Center for Biotechnology and Biomedicine

Leipzig, Germany

Center for Biotechnology and Biomedicine

Leipzig, Germany
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PubMed | University of Leipzig, University of Cologne, RWTH Aachen, The Interdisciplinary Center and 3 more.
Type: Journal Article | Journal: The Journal of cell biology | Year: 2015

Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation.


Jahnke H.-G.,Center for Biotechnology and Biomedicine | Poenick S.,Center for Biotechnology and Biomedicine | Maschke J.,University of Leipzig | Kendler M.,University of Leipzig | And 2 more authors.
Cancer Research | Year: 2014

Stage III/IV melanoma remains incurable in most cases due to chemotherapeutic resistance. Thus, predicting and monitoring chemotherapeutic responses in this setting offer great interest. To overcome limitations of existing assays in evaluating the chemosensitivity of dissociated tumor cells, we developed a label-free monitoring system to directly analyze the chemosensitivity of undissociated tumor tissue. Using a preparation of tumor micro-fragments (TMF) established from melanoma biopsies, we characterized the tissue organization and biomarker expression by immunocytochemistry. Robust generation of TMF was established successfully and demonstrated on a broad range of primary melanoma tumors and tumor metastases. Organization and biomarker expression within the TMF were highly comparable with tumor tissue, in contrast to dissociated, cultivated tumor cells. Using isolated TMF, sensitivity to six clinically relevant chemotherapeutic drugs (dacarbazine, doxorubicin, paclitaxel, cisplatin, gemcitabine, and treosulfan) was determined by impedance spectroscopy in combination with a unique microcavity array technology we developed. In parallel, comparative analyses were performed on monolayer tumor cell cultures. Lastly, we determined the efficacy of chemotherapeutic agents on TMF by impedance spectroscopy to obtain individual chemosensitivity patterns. Our results demonstrated nonpredictable differences in the reaction of tumor cells to chemotherapy in TMF by comparison with dissociated, cultivated tumor cells. Our direct impedimetric analysis of melanoma biopsies offers a direct ex vivo system to more reliably predict patient-specific chemosensitivity patterns and to monitor antitumor efficacy. © 2014 American Association for Cancer Research.


Jahnke H.-G.,Center for Biotechnology and Biomedicine | Steel D.,Cellectis | Fleischer S.,Center for Biotechnology and Biomedicine | Seidel D.,Center for Biotechnology and Biomedicine | And 5 more authors.
PLoS ONE | Year: 2013

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks. © 2013 Jahnke et al.


Wu S.,Tsinghua University | Wu S.,Cooperative Innovation Center for High Performance Computing | Wu S.,Center for Biotechnology and Biomedicine | Ge Y.,Cooperative Innovation Center for High Performance Computing | And 5 more authors.
Cell Cycle | Year: 2014

Telomerase is often upregulated during initiation and/or progression of human tumors, suggesting that repression of telomerase might inhibit cancer growth or progression. Here, we report that BRG1, the ATPase subunit of the SWI/ SNF chromatin remodeling complex, is a general suppressor of hTERT transcription in human cancer cells. While overexpression of BRG1 inhibits hTERT transcription, depletion of BRG1 stimulates transcription of hTERT, leading to higher telomerase activity and longer telomeres. Chromatin-immunoprecipitation assays revealed that BRG1 binds to the transcription start site (TSS) of the hTERT promoter and forms a ternary complex with histone deacetylase 2 (HDAC2). BRG1 remodels chromatin structure to facilitate the action of HDAC2, leading to deacetylation of H3K9ac and H4ac at the TSS and suppression of hTERT transcription. On the other hand, β-catenin binds to the TSS and stimulates hTERT transcription. Thus, BRG1/HDAC2 and β-catenin constitute a manipulative apparatus at the TSS to play opposite but complementary roles in regulating hTERT expression. These results uncover a yin-yang mechanism in modulating hTERT transcription and provide explanation for limited transcription of hTERT in human cancer cells. BRG1/ HDAC2 may have a potential as an anti-cancer therapeutic and/or for reactivating cellular proliferative capacity in the context of in vitro tissue engineering. © 2014 Taylor & Francis Group, LLC.


Jahnke H.-G.,Center for Biotechnology and Biomedicine | Braesigk A.,Center for Biotechnology and Biomedicine | Mack T.G.A.,German Center for Neurodegenerative Diseases | Mack T.G.A.,KeyNeurotek Pharmaceuticals AG | And 4 more authors.
Biosensors and Bioelectronics | Year: 2012

Alzheimer's disease (AD) and other tauopathies comprise death of cell bodies, synapses and neurites but there is surprising little knowledge of the temporal sequence and the causal relationships among these events. Here, we present a novel biosensoric approach to monitor retrograde neurite degeneration before cell death occurs. We induced tau hyperphosphorylation in organotypic hippocampal slice cultures (OHSC) and applied marker-independent real-time electrical impedance spectroscopy (EIS) for cellular real-time pathology monitoring. Using this approach, we were able to define two distinct phases of neurite degeneration, first a rapid swelling of axonal processes that manifests itself in relative impedance above control levels followed by a slower phase of collapse and subsequent fragmentation indicated by decreased relative impedance below control levels. Initial axon swelling is strictly dose-dependent and swelling intensity correlates with second phase impedance decrease implicating a causative link between both degenerative mechanisms. Moreover, suppressing tau hyperphosphorylation by kinase inhibition nearly prevented both phases of axon degeneration. Our findings demonstrate that the temporal sequence of tau-triggered neurite degeneration can be directly visualized by EIS-based, non-invasive and label-free monitoring. We therefore suggest this approach as a powerful extension of high content applications to study mechanisms of neurite degeneration and to exploit therapeutic options against AD and tau-related disorders. © 2011 Elsevier B.V.


Jahnke H.-G.,Center for Biotechnology and Biomedicine | Heimann A.,Medical University Mainz | Azendorf R.,Center for Biotechnology and Biomedicine | Mpoukouvalas K.,Fraunhofer Institute of Microtechnology Mainz | And 4 more authors.
Biosensors and Bioelectronics | Year: 2013

Until today, brain tumors especially glioblastoma are difficult to treat and therefore, results in a poor survival rate of 0-14% over five years. To overcome this problem, the development of novel therapeutics as well as optimization of neurosurgical procedures to remove the tumor tissue are subject of intensive research. The main problem of the tumor excision, as the primary clinical intervention is the diffuse infiltration of the tumor cells in unaltered brain tissue that complicates the complete removal of residual tumor cells. In this context, we are developing novel approaches for the label-free discrimination between tumor tissue and unaltered brain tissue in real-time during the surgical process. Using our impedance spectroscopy-based measurement system in combination with flexible microelectrode arrays we could successfully demonstrate the discrimination between a C6-glioma and unaltered brain tissue in an in vivo rat model. The analysis of the impedance spectra revealed specific impedance spectrum shape characteristics of physiologic neuronal tissue in the frequency range of 10-500. kHz that were significantly different from the tumor tissue. Moreover, we used an adapted equivalent circuit model to get a deeper understanding for the nature of the observed effects. The impedimetric label-free and real-time discrimination of tumor from unaltered brain tissue offers the possibility for the implementation in surgical instruments to support surgeons to decide, which tissue areas should be removed and which should be remained. © 2013 Elsevier B.V.


Heine C.,University of Leipzig | Sygnecka K.,University of Leipzig | Scherf N.,TU Dresden | Grohmann M.,University of Leipzig | And 2 more authors.
Neuropharmacology | Year: 2015

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R-/-) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration. © 2015 Elsevier Ltd. All rights reserved.


PubMed | University of Leipzig and Center for Biotechnology and Biomedicine
Type: Journal Article | Journal: Cancer research | Year: 2014

Stage III/IV melanoma remains incurable in most cases due to chemotherapeutic resistance. Thus, predicting and monitoring chemotherapeutic responses in this setting offer great interest. To overcome limitations of existing assays in evaluating the chemosensitivity of dissociated tumor cells, we developed a label-free monitoring system to directly analyze the chemosensitivity of undissociated tumor tissue. Using a preparation of tumor micro-fragments (TMF) established from melanoma biopsies, we characterized the tissue organization and biomarker expression by immunocytochemistry. Robust generation of TMF was established successfully and demonstrated on a broad range of primary melanoma tumors and tumor metastases. Organization and biomarker expression within the TMF were highly comparable with tumor tissue, in contrast to dissociated, cultivated tumor cells. Using isolated TMF, sensitivity to six clinically relevant chemotherapeutic drugs (dacarbazine, doxorubicin, paclitaxel, cisplatin, gemcitabine, and treosulfan) was determined by impedance spectroscopy in combination with a unique microcavity array technology we developed. In parallel, comparative analyses were performed on monolayer tumor cell cultures. Lastly, we determined the efficacy of chemotherapeutic agents on TMF by impedance spectroscopy to obtain individual chemosensitivity patterns. Our results demonstrated nonpredictable differences in the reaction of tumor cells to chemotherapy in TMF by comparison with dissociated, cultivated tumor cells. Our direct impedimetric analysis of melanoma biopsies offers a direct ex vivo system to more reliably predict patient-specific chemosensitivity patterns and to monitor antitumor efficacy.


PubMed | Martin Luther University of Halle Wittenberg, University of Nottingham and Center for Biotechnology and Biomedicine
Type: Journal Article | Journal: Journal of applied toxicology : JAT | Year: 2015

Di(2-ethylhexyl)phthalate (DEHP) is the most common plasticizer in plastic devices of everyday use. It is a ubiquitous environmental contaminant and primarily known to impair male gonadal development and fertility. Studies concerning the long-term effects of prenatal DEHP exposure on certain diseases [The Developmental Origins of Health and Disease paradigm (DOHaD) hypothesis] are scarce although it is proven that DEHP crosses the placenta. Rising environmental pollution during the last centuries coincides with an increasing prevalence of cardiovascular and metabolic diseases. We have investigated the effects of an early embryonic DEHP exposure at different developmental stages on cardiomyogenesis. We used an in-vitro model, the murine P19 embryonic carcinoma cell line (P19 ECC), mimicking early embryonic stages up to differentiated beating cardiomyocytes. P19 ECC were exposed to DEHP (5, 50, 100gml(-1)) at the undifferentiated stage for 5days and subsequently differentiated to beating cardiomyocytes. We analyzed the expression of metabolic (Pparg1, Fabp4 and Glut4), cardiac (Myh6, Gja1) and methylation (Dnmt1, Dnmt3a) marker genes by quantitative real-time PCR (qRT-PCR), beating rate and the differentiation velocity of the cells. The methylation status of Pparg1, Ppara and Glut4 was investigated by pyrosequencing. DEHP significantly altered the expression of all investigated genes. The beating rate and differentiation velocity were accelerated. Exposure to DEHP led to small but statistically significant increases in methylation of specific CpGs within Ppara and Pparg1, which otherwise were generally hypomethylated, but methylation of Glut4 was unaltered. Early DEHP exposure of P19 ECC alters the expression of genes associated with cellular metabolism and the functional features of cardiomyocytes.


PubMed | TU Dresden, University of Leipzig and Center for Biotechnology and Biomedicine
Type: | Journal: Neuropharmacology | Year: 2015

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R(-/-)) mice, the ADPS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration.

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