Identifizierung und Charakterisierung kleiner nicht-kodierender RNA Moleküle aus dem Zellkern muriner Embryonaler Stammzellen und Embryonic Bodies

In den vergangenen Jahren hat sich unser Verständnis von der funktionellen Diversität der RNA erheblich vergrößert. Besonders konnte eine große Anzahl nicht-kodierender RNA (ncRNA) Moleküle identifiziert werden, die entscheidende Schritte in der zellulären Differenzierung steuern. Angesichts der komplexen Natur der neuronalen Entwicklung wächst zunehmend die Erkenntnis, dass nicht-kodierende RNA eine bedeutende und bislang noch nicht aufgeklärte Rolle in der Kontrolle der zugrunde liegenden zellulären Differenzierungsprozesse spielen könnte. Zusätzlich zu ihrer Wichtigkeit für die neuronale Entwicklung könnte nicht-kodierende RNA auch an der Ausbildung neurodegenerativer Erkrankungen beteiligt sein. Um neue nicht-kodierende RNA aufklären zu können, die an diesen Prozessen beteiligt sind, wurde ein in vitro Modell der frühen neuronalen Differenzierung verwendet und aus zwei unterschiedlichen Differenzierungsstadien kleine nukleare RNA isoliert. Ausgehend vom Embryonalen Stammzellen (ES Zellen) der Maus wurden Spheroblasten acht Tage alter Embryonic Bodies (EBs) erzeugt. Aus beiden Entwicklungsstadien, ES Zellen und EBs, wurde RNA isoliert und dazu verwendet cDNA Bibliotheken aus kleiner nuklearer RNA herzustellen. Ein Aliquot der isolierten RNA wurde mittels in vitro Capping mit einer m7G-Cap-Struktur versehen. Hierdurch konnte ein spezifisches Muster der kleinen nuklearen RNA innerhalb der beiden Differenzierungsstadien aufgedeckt werden. RNA entsprechender Größe wurde dazu verwendet um cDNA Bibliotheken der beiden Entwicklungsstadien herzustellen. Insgesamt konnten 14 neue nukleare nicht-kodierende RNA Kandidaten in den beiden cDNA Bibliotheken identifiziert werden. Vier davon entfielen auf die cDNA Bibliothek aus ES Zellen, zehn auf die der EBs. Neun der neuen ncRNA Kandidaten Genen konnte in intronischen Bereichen des Genoms lokalisiert werden, nur zwei ncRNA Kandidaten konnten exonischen Regionen zugeordnet werden. Die Expression eines ncRNA Kandidaten (2-57) konnte im Northern Blot bestätigt werden. 2-57 zeigte eine ausgeprägte Expression vor allem in EBs und AtT-20 Zellen, während er in ES Zellen nicht exprimiert war. Neben den neuen ncRNA Kandidaten wurden auch bekannte ncRNAs, die in den cDNA Bibliotheken nachgewiesen werden konnten, auf ihre Expression untersucht. Pre-mi-690 und Y3 scRNA zeigten hierbei eine geregelte Expression in den beiden untersuchten Dif-ferenzierungsstadien. Beide waren in EBs schwächer exprimiert, als in ES Zellen und den untersuchte Zelllinien

Individualized versus Standardized Risk Assessment in Patients at High Risk for Adverse Drug Reactions (The IDrug Randomized Controlled Trial)–Never Change a Running System?

The aim of this study was to compare effects of an individualized with a standardized risk assessment for adverse drug reactions to improve drug treatment with antithrombotic drugs in older adults. A randomized controlled trial was conducted in general practitioner (GP) offices. Patients aged 60 years and older, multi-morbid, taking antithrombotic drugs and at least one additional drug continuously were randomized to individualized and standardized risk assessment groups. Patients were followed up for nine months. A composite endpoint defined as at least one bleeding, thromboembolic event or death reported via a trigger list was used. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. In total, N = 340 patients were enrolled from 43 GP offices. Patients in the individualized risk assessment group met the composite endpoint more often than in the standardized group (OR 1.63 [95%CI 1.02–2.63]) with multiple adjustments. The OR was higher in patients on phenprocoumon treatment (OR 1.99 [95%CI 1.05–3.76]), and not significant on DOAC treatment (OR 1.52 [95%CI 0.63–3.69]). Pharmacogenenetic variants of CYP2C9, 2C19 and VKORC1 were not observed to be associated with the composite endpoint. The results of this study may indicate that the time point for implementing individualized risk assessments is of importance

Molecular Genetic Techniques in Biomarker Analysis Relevant for Drugs Centrally Approved in Europe

On the basis of scientific evidence, information on the option, recommendation or requirement to test for pharmacogenetic or pharmacogenomic biomarkers is incorporated in the Summary of Product Characteristics of an increasing number of drugs in Europe. A screening of the Genetic Testing Registry (GTR) showed that a variety of molecular genetic testing methods is currently offered worldwide in testing services with regard to according drugs and biomarkers. Thereby, among the methodology indicated in the screened GTR category ‘Molecular Genetics’, next-generation sequencing is applied for identification of the largest proportion of evaluated biomarkers that are relevant for therapeutic management of centrally approved drugs in Europe. However, sufficient information on regulatory clearances, clinical utility, analytical and clinical validity of applied methods is rarely provided

Cytogenetic and Biochemical Genetic Techniques for Personalized Drug Therapy in Europe

For many authorized drugs, accumulating scientific evidence supports testing for predictive biomarkers to apply personalized therapy and support preventive measures regarding adverse drug reactions and treatment failure. Here, we review cytogenetic and biochemical genetic testing methods that are available to guide therapy with drugs centrally approved in the European Union (EU). We identified several methods and combinations of techniques registered in the Genetic Testing Registry (GTR), which can be used to guide therapy with drugs for which pharmacogenomic-related information is provided in the European public assessment reports. Although this registry provides information on genetic tests offered worldwide, we identified limitations regarding standard techniques applied in clinical practice and the information on test validity rarely provided in the according sections

Transcriptional profiling of breast cancer-associated lymphatic vessels reveals VCAM-1 as regulator of lymphatic invasion and permeability

Tumor-associated lymphangiogenesis and lymphatic invasion of tumor cells correlate with poor outcome in many tumor types, including breast cancer. Various explanations for this correlation have been suggested in the past, including the promotion of lymphatic metastasis and an immune-inhibitory function of lymphatic endothelial cells (LECs). However, the molecular features of tumor-associated lymphatic vessels and their implications for tumor progression have been poorly characterized. Here, we report the first transcriptional analysis of tumor-associated LECs directly isolated from the primary tumor in an orthotopic mouse model of triple negative breast cancer (4T1). Gene expression analysis showed a strong upregulation of inflammation-associated genes, including endothelial adhesion molecules such as VCAM-1, in comparison to LECs derived from control tissue. In vitro experiments demonstrated that VCAM-1 is not involved in the adhesion of tumor cells to LECs but unexpectedly promoted lymphatic permeability by weakening of lymphatic junctions, most likely through a mechanism triggered by interactions with integrin α4 which was also induced in tumor-associated LECs. In line with this, in vivo blockade of VCAM-1 reduced lymphatic invasion of 4T1 cells. Taken together, our findings suggest that disruption of lymphatic junctions and increased permeability via tumor-induced lymphatic VCAM-1 expression may represent a new target to block lymphatic invasion and metastasis

Distinct and overlapping gene regulatory networks in BMP- and HDAC-controlled cell fate determination in the embryonic forebrain

<p>Abstract</p> <p>Background</p> <p>Both bone morphogenetic proteins (BMPs) and histone deacetylases (HDACs) have previously been established to play a role in the development of the three major cell types of the central nervous system: neurons, astrocytes, and oligodendrocytes. We have previously established a connection between these two protein families, showing that HDACs suppress BMP-promoted astrogliogenesis in the embryonic striatum. Since HDACs act in the nucleus to effect changes in transcription, an unbiased analysis of their transcriptional targets could shed light on their downstream effects on BMP-signaling.</p> <p>Results</p> <p>Using neurospheres from the embryonic striatum as an <it>in vitro</it> system to analyze this phenomenon, we have performed microarray expression profiling on BMP2- and TSA-treated cultures, followed by validation of the findings with quantitative RT-PCR and protein analysis. In BMP-treated cultures we first observed an upregulation of genes involved in cell-cell communication and developmental processes such as members of BMP and canonical Wnt signaling pathways. In contrast, in TSA-treated cultures we first observed an upregulation of genes involved in chromatin modification and transcription. Interestingly, we could not record direct changes in the protein levels of canonical members of BMP2 signaling, but we did observe an upregulation of both the transcription factor STAT3 and its active isoform phospho-STAT3 at the protein level.</p> <p>Conclusions</p> <p>STAT3 and SMAD1/5/8 interact synergistically to promote astrogliogenesis, and thus we show for the first time that HDACs act to suppress BMP-promoted astrogliogenesis by suppression of the crucial partner STAT3.</p

Real-Time Monitoring of Cisplatin-Induced Cell Death

Since the discovery of cisplatin more than 40 years ago and its clinical introduction in the 1970s an enormous amount of research has gone into elucidating the mechanism of action of cisplatin on tumor cells. With a novel cell biosensor chip system allowing continuous monitoring of respiration, glycolysis, and impedance we followed cisplatin treatment of different cancer cell lines in real-time. Our measurements reveal a first effect on respiration, in all cisplatin treated cell lines, followed with a significant delay by interference with glycolysis in HT-29, HCT-116, HepG2, and MCF-7 cells but not in the cisplatin-resistant cell line MDA-MB-231. Most strikingly, cell death started in all cisplatin-sensitive cell lines within 8 to 11 h of treatment, indicating a clear time frame from exposure, first response to cisplatin lesions, to cell fate decision. The time points of most significant changes were selected for more detailed analysis of cisplatin response in the breast cancer cell line MCF-7. Phosphorylation of selected signal transduction mediators connected with cellular proliferation, as well as changes in gene expression, were analyzed in samples obtained directly from sensor chips at the time points when changes in glycolysis and impedance occurred. Our online cell biosensor measurements reveal for the first time the time scale of metabolic response until onset of cell deat

Effects of genetic variability of CYP2D6 on neural substrates of sustained attention during on-task activity

The polymorphic drug-metabolizing enzyme CYP2D6, which is responsible for the metabolism of most psychoactive compounds, is expressed not only in the liver, but also in the brain. The effects of its marked genetic polymorphism on the individual capacity to metabolize drugs are well known, but its role in metabolism of neural substrates affecting behavior personality or cognition, suggested by its CNS expression, is a long-standing unresolved issue. To verify earlier findings suggesting a potential effect on attentional processes, we collected functional imaging data, while N = 415 participants performed a simple task in which the reward for correct responses varied. CYP2D6 allelic variants predicting higher levels of enzymatic activity level were positively associated with cortical activity in occipito-parietal areas as well as in a right lateralized network known to be activated by spatial attentional tasks. Reward-related modulation of activity in cortical areas was more pronounced in poor metabolizers. In conjunction with effects on reaction times, our findings provide evidence for reduced cognitive efficiency in rapid metabolizers compared to poor metabolizers in on-task attentional processes manifested through differential recruitment of a specific neural substrate