Protein unlocking procedures of formalin-fixed paraffin-embedded tissues: application to MALDI-TOF imaging MS investigations

Archival formalin-fixed paraffin-embedded (FFPE) tissues are a powerful tool for examining the clinical course of diseases. These specimens represent an incredible mine of valuable clinical and biological information for proteomic investigation. MALDI-TOF imaging MS (MALDI-IMS) is a protein profiling technique which enables the direct sampling of histological section; however, the quality of molecular data are strongly influenced by the tissue preparation condition. In fact, in previous years most of the studies employing such a technological platform have been conducted using cryo-preserved tissues. We have developed an in vitro approach using "tissue surrogate" samples in order to explore different protein unlocking procedures which might enable a suitable recovery of polypeptides for MS analysis. The developed protocols have been compared both by MALDI-TOF MS and nLC-MS(E) analysis either on surrogate samples or on FFPE specimen from human breast cancer. The collected evidence has been applied for the preparation of FFPE tissue sections following MALDI-IMS analysis. Our results outline the possibility to obtain valuable peptide mass spectra profiles form FFPE preparations by applying a combined two steps procedure of heat induced antigen retrieval (HIAR) in presence of EDTA and on target trypsin hydrolysis. A multivariate statistical evaluation is presented and discussed according to molecular spatial distributions and tissue morphology.     

Distribution of class I, III and IV alcohol dehydrogenase mRNAs in the adult rat, mouse and human brain.

The localization of different classes of alcohol dehydrogenases (ADH) in the brain is of great interest because of their role in both ethanol and retinoic acid metabolism. Conflicting data have been reported in the literature. By Northern blot and enzyme activity analyses only class III ADH has been detected in adult brain specimens, while results from riboprobe in situ hybridization indicate class I as well as class IV ADH expression in different regions of the rat brain. Here we have studied the expression patterns of three ADH classes in adult rat, mouse and human tissues using radioactive oligonucleotide in situ hybridization. Specificity of probes was tested on liver and stomach control tissue, as well as tissue from class IV ADH knock-out mice. Only class III ADH mRNA was found to be expressed in brain tissue of all three investigated species. Particularly high expression levels were found in neurons of the red nucleus in human tissue, while cortical neurons, pyramidal and granule cells of the hippocampus and dopamine neurons of substantia nigra showed moderate expression levels. Purkinje cells of cerebellum were positive for class III ADH mRNA in all species investigated, whereas granular layer neurons were positive only in rodents. The choroid plexus was highly positive for class III ADH, while no specific signal for class I or class IV ADH was detected. Our results thus support the notion that the only ADH expressed in adult mouse, rat and human brain is class III ADH.     

Lysosomal storage diseases as disorders of autophagy

The cellular turnover of proteins and organelles requires cooperation between the autophagic and the lysosomal degradation pathways. A crucial step in this process is the fusion of the autophagosome with the lysosome. In our study we demonstrate that in Lysosomal Storage Disorders (LSDs) accumulation of undegraded substrates in lysosomes, due to deficiency of specific lysosomal enzymes, impairs the fusion between autophagosomes and lysosomes. This, in turn, leads to a progressive accumulation of poly-ubiquitinated protein aggregates and of dysfunctional mitochondria. These findings suggest that neurodegeneration in LSDs may share some mechanisms with late-onset neurodegenerative disorders in which the accumulation of protein aggregates is a prominent feature.     

The Holocene treeline in the northern Andes (Ecuador): First evidence from soil charcoal

Indications for the speed and timing of past altitudinal treeline shifts are often contradictory. Partly, this may be due to interpretation difficulties of pollen records, which are generally regional rather than local proxies. We used pedoanthracology, the identification and dating of macroscopic soil charcoal, to study vegetation history around the treeline in the northern Ecuadorian Andes. Pedoanthracology offers a complementary method to pollen-based vegetation reconstructions by providing records with high spatial detail on a local scale. The modern vegetation is tussock grass páramo (tropical alpine vegetation) and upper montane cloud forest, and the treeline is located at ca. 3600 m. Charcoal was collected from soils in the páramo (at 3890 and 3810 m) and in the forest (at 3540 m), and represents a sequence for the entire Holocene.The presence of páramo taxa throughout all three soil profiles, especially in combination with the absence of forest taxa, shows that the treeline in the study area has moved up to its present position only late in the Holocene (after ca. 5850 cal years BP). The treeline may have been situated between 3600 m and 3800 m at some time after ca. 4900 cal years BP, or it may never have been higher than it is today. The presence of charcoal throughout the profiles also shows that fires have occurred in this area at least since the beginning of the Holocene.These results contradict interpretations of palaeological data from Colombia, which suggest a rapid treeline rise at the Pleistocene–Holocene transition. They also contradict the hypothesis that man-made fires have destroyed large extents of forest above the modern treeline. Instead, páramo fires have probably contributed to the slowness of treeline rise during the Holocene.     

Development and assessment of simple PCR markers for SNP genotyping in barley

Simple molecular marker assays underpin routine plant breeding and research activities in many laboratories worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important crop plants, the availability of routine, low-tech marker assays for genotyping SNPs is of increased importance. In this study, we demonstrate that temperature-switch PCR (TSP) supports the rapid development of robust, allele-specific PCR markers for codominant SNP genotyping on agarose gel. A total of 87 TSP markers for assessing gene diversity in barley were developed and used to investigate the efficacy for marker development, assay reliably and genotyping accuracy. The TSP markers described provide good coverage of the barley genome, are simple to use, easy to interpret and score, and are amenable to assay automation. They provide a resource of informative SNP markers for assessing genetic relationships among individuals, populations and gene pools of cultivated barley (Hordeum vulgare L.) and its wild relative H. spontaneum K. Koch. TSP markers provide opportunities to use available SNP resources for marker-assisted breeding and plant genetic research, and to generate information that can be integrated with SNP data from different sources and studies. TSP markers are expected to provide similar advantages for any animal or plant species. M. J. Hayden and T. Tabone contributed equally to this work.