BioM BioTech Cluster Development GmbH
BioM BioTech Cluster Development GmbH
Wolf C.,Hoffmann-La Roche |
Wolf C.,BioM BioTech Cluster Development GmbH |
Jarutat T.,Hoffmann-La Roche |
Vega Harring S.,Hoffmann-La Roche |
And 6 more authors.
Histopathology | Year: 2014
Aims: For selection of patients who will benefit from targeted therapies, identification of biomarkers predictive of treatment response is desirable. Activation of the targeted pathway becomes apparent by protein phosphorylation. Determination of this phenomenon is therefore considered a promising biomarker approach. To date, however, it is unclear whether routinely collected tissue specimens allow determination of in-vivo phosphorylation states. Methods and results: To investigate whether routinely collected tissue specimens retain the true phosphorylation states of a tumour's proteins, we compared protein phosphorylation states between matched tumour samples that were subjected to different ischaemic times by immunohistochemistry. The influence of formalin fixation and paraffin-embedding on phosphorylation states was investigated by comparison of matched fresh frozen and formalin-fixed paraffin-embedded surgical specimens as well as small biopsies. We show that ischaemia influences protein phosphorylation in a tumour-specific, unpredictable manner. Formalin fixation and paraffin-embedding lead to a decrease in detectable protein phosphorylation in larger surgical specimens, but not in small biopsies. Conclusions: Determination of protein phosphorylation using routinely collected surgical specimens results in artefacts which do not reflect a tumour's true states of pathway activation. Valid measurement of phosphorylated biomarkers requires that tissue collection procedures are tightly controlled, avoiding ischaemia and large-specimen fixation. © 2013 John Wiley & Sons Ltd.
Birke H.,University of Heidelberg |
Birke H.,CSIRO |
Heeg C.,University of Heidelberg |
Heeg C.,BioM BioTech Cluster Development GmbH |
And 2 more authors.
Plant Physiology | Year: 2013
The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant. © 2013 American Society of Plant Biologists. All Rights Reserved.
Aumiller V.,Ludwig Maximilians University of Munich |
Aumiller V.,Comprehensive Pneumology Center |
Graebsch A.,Ludwig Maximilians University of Munich |
Graebsch A.,Helmholtz Center for Environmental Research |
And 5 more authors.
RNA Biology | Year: 2012
Pur-α was identified as a DNA-binding protein with high affinity for the single-stranded PUR-motif (GGN)n. Bound to DNA, Pur-α can both activate and repress transcription. In addition, Pur-α binds to RNA and may participate in nuclear RNA export as well as transport of cytoplasmic neuronal mRNP granules. The heritable trinucleotide-repeat expansion disease Fragile X associated Tremor and Ataxia Syndrome (FXTAS) leads to interaction of Pur-α with mutant, abnormally long r(CGG)n stretches, which appears to titrate the protein away from its physiologic mRNA targets into nuclear RNA-protein aggregates. We examined the function of Drosophila Pur-α and demonstrate that the protein accumulates in the growing oocyte early in oogenesis. Co-purifying proteins reveal that Pur-α is part of transported mRNP complexes, analogous to its reported role in nerve cells. We analyzed the subcellular localization of mutant GFP-Pur-α fusion proteins where either nucleic acid binding or dimerization, or both, were prevented. We propose that association with mRNAs occurs in the nucleus and is required for nuclear export of the complex. Furthermore, efficient translocation into the oocyte also requires RNA binding as well as dimerization. RNA binding assays demonstrate that recombinant Drosophila Pur-α can bind r(CGG)4 with higher affinity than previously thought. Related sequences, such as r(CAG)4 and the consensus sequence of the opa-repeat r(CAG) 3CAA, can also associate with Pur-α in vitro and in vivo. The mRNA target spectrum of Pur-α may therefore be larger than previously anticipated. © 2012 Landes Bioscience.
Gundisch S.,TU Munich |
Gundisch S.,BioM BioTech Cluster Development GmbH |
Grundner-Culemann K.,Evotec |
Grundner-Culemann K.,BioM BioTech Cluster Development GmbH |
And 17 more authors.
Journal of Proteome Research | Year: 2013
Protein phosphorylation controls the activity of signal transduction pathways regulated by kinases and phosphatases. Little is known, however, about the impact of preanalytical factors, for example, delayed times to tissue fixation, on global phosphoprotein levels in tissues. The aim of this study was to characterize the potential effects of delayed tissue preservation (cold ischemia) on the levels of phosphoproteins using targeted and nontargeted proteomic approaches. Rat and murine liver samples were exposed to different cold ischemic conditions ranging from 10 to 360 min prior to cryopreservation. The phosphoproteome was analyzed using reverse phase protein array (RPPA) technology and phosphoprotein-enriched quantitative tandem mass spectrometry (LC-MS/MS). RPPA analysis of rat liver tissues with long (up to 360 min) cold ischemia times did not reveal statistically significant alterations of specific phosphoproteins even though nonphosphorylated cytokeratin 18 (CK18) showed increased levels after 360 min of delay to freezing. Keeping the samples on ice prior to cryopreservation prevented this effect. LC-MS/MS-based quantification of 1684 phosphorylation sites in rat liver tissues showed broadening of their distribution compared to time point zero, but without reaching statistical significance for individual phosphosites. Similarly, RPPA analysis of mouse liver tissues with short (<60 min) cold ischemia times did not reveal directed or predictable changes of protein and phosphoprotein levels. Using LC-MS/MS and quantification of 791 phosphorylation sites, we found that the distribution of ratios compared to time point zero broadens with prolonged ischemia times, but these were rather undirected and diffuse changes, as we could not detect significant alterations of individual phosphosites. On the basis of our results from RPPA and LC-MS/MS analysis of rat and mouse liver tissues, we conclude that prolonged cold ischemia results in unspecific phosphoproteome changes that can be neither predicted nor assigned to individual proteins. On the other hand, we identified a number of phosphosites which were extraordinarily stable even after 360 min of cold ischemia and, therefore, may be used as general reference markers for future companion diagnostics for kinase inhibitors. © 2013 American Chemical Society.
Thasler W.E.,Ludwig Maximilians University of Munich |
Thasler R.M.K.,Ludwig Maximilians University of Munich |
Thasler R.M.K.,BioM BioTech Cluster Development GmbH |
Schelcher C.,Ludwig Maximilians University of Munich |
Jauch K.-W.,Ludwig Maximilians University of Munich
Langenbeck's Archives of Surgery | Year: 2013
Background: High-quality biospecimens of human origin with annotated clinical and procedural data are an important tool for biomedical research, not only to map physiology, pathophysiology and aetiology but also to go beyond in translational research. This has opened a new special field of research known as 'biobanking', which focuses on how to collect, store and provide these specimens and data, and which is substantially supported by national and European funding. Purpose: An overview on biobanking is given, with a closer look on a clinical setting, concerning a necessary distinction from clinical trials and studies as well as a comparison of prospective sample collection with secondary use of archived samples from diagnostics. Based on a summary of possible use and scientific impact of human tissue in research, it is shown how surgical expertise boosts the scientific value of specimens and data. Finally, an assessment of legal and ethical issues especially from a surgical perspective is given, followed by a model of interdisciplinary biobanking within a joint 'centre' that as synergistic structure merges essential input from surgery as well as laboratory medicine, pathology and biometry. Conclusion: Within the domain of biobanking, surgeons have to develop a better awareness of their role within translational research, not only on the level of medical faculties but also as nationally and internationally funded initiatives. Therefore, the authors suggest a platform for biobanking within the German association of surgeons in analogy to the existing special interest group for clinical trials. © 2013 Springer-Verlag Berlin Heidelberg.
Lee S.M.L.,Ludwig Maximilians University of Munich |
Schelcher C.,Ludwig Maximilians University of Munich |
Gashi S.,Ludwig Maximilians University of Munich |
Schreiber S.,Ludwig Maximilians University of Munich |
And 3 more authors.
Molecular Biotechnology | Year: 2013
The accuracy of information garnered by real-time quantitative polymerase chain reaction (RT-qPCR), an important technology for elucidating molecular mechanisms of disease, is dependent on tissue quality. Thus, this study aimed to determine the effects of intra-operative manipulation, extended processing times, different temperatures or storage in RNAlater on RNA quality in liver samples for tissue banking. Liver samples, flash-frozen or in RNAlater, were collected over a time course (during surgery before blood arrest up to 1 day after surgery) with samples kept either at room temperature (RT) or on ice. This study showed that at the longest time-point at RT, the RNA quality decreased significantly by 20%. However, relative gene expressions of FOS, GUSB, MYC, HIF1α and GFER were in general not significantly different when the time-points were compared. In conclusion, samples should be kept on ice during processing, and either RNAlater or snap-freezing should be utilised for storage. Further, intra-operative manipulation and extended postoperative processing time generally does not change relative gene expression levels for the 5 genes studied, making such sampling suitable for RT-qPCR analysis. Thus, if relative gene expression of a gene of interest is stable, these guidelines will lead to increased accrual of samples to the tissue bank. © 2012 Springer Science+Business Media, LLC.