ABSTRACT
Collecting biological tissue samples in a biobank grants a unique opportunity to validate diagnostic and therapeutic strategies for translational and clinical research. In the present work, we provide our long-standing experience in establishing and maintaining a biobank of vascular tissue samples, including the evaluation of tissue quality, especially in formalin-fixed paraffin-embedded specimens (FFPE). Our Munich Vascular Biobank includes, thus far, vascular biomaterial from patients with high-grade carotid artery stenosis (n = 1567), peripheral arterial disease (n = 703), and abdominal aortic aneurysm (n = 481) from our Department of Vascular and Endovascular Surgery (January 2004â»December 2018). Vascular tissue samples are continuously processed and characterized to assess tissue morphology, histological quality, cellular composition, inflammation, calcification, neovascularization, and the content of elastin and collagen fibers. Atherosclerotic plaques are further classified in accordance with the American Heart Association (AHA), and plaque stability is determined. In order to assess the quality of RNA from FFPE tissue samples over time (2009â»2018), RNA integrity number (RIN) and the extent of RNA fragmentation were evaluated. Expression analysis was performed with two housekeeping genes-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB)-using TaqMan-based quantitative reverse-transcription polymerase chain reaction (qRT)-PCR. FFPE biospecimens demonstrated unaltered RNA stability over time for up to 10 years. Furthermore, we provide a protocol for processing tissue samples in our Munich Vascular Biobank. In this work, we demonstrate that biobanking is an important tool not only for scientific research but also for clinical usage and personalized medicine.
ABSTRACT
For an understanding of metabolic interactions in ectomycorrhizal associations it is essential to distinguish enzyme activities of the symbionts. For the ATP-dependent phosphofructokinase (PFK) from ectomycorrhizas of fly agaric (Amanita muscaria (L. ex Fr.) Hooker) on Norway spruce (Picea abies (L.) Karst.) we were able to achieve a symbiont-specific differentiation by special assay conditions. Substrate concentrations, Mg2+ : ATP ratio and pH values which were optimum for the fly agaric PFK completely suppressed the PFK activity of spruce roots. On the other hand, under the optimum assay conditions for the spruce root PFK, the fungal PFK activity was reduced by more than 90%. The most pronounced difference between the enzymes of both organisms was the response towards fructose-2,6-bisphosphate (F26BP); whereas F26BP had no influence on the spruce PFK activity, the fly agaric PFK activity was strongly enhanced by very low levels of F26BP. The distinction of the partner-specific PFK activities illustrated that mycorrhiza formation exerted partner-specific effects. On the basis of host d. wt of the mycorrhizas, the host-specific PFK activity was more than doubled compared with that of the non-mycorrhizal short roots. By contrast, the fungal PFK activity, on a fungal d.wt basis, was reduced in the mycorrhizas to less than 1/4 of the activity of the free-living mycelium.
ABSTRACT
Phosphoenolpyruvate carboxylase (PEPC) activity, fructose 2, 6-bisphosphate (F26BP), starch and soluble sugar contents were determined m needles and roots of Norway spruce seedlings grown in a semi-hydroponic cultivation system under different nutrient regimes, tn needles, a surplus of nitrogen caused an increase in specific PEPC activity (up to six times control activity) and F26BP content (up to three times control level) while starch content was reduced. Sucrose contents were not affected. Basically, the responses in root samples were similar. Here, PEPC was highest at an imbalance in nutrition (+ N/ -P) F26BP, with root contents being 3- to 11 -times higher than those in needles, significantly exceeded control values at + N/+ P. The results show that alteration of nitrogen supply leads to marked changes in allocation of carbon between pathways, which is also influenced by P-nutrition. Pool sizes of F26BP and activity of PEPC are indicators for these changes in leaf as well as in root tissues of Norway spruce.
