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Martinsried, Germany

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Meyer S.U.,TU Munich | Thirion C.,SIRION BIOTECH | Polesskaya A.,University Paris - Sud | Bauersachs S.,Ludwig Maximilians University of Munich | And 4 more authors.
Cell Communication and Signaling | Year: 2015

Background: Elevated levels of the inflammatory cytokine TNF-α are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-α and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-α or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation. Results: In this study, we analyzed the impact of TNF-α or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance. Conclusions: The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1. This study indicates thatmiRNAs aremediators of the inhibitory effect of TNF-α on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-α by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-α or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets. © 2015 Meyer et al.; licensee BioMed Central.


Meyer S.U.,TU Munich | Meyer S.U.,Ludwig Maximilians University of Munich | Kaiser S.,Ludwig Maximilians University of Munich | Wagner C.,IMGM Laboratories GmbH | And 2 more authors.
PLoS ONE | Year: 2012

Background: Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. Methodology/Principal Findings: We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. Conclusions/Significance: We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments. © 2012 Meyer et al.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH.2011.2.4.3-2 | Award Amount: 8.01M | Year: 2011

A reduction in functional pancreatic beta cell mass is the key feature of type 1 and type 2 diabetes. Beta cell failure is exacerbated in the context of obesity and insulin resistance. Brown adipose tissue (BAT) is a highly metabolic organ, mediating energy dissipation and glucose disposal, thus contributing to maintain energy balance. BAT dysfunction contributes to obesity and impaired glucose metabolism, increasing functional demand on the beta cells. The cellular dysfunction of beta cells and BAT in diabetes is the fruit of defective signal transduction and organelle function, and the vulnerability to these molecular defects may be modulated by diabetes genes. To identify the crosstalk and pathways responsible for beta cell and BAT dysfunction and beta cell apoptosis in diabetes at its real level of complexity, this consortium will use cutting edge genetics, functional (epi)genomics, molecular biology and computational tools to reach an accurate organelle and cell diagnosis. This diagnosis will be used to define novel targets for intervention to prevent dysfunction and facilitate recovery of functionally impaired metabolic tissues. Some of these targets have already been identified by consortium members, including endoplasmic reticulum (ER) stress and mitochondrial dysfunction and apoptosis. Since many of these targets are related to dysfunction of specific organelles, we will focus on the novel concept of organelle therapy, aiming to preserve mitochondrial and ER function by the use of viral vectors and small molecule chemical probes. We will thus follow a two-pronged approach, namely a focused approach to restore known and specific cellular/organelle dysfunctions and a systems biology approach to identify novel targets for intervention. This, in combination with high throughput screening of large compound libraries, will translate discoveries into innovative therapeutic strategies that halt destruction and facilitate recovery of cell dysfunction in diabetes.


Grant
Agency: European Commission | Branch: H2020 | Program: RIA | Phase: PHC-02-2015 | Award Amount: 6.00M | Year: 2016

Type 2 diabetes (T2D) is a major public health problem, affecting 55 million European citizens. T2D ensues in individuals who develop a progressive pancreatic beta cell failure. T2D probably comprises a heterogeneous group of diseases. A new molecular taxonomy of T2D is essential for the development of medical care that is predictive, preventive and personalized. Currently available T2D therapies are not disease modifying: they treat hyperglycaemia without addressing its underlying cause, i.e. beta cell failure. In this proposal we seek to identify pathogenic molecular events that operate in the diseased tissue, i.e. the failing human beta cell, at their true level of complexity. T2DSystems will accomplish this ambitious goal by integrating human islet genetic and epigenetic data with disease-relevant environmental perturbations, metabolomics and functional studies, and use this knowledge to identify distinct human islet phenotypes in subgroups of patients. In closely interacting work packages, we will achieve the following goals: Compile and expand existing European bio-banks and datasets to create the Translational human pancreatic Islet Genotype tissue-Expression Resource (TIGER), a T2D systems biomedicine resource of unprecedented scale; Develop large-scale data analysis tools and both data driven and mechanistic probabilistic modelling frameworks to exploit TIGER towards system level biological insight; Translate these findings to identify stratified beta cell phenotypes in human cohorts. This will provide understanding of beta cell pathophysiology in vivo and enable stratified prevention and therapeutics. T2DSystems will enable the development of personalized diagnostic tests, taking into account individual environmental and genetic risk factors. The newly identified molecular disease mechanisms will provide the basis for development of novel therapies and for patient stratification to test individualized therapies.


Hochheimer A.,BRAIN AG | Hochheimer A.,Amgen Research GmbH | Krohn M.,BRAIN AG | Rudert K.,BRAIN AG | And 4 more authors.
Chemical Senses | Year: 2014

Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities. © The Author 2014. Published by Oxford University Press.


Patent
Sirion Biotech | Date: 2013-02-28

The present invention relates to a method for transducing a target cell, the method comprising the step of contacting a target cell with a retroviral vector and a poloxamer having a molecular weight of 12.8 kDa to about 15 kDa. Further, the invention relates to the use of a poloxamer as defined herein, optionally in combination with a polycationic substance as defined herein, for transducing a target cell with a retroviral vector and a kit comprising a retroviral vector, a poloxamer as defined herein and, optionally, instructions for use.


Patent
Sirion Biotech | Date: 2015-01-09

The present invention relates to a nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the vesicular stomatitis virus envelope glycoprotein (VSV-G) linked to a (poly)peptide comprising or consisting of a cell membrane-binding domain, said nucleic acid sequence comprising in 5 to 3 direction (a) a first sequence segment encoding an endoplasmic reticulum (ER) signal sequence; (b) a second sequence segment encoding said (poly)peptide comprising or consisting of a cell membrane-binding domain; (c) a third sequence segment encoding a linker; and (d) a fourth sequence segment encoding said VSV-G. Further, the invention relates to a vector comprising the nucleic acid molecule of the invention, a host cell comprising said vector or nucleic acid molecule, the polypeptide encoded by said nucleic acid molecule and a method of producing the polypeptide encoded by said nucleic acid molecule. In addition, the invention relates to a pseudotyped lentiviral vector particle, a method of transducing a cell as well as a kit comprising various combinations of the nucleic acid molecule, vectors, polypeptides and host cells of the invention.


Patent
Sirion Biotech | Date: 2013-07-04

The invention relates in a first embodiment to a method for increasing the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region, wherein said adenoviruses are generated in a production cell, the method comprising the steps of: (a) expressing in said production cell an adenoviral pIX polypeptide from a nucleic acid sequence encoding said adenoviral pIX polypeptide under the control of (i) at least a minimal endogenous pIX promoter and a heterologous promoter; or (ii) a heterologous promoter; and (b) expressing in said production cell the elements necessary for the production and assembly of said adenoviruses from corresponding coding sequences, thereby increasing the yield of said adenoviruses generated in said production cell in comparison to the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region generated in said production cell in the absence of said nucleic acid sequence encoding said adenoviral pIX polypeptide. In another embodiment, the invention relates to a method for constructing an adenovirus library, a production cell as well as the use of an adenoviral pIX polypeptide for increasing the yield of replication-incompetent adenoviruses having at least a partial deletion in the E1-region.


The present invention is related to a nucleic acid molecule, which is also referred to as third nucleic acid molecule, wherein the third nucleic acid molecule comprises


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