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Doppel (Dpl) is a membrane-bound glycoprotein mainly expressed in the testis of adult healthy people. It is generally absent in the central nervous system, but its coding gene sequence is ectopically expressed in astrocytoma specimens and in derived cell lines. In this paper, we investigated the expression and the biochemical features of Dpl in a panel of 49 astrocytoma specimens of different WHO malignancy grades. As a result, Dpl was expressed in the majority of the investigated specimens (86%), also including low grade samples. Importantly, Dpl exhibited different cellular localizations and altered glycan moieties composition, depending on the tumor grade. Most low-grade astrocytomas (83%) showed a membrane-bound Dpl, like human healthy testis tissue, whereas the majority of high-grade astrocytomas (75%) displayed a cytosolic Dpl. Deglycosylation studies with N-glycosidase F and/or neuraminidase highlighted defective glycan moieties and an unexpected loss of sialic acid. To find associations between glial tumor progression and Dpl biochemical features, predictive bioinformatics approaches were produced. In particular, Decision tree and Nomogram analysis showed well-defined Dpl-based criteria that separately clustered low-and high-grade astrocytomas. Taken together, these findings show that in astrocytomas, Dpl undergoes different molecular processes that might constitute additional helpful tools to characterize the glial tumor progression.

Protein interactions are crucial in most biological processes. Several in silico methods have been recently developed to predict them. This paper describes a bioinformatics method that combines sequence similarity and structural information to support experimental studies on protein interactions. Given a target protein, the approach selects the most likely interactors among the candidates revealed by experimental techniques, but not yet in vivo validated. The sequence and the structural information of the in vivo confirmed proteins and complexes are exploited to evaluate the candidate interactors. Finally, a score is calculated to suggest the most likely interactors of the target protein. As an example, we searched for GRB2 interactors. We ranked a set of 46 candidate interactors by the presented method. These candidates were then reduced to 21, through a score threshold chosen by means of a cross-validation strategy. Among them, the isoform 1 of MAPK14 was in silico confirmed as a GRB2 interactor. Finally, given a set of already confirmed interactors of GRB2, the accuracy and the precision of the approach were 75% and 86%, respectively. In conclusion, the proposed method can be conveniently exploited to select the proteins to be experimentally investigated within a set of potential interactors.

In this study the mRNA levels of fiveEGFRindirectly related genes,EGFR,HB-EGF,ADAM17,PTEN, andMMP9, have been assessed by Real-time PCR in a panel of 37 glioblastoma multiforme specimens and in 5 normal brain samples; as a result, in glioblastoma,ADAM17andPTENexpression was significantly lower than in normal brain samples, and, in particular, a statistically significant inverse correlation was found betweenPTENandMMP9mRNA levels. To verify if this correlation was conserved in gliomas,PTENandMMP9expression was further investigated in an additional panel of 16 anaplastic astrocytoma specimens and, in parallel, in different human normal and astrocytic tumor cell lines. In anaplastic astrocytomasPTENexpression was significantly higher than in glioblastoma multiforme, but no significant correlation was found betweenPTENandMMP9expression.PTENandMMP9mRNA levels were also employed to identify subgroups of specimens within the different glioma malignancy grades and to define a gene expression-based diagnostic classification scheme. In conclusion, this gene expression survey highlighted that the combined measurement ofPTENandMMP9transcripts might represent a novel reliable tool for the differential diagnosis of high-grade gliomas, and it also suggested a functional link involving these genes in glial tumors.