Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr¹⁶⁰ phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr^152–156, Tyr⁵⁶⁷, and a proline-rich region, Pro^415–417. Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.     

Robust determinants of thermostability highlighted by a codon frequency index capable of discriminating thermophilic from mesophilic genomes

Can genome analysis tell us about the lifestyle of an organism? We ask this question considering a thorough cross comparison of thermophilic and mesophilic genomes, since presently the number of available genomes is enough to ensure statistical significance of the results. We analyze, by means of principal component analysis (PCA), the codon composition of a database comprising 116 genomes, selected so as to include one species for each genus and show that a cross genomic approach can allow the extraction of common determinants of thermostability at the genome level. The results of our analysis indicate that all the known features of thermostability can be found in the 64 component loadings of the second principal axis of PCA. By this, we develop an index of thermostability whose discriminative power between mesophiles and thermophiles scores with 98% accuracy at the genome level and with 95% accuracy at the protein sequence level. We also prove that these results are not due to phylogenetic differences between archaea and bacteria.     

Evolutionary and clinical neocentromeres: two faces of the same coin?

It has been hypothesized that human clinical neocentromeres and evolutionary novel centromeres (ENC) represent two faces of the same phenomenon. However, there are only two reports of loci harboring both a novel centromere and a clinical neocentromere. We suggest that only the tip of the iceberg has been scratched because most neocentromerization events have a very low chance of being observed. In support of this view, we report here on a neocentromere at 9q33.1 that emerged in a ring chromosome of about 12 Mb. The ring was produced by a balanced rearrangement that was fortuitously discovered because of its malsegregation in the propositus. Chromatin-immunoprecipitation-on-chip experiments using anti-centromere protein (CENP)-A and anti-CENP-C antibodies strongly indicated that a novel centromeric domain was present in the ring, in a chromosomal domain where an ENC emerged in the ancestor to Old World monkeys.     

Challenging popular tools for the annotation of genetic variations with a real case,pathogenic mutations of lysosomal alpha-galactosidase

Background

Severity gradation of missense mutations is a big challenge for exome annotation. Predictors of deleteriousness that are most frequently used to filter variants found by next generation sequencing, produce qualitative predictions, but also numerical scores. It has never been tested if these scores correlate with disease severity.

Results

wANNOVAR, a popular tool that can generate several different types of deleteriousness-prediction scores, was tested on Fabry disease. This pathology, which is caused by a deficit of lysosomal alpha-galactosidase, has a very large genotypic and phenotypic spectrum and offers the possibility of associating a quantitative measure of the damage caused by mutations to the functioning of the enzyme in the cells. Some predictors, and in particular VEST3 and PolyPhen2 provide scores that correlate with the severity of lysosomal alpha-galactosidase mutations in a statistically significant way.

Conclusions

Sorting disease mutations by severity is possible and offers advantages over binary classification. Dataset for testing and training in silico predictors can be obtained by transient transfection and evaluation of residual activity of mutants in cell extracts. This approach consents to quantitative data for severe, mild and non pathological variants.

     

IL-18 gene promoter polymorphism is involved in HIV-1 infection in a Brazilian pediatric population

In our study, we identified a polymorphism (C-607A) in the promoter region of the IL-18 gene that shows different frequencies between human immunodeficiency virus (HIV)-1-infected children and healthy controls in a pediatric Brazilian population. The presence of the −607 C allele correlates to HIV-1 infection and confers an increased risk of infection in subjects carrying the single nucleotide polymorphism.     

Synthesis and biological evaluation of piperazinyl carbamates and ureas as fatty acid amide hydrolase (FAAH) and transient receptor potential (TRP) channel dual ligands

The evaluation of a series of piperazinyl carbamates and ureas, designed on the basis of previously reported TRPV1 antagonists and FAAH inhibitors, led to the identification of some ‘dual-action’ compounds targeting both FAAH and TRPV1 or TRPA1 receptors.     

Rules for gene usage inferred from a comparison of large-scale gene expression profiles of T and B lymphocyte development

Ribonucleic acid expression profiles of seven consecutive stages of mouse thymocyte development were generated on high density oligonucleotide arrays. Previously known expression patterns of several genes were confirmed. Ten percent (1,304 of more than 13,000) of the monitored genes were found with 99% confidence to be differentially expressed across all T cell developmental stages. When compared with 1,204 genes differentially expressed in five consecutive B lineage developmental stages of bone marrow, >40% (546 genes) appeared to be shared by both lineages. However, when four pools of functionally distinct cell stages were compared between B and T cell development, DJ-rearranged precursor cells and resting immature precursor cells before and after surface Ag receptor expression shared less than 10%, mature resting lymphocytes between 15 and 20%, and only cycling precursors responding to precursor lymphocyte receptor deposition shared >50% of these differentially expressed genes. Three general rules emerge from these results: 1) proliferation of cells at comparable stages is in majority executed by the same genes; 2) intracellular signaling and intercellular communication are effected largely by different genes; and 3) most genes are not used strictly at comparable, but rather at several, stages, possibly in different functional contexts.