Untersuchung von Membran-DNA Komplexen in äußeren elektrischen Feldern

Untersuchung von Membran-DNA Komplexen in äußeren elektrischen Feldern Die vorliegende Arbeit beschreibt die Entwicklung, Optimierung und Automatisierung einer neuartigen Methode zum Transferieren von DNA-Molekülen in Polyacrylamidgele. Zuerst wird die DNA-Lösung durch Kapillarkräfte in geeignete poröse Membranen absorbiert. Danach wird ein äußeres elektrisches Feld angelegt. Die Bindungskräfte zwischen der Membran und der DNA überlagern sich mit der elektrischen Feldkraft derart, dass eine resultierende Kraft auf die DNA-Moleküle in Richtung des Gels wirkt. Die praktischen Arbeiten wurden auf der am EMBL entwickelten automatischen DNA-Sequenziereinheit Arakis durchgeführt. Über die automatische Detektion und Analyse der DNA-Fragmente hinaus konnte mit dieser Methode das Auftragen der Proben vollständig automatisiert werden. Membrangebundene Proben trugen dazu bei den Durchsatz auf Arakis, bei gleichbleibender Qualität der Sequenzdaten, um mehr als den Faktor 4 zu erhöhen. Die Methode wurde erfolgreich auf das Sequenziersystem ABI PRISM 377 der Firma 'Applied Biosystems' übertragen. Zur Kommerzialisierung wurde eine Lizenz an die Firma 'MWG-Biotech' vergeben

Normalizing for individual cell population context in the analysis of high-content cellular screens

<p>Abstract</p> <p>Background</p> <p>High-content, high-throughput RNA interference (RNAi) offers unprecedented possibilities to elucidate gene function and involvement in biological processes. Microscopy based screening allows phenotypic observations at the level of individual cells. It was recently shown that a cell's population context significantly influences results. However, standard analysis methods for cellular screens do not currently take individual cell data into account unless this is important for the phenotype of interest, i.e. when studying cell morphology.</p> <p>Results</p> <p>We present a method that normalizes and statistically scores microscopy based RNAi screens, exploiting individual cell information of hundreds of cells per knockdown. Each cell's individual population context is employed in normalization. We present results on two infection screens for hepatitis C and dengue virus, both showing considerable effects on observed phenotypes due to population context. In addition, we show on a non-virus screen that these effects can be found also in RNAi data in the absence of any virus. Using our approach to normalize against these effects we achieve improved performance in comparison to an analysis without this normalization and hit scoring strategy. Furthermore, our approach results in the identification of considerably more significantly enriched pathways in hepatitis C virus replication than using a standard analysis approach.</p> <p>Conclusions</p> <p>Using a cell-based analysis and normalization for population context, we achieve improved sensitivity and specificity not only on a individual protein level, but especially also on a pathway level. This leads to the identification of new host dependency factors of the hepatitis C and dengue viruses and higher reproducibility of results.</p

Dicer and microRNAs protect adult dopamine neurons

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (Dicer(HET)) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (Dicer(CKO)). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.Peer reviewe

Reciprocal Effects of Fibroblast Growth Factor Receptor Signaling on Dengue Virus Replication and Virion Production

International audienceDengue virus (DENV) is a human arboviral pathogen accounting for 390 million infections every year. The available vaccine has limited efficacy, and DENV-specific drugs have not been generated. To better understand DENV-host cell interaction, we employed RNA interference-based screening of the human kinome and identified fibroblast growth factor receptor 4 (FGFR4) to control the DENV replication cycle. Pharmacological inhibition of FGFR exerts a reciprocal effect by reducing DENV RNA replication and promoting the production of infectious virus particles. Addressing the latter effect, we found that the FGFR signaling pathway modulates intracellular distribution of DENV particles in a PI3K-dependent manner. Upon FGFR inhibition, virions accumulate in the trans-Golgi network compartment, where they undergo enhanced maturation cleavage of the envelope protein precursor membrane (prM), rendering virus particles more infectious. This study reveals an unexpected reciprocal role of a cellular receptor tyrosine kinase regulating DENV RNA replication and the production of infectious virions

Machine learning on large scale perturbation screens for SARS-CoV-2 host factors identifies β-catenin/CBP inhibitor PRI-724 as a potent antiviral

Expanding antiviral treatment options against SARS-CoV-2 remains crucial as the virus evolves under selection pressure which already led to the emergence of several drug resistant strains. Broad spectrum host-directed antivirals (HDA) are promising therapeutic options, however the robust identification of relevant host factors by CRISPR/Cas9 or RNA interference screens remains challenging due to low consistency in the resulting hits. To address this issue, we employed machine learning, based on experimental data from several knockout screens and a drug screen. We trained classifiers using genes essential for virus life cycle obtained from the knockout screens. The machines based their predictions on features describing cellular localization, protein domains, annotated gene sets from Gene Ontology, gene and protein sequences, and experimental data from proteomics, phospho-proteomics, protein interaction and transcriptomic profiles of SARS-CoV-2 infected cells. The models reached a remarkable performance suggesting patterns of intrinsic data consistency. The predicted HDF were enriched in sets of genes particularly encoding development, morphogenesis, and neural processes. Focusing on development and morphogenesis-associated gene sets, we found β-catenin to be central and selected PRI-724, a canonical β-catenin/CBP disruptor, as a potential HDA. PRI-724 limited infection with SARS-CoV-2 variants, SARS-CoV-1, MERS-CoV and IAV in different cell line models. We detected a concentration-dependent reduction in cytopathic effects, viral RNA replication, and infectious virus production in SARS-CoV-2 and SARS-CoV-1-infected cells. Independent of virus infection, PRI-724 treatment caused cell cycle deregulation which substantiates its potential as a broad spectrum antiviral. Our proposed machine learning concept supports focusing and accelerating the discovery of host dependency factors and identification of potential host-directed antivirals

PITX1 is a regulator of TERT expression in prostate cancer with prognostic power

Simple Summary Most prostate cancer is of an indolent form and is curable. However, some prostate cancer belongs to rather aggressive subtypes leading to metastasis and death, and immediate therapy is mandatory. However, for these, the therapeutic options are highly invasive, such as radical prostatectomy, radiation or brachytherapy. Hence, a precise diagnosis of these tumor subtypes is needed, and the thus far applied diagnostic means are insufficient for this. Besides this, for their endless cell divisions, prostate cancer cells need the enzyme telomerase to elongate their telomeres (chromatin endings). In this study, we developed a gene regulatory model based on large data from transcription profiles from prostate cancer and chromatin-immuno-precipitation studies. We identified the developmental regulator PITX1 regulating telomerase. Besides observing experimental evidence of PITX1′s functional role in telomerase regulation, we also found PITX1 serving as a prognostic marker, as concluded from an analysis of more than 15,000 prostate cancer samples. Abstract The current risk stratification in prostate cancer (PCa) is frequently insufficient to adequately predict disease development and outcome. One hallmark of cancer is telomere maintenance. For telomere maintenance, PCa cells exclusively employ telomerase, making it essential for this cancer entity. However, TERT, the catalytic protein component of the reverse transcriptase telomerase, itself does not suit as a prognostic marker for prostate cancer as it is rather low expressed. We investigated if, instead of TERT , transcription factors regulating TERT may suit as prognostic markers. To identify transcription factors regulating TERT , we developed and applied a new gene regulatory modeling strategy to a comprehensive transcriptome dataset of 445 primary PCa. Six transcription factors were predicted as TERT regulators, and most prominently, the developmental morphogenic factor PITX1. PITX1 expression positively correlated with telomere staining intensity in PCa tumor samples. Functional assays and chromatin immune-precipitation showed that PITX1 activates TERT expression in PCa cells. Clinically, we observed that PITX1 is an excellent prognostic marker, as concluded from an analysis of more than 15,000 PCa samples. PITX1 expression in tumor samples associated with (i) increased Ki67 expression indicating increased tumor growth, (ii) a worse prognosis, and (iii) correlated with telomere length

Systematic RNA-interference in primary human monocyte-derived macrophages: A high-throughput platform to study foam cell formation

Macrophage-derived foam cells are key regulators of atherogenesis. They accumulate in atherosclerotic plaques and support inflammatory processes by producing cytokines and chemokines. Identifying factors that regulate macrophage lipid uptake may reveal therapeutic targets for coronary artery disease (CAD). Here, we establish a high-throughput screening workflow to systematically identify genes that impact the uptake of DiI-labeled low-density lipoprotein (LDL) into monocyte-derived primary human macrophages. For this, monocytes isolated from peripheral blood were seeded onto 384-well plates, solid-phase transfected with siRNAs, differentiated in vitro into macrophages, and LDL-uptake per cell was measured by automated microscopy and quantitative image analysis. We applied this workflow to study how silencing of 89 genes impacts LDL-uptake into cells from 16 patients with CAD and 16 age-matched controls. Silencing of four novel genes (APOC1, CMTM6, FABP4, WBP5) reduced macrophage LDL-uptake. Additionally, knockdown of the chemokine receptor CXCR4 reduced LDL-uptake, most likely through a G-protein coupled mechanism that involves the CXCR4 ligand macrophage-induced factor (MIF), but is independent of CXCL12. We introduce a high-throughput strategy to systematically study gene function directly in primary CAD-patient cells. Our results propose a function for the MIF/CXCR4 signaling pathway, as well as several novel candidate genes impacting lipid uptake into human macrophages

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI