiller mig for faget. (...

ATC-ICD: enabling domain experts to explore and evaluate machine learning models estimating diagnoses from filled predictions

Introduction Administrative and reimbursement data from the Austrian health care system is linked and utilized for research and to support policy makers. Lacking standardized, reliable and systematic coding of diagnoses in the outpatient sector, statistical and machine learning models are developed to estimate individual diagnoses coded as ICD-10 based on filled prescriptions (ATC codes), hence called “ATC->ICD models”. Evaluating the performance of such models, presenting predictions on a global as well as individual level, comparing different technological approaches and establishing trust by providing an intuitive insight into results for non-technical users are the aim of this project. Method ATC->ICD models are presented utilizing interactive web interfaces based on the R shiny package. As one size does not fit all, customized applications are required for different models and points of view. Applying modularization of reoccurring functionality and retaining design principles like a common dashboard layout facilitates the development and training of users. Software containers and centralized infrastructure providing e.g. backup, encryption and authentication enables efficient deployment of new application and their maintenance. Results We developed interactive web-based dashboards enabling experts to explore the prediction of single ATC->ICD models and compare the output of different approaches. The possibility to export and annotate results allows us to collect expert opinions, enhance understanding and gain acceptance conveniently. The combination of various dynamic controls, e.g. to filter, search, sort and cluster results, provides flexible access to complex models and large datasets. Linked and interactive graphs and tables help to understand valid and identify erroneous results much faster than with raw output and printed reports. Conclusion Presenting ATC->ICD models renders them accessible to data scientists and domain experts. It allows us to collect valuable feedback and gain trust in complex, hard to understand methodologies and results

(E)-Methyl 2-[(2S,3S,12bR)-3-ethyl-8-meth­oxy-1,2,3,4,6,7,12,12b-octa­hydro­indolo[2,3-a]quinolizin-2-yl]-3-methoxy­acrylate ethanol solvate

In the title compound, C23H30N2O4·C2H6O, the indole derivative has four fused rings, forming an indolo[2-3a]quinolizine system, in which one six-membered ring is directly connected to the indole unit and has a distorted chair conformation. The fourth ring is also a six-membered ring, depicting a regular chair conformation. In the crystal, the mol­ecules are linked by N—H⋯O and O—H⋯N inter­actions, forming a C(7) chain

The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by hydrogen peroxide to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Specificity constants indicate that SCN− is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. While HOCl and HOBr are powerful oxidizing agents, HOSCN is a less reactive, but more specific, oxidant which targets thiols and especially low pKa species. In the present study we show that HOSCN targets cysteine residues present in PTPs (protein tyrosine phosphatases) with this resulting in a loss of PTP activity for the isolated enzyme, in cell lysates and intact J774A.1 macrophage-like cells. Inhibition also occurs with MPO-generated HOCl and HOBr, but is more marked with MPO-generated HOSCN, particularly at longer incubation times. This inhibition is reversed by dithiothreitol, particularly at early time points, consistent with the reversible oxidation of the active site cysteine residue to give either a cysteine–SCN adduct or a sulfenic acid. Inhibition of PTP activity is associated with increased phosphorylation of p38a and ERK2 (extracellular-signal-regulated kinase 2) as detected by Western blot analysis and phosphoprotein arrays, and results in altered MAPK (mitogen-activated protein kinase) signalling. These data indicate that the highly selective targeting of some protein thiols by HOSCN can result in perturbation of cellular phosphorylation and altered cell signalling. These changes occur with (patho)physiological concentrations of SCN− ions, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage, particularly in smokers who have elevated plasma levels of SCN−

Sin3b interacts with Myc and decreases Myc levels

Myc expression is deregulated in many human cancers. A yeast two-hybrid screen has revealed that the transcriptional repressor Sin3b interacts with Myc protein. Endogenous Myc and Sin3b co-localize and interact in the nuclei of human and rat cells, as assessed by co-immunoprecipitation, immunofluorescence, and proximity ligation assay. The interaction is Max-independent. A conserved Myc region (amino acids 186-203) is required for the interaction with Sin3 proteins. Histone deacetylase 1 is recruited to Myc-Sin3b complexes, and its deacetylase activity is required for the effects of Sin3b on Myc. Myc and Sin3a/b co-occupied many sites on the chromatin of human leukemia cells, although the presence of Sin3 was not associated with gene down-regulation. In leukemia cells and fibroblasts, Sin3b silencing led to Myc up-regulation, whereas Sin3b overexpression induced Myc deacetylation and degradation. An analysis of Sin3b expression in breast tumors revealed an association between low Sin3b expression and disease progression. The data suggest that Sin3b decreases Myc protein levels upon Myc deacetylation. As Sin3b is also required for transcriptional repression by Mxd-Max complexes, our results suggest that, at least in some cell types, Sin3b limits Myc activity through two complementary activities: Mxd-dependent gene repression and reduction of Myc levels

Different states of integrin LFA-1 aggregation are controlled through its association with tetraspanin CD9

This is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta - Mollecular Cell Research. A definitive version was subsequently published in Biochimica et Biophysica Acta - Mollecular Cell Research, 1853.10 (2015): 2464-2480 DOI: 10.1016/j.bbamcr.2015.05.018The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on coimmunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilisation conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggests a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregationThis work was supported by grant SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECO», (to C.C.). R.R.M. salary is supported by a «Profesor Ayudante» position from Departamento de Biología, Facutad de Ciencias, Universidad Autónoma de Madri