Combining laser microdissection and microRNA expression profiling to unmask microRNA signatures in complex tissues

Neglecting tissue heterogeneity during the analysis of microRNA (miRNA) levels results in average signals from an unknown mixture of different cell types that are difficult to interpret. Here we demonstrate the technical requirements needed to obtain high-quality, quantitative miRNA expression infor- mation from tumor tissue compartments obtained by laser microdissection (LMD). Furthermore, we show the significance of disentangling tumor tissue heterogeneity by applying the newly developed protocols for combining LMD of tumor tissue compartments with RT-qPCR analysis to reveal compartment- specific miRNA expression signatures. An important advantage of this strategy is that the miRNA signature can be directly linked to histopatho logy. In summary, combining LMD and RT-qPCR is a powerful approach for spatial miRNA expression analysis in complex tissues, enabling discovery of disease mechanisms, biomarkers and drug candidates

Induction of autophagy by spermidine promotes longevity.

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MicroRNAs and toxicology: A love marriage

With the dawn of personalized medicine, secreted microRNAs (miRNAs) have come into the very focus of biomarker development for various diseases. MiRNAs fulfil key requirements of diagnostic tools such as i) non or minimally invasive accessibility, ii) robust, standardized and non-expensive quantitative analysis, iii) rapid turnaround of the test result and iv) most importantly because they provide a comprehensive snapshot of the ongoing physiologic processes in cells and tissues that package and release miRNAs into cell-free space. These characteristics have also established circulating miRNAs as promising biomarker candidates for toxicological studies, where they are used as biomarkers of drug-, or chemical-induced tissue injury for safety assessment. The tissue-specificity and early release of circulating miRNAs upon tissue injury, when damage is still reversible, are main factors for their clinical utility in toxicology. Here we summarize in brief, current knowledge of this field. Keywords: microRNAs, Biomarker, Toxicology, Minimal-invasive, DIL

Potentiation of liver X receptor transcriptional activity by peroxisome-proliferator-activated receptor gamma co-activator 1 alpha.

Peroxisome-proliferator-activated receptor (PPAR) gamma co-activator 1 alpha (PGC-1 alpha/PPARGC1) plays an important role in energy metabolism by co-ordinating transcriptional programmes of mitochondrial biogenesis, adaptive thermogenesis and fatty acid beta-oxidation. PGC-1 alpha has also been identified to play a role in the intermediary metabolism by co-activating key transcription factors of hepatic gluconeogenesis and glucose uptake in muscles. In the present study, we show that PGC-1 alpha serves as a co-activator for the liver X receptor (LXR) alpha, known to contribute to the regulation of cellular cholesterol homoeostasis. In transient transfection studies, PGC-1 alpha amplified the LXR-mediated autoregulation of the LXR alpha promoter in a human brown adipocyte line and in 3T3-L1 cells via an LXR response element described previously. LXR-mediated transactivation via a natural LXR response element from the cholesteryl ester transfer-protein gene promoter was also enhanced by PGC-1 alpha in a ligand-dependent manner. Mutational analysis showed that the LXXLL signature motif (L2) of PGC-1 alpha was essential for co-activation of LXR-mediated transcriptional responses. This motif is located in the vicinity of the binding region for a putative repressor described previously. The repressor sequesters PGC-1 alpha from PPAR alpha and the glucocorticoid receptor, and this repressor did not interfere with PGC-1 alpha-mediated co-activation of LXR-dependent gene transcription. Moreover, inhibition of p38 mitogen-activated protein kinase signalling, shown to abolish the co-activation of PPAR alpha by PGC-1 alpha, had only a moderate inhibitory effect on the co-activation of LXR. These results identify PGC-1 alpha as a bona fide LXR co-activator and implicate distinct interfaces of PGC-1 alpha and/or additional cofactors in the modulation of LXR and PPAR alpha transcriptional activities

Cancer Sample Biobanking at the Next Level: Combining Tissue With Living Cell Repositories to Promote Precision Medicine

Biorespositories of formalin-fixed and paraffin-embedded (FFPE) or fresh frozen human tissues from malignant diseases generated as integral part of the diagnostic workup in many pathology departments have been pivotal resources for translational cancer studies. However, such tissue biobanks have traditionally contained only non-viable specimens and thus cannot enable functional assays for the discovery and validation of therapeutic targets or the assessment of drug responses and resistance to treatment. To overcome these limitations, we have developed a next-generation comprehensive biobanking platform that includes the generation of patient-derived in vitro cell models from colorectal, pancreatic and kidney cancers among others. As such patient-derived cell (PDC) models retain important features of the original human tumors, they have emerged as relevant tools for more dynamic clinical and experimental analyses of cancer. Here, we describe details of the complex processes of acquisition and processing of patient-derived samples, propagation, annotation, characterization and distribution of resulting cell models and emphasize the requirements of quality assurance, organizational considerations and investment into resources. Taken together, we show how clinical tissue collections can be taken to the next level thus promising major new opportunities for understanding and treating cancer in the context of precision medicine

lin–Sca-1+ Cells and Age-Dependent Changes of Their Proliferation Potential Are Reliant on Mesenchymal Stromal Cells and Are Leukemia Inhibitory Factor Dependent

Aging as a process is paralleled by a variety of hematological alterations. Characteristic features are a diminished homeostatic control of blood cell production and a decline in immune functions. It is generally accepted that stromal cells play a basal role in hematopoiesis by providing survival and differentiation signals, by secreting cytokines, or through direct contact with hematopoietic stem cells, thereby supporting the generation and replenishment of hematopoietic progenitor cells (HPC). Here we demonstrated that HPC-related colony formation is positively influenced by mesenchymal stromal cells (MSCs) when grown in co-culture, in particular regarding the number of primary granulocyte/macrophage colony-forming units as well as with respect to the average size of the formed colonies. These effects were more pronounced when the MSCs originated from young donors than from old ones. Because leukemia inhibitory factor (LIF) plays an important role during hematopoiesis, properties of lin-- Sca-1+ cells and MSCs derived from LIF-deficient mice (LIF--/--) were determined both ex vivo and in vitro. LIF--/-- animals contain a significantly reduced number of lin-- Sca-1+ cells, nevertheless the replating capacity of LIF--/-- HPCs was found to be generally unchanged when compared to those from LIF+/+ animals. However, when cocultured with MSCs, LIF--/-- lin-- Sca-1+ cells exhibited comparable characteristics to HPCs derived from old wild-type animals