Previous studies demonstrated that no significant relationships exist between salivary and serum IL-6 in resting conditions and following exercise and that appropriate saliva collection procedures allow to avoid analytical drawbacks. This investigation aimed to: (a) compare the effects of two methods of saliva collection on IL-6 assay; (b) search for correlation between salivary and serum IL-6 in resting and post-exercise conditions; (c) evaluate the IL-6 response to isometric contractions. Seventeen sedentary subjects and fifteen athletes underwent one blood and two salivary draws: saliva was collected chewing on cotton salivettes and using a plastic straw (SA method and ST method, respectively). Afterwards, the athletes only completed a fatiguing isometric exercise of the knee extensors and blood and saliva were sampled after the exercise. In the entire group (n=32), ST method produced higher IL-6 levels than SA method and serum sampling. The exercise elicited significant responses of lactate, serum IL-6, salivary IL-6 (by ST method): salivary IL-6 values using the ST collection method were higher at each sampling point than with the SA method. The correlation analyses applied to both resting levels in the entire group and absolute changes above baseline in the athlete group showed that: (1) no significant relationships exist between serum and salivary IL-6 levels; (2) the greater the salivary IL-6 measurement, the higher the resultant inaccuracy of the SA method; (3) significant correlations exist between isometric force and mechanical fatigue during exercise and peaks of lactate and serum IL-6. These data provided demonstration of a cotton-interference effect for the results of salivary IL-6 assay and confirmed the lack of significant correlation between salivary and serum IL-6 in resting and post-exercise conditions.

We describe rblA, the Dictyostelium ortholog of the retinoblastoma susceptibility gene Rb. In the growth phase, rblA expression is correlated with several factors that lead to 'preference' for the spore pathway. During multicellular development, expression increases 200-fold in differentiating spores. rblA-null strains differentiate stalk cells and spores normally, but in chimeras with wild type, the mutant shows a strong preference for the stalk pathway. rblA-null cells are hypersensitive to the stalk morphogen DIF, suggesting that rblA normally suppresses the DIF response in cells destined for the spore pathway. rblA overexpression during growth leads to G1 arrest, but as growing Dictyostelium are overwhelmingly in G2 phase, rblA does not seem to be important in the normal cell cycle. rblA-null cells show reduced cell size and a premature growth-development transition; the latter appears anomalous but may reflect selection pressures acting on social ameba.

The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) were detected in 56–88% of JAK2/MPL-negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs.We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene.

BackgroundMutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABLT315I mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation.ResultsThe PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors.ConclusionsWe present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.

Introduction Tyrosine kinases (TKs) play different roles either in physiological or in pathological cellular processes ranging from the regulation of intracellular signaling pathways leading for example to proliferation, differentiation and/or DNA syntheisis. The whole human genome encodes for approximately 95 TKs classified in two major families: transmembrane and non-transmembrane TKs. Transmembrane TKs are further splitted in two groups: Receptor Tyrosine Kinases (RTKs) and Non-Receptor Transmembrane Tyrosine Kinases. Despite in the last decade the role of RTKs has been deeply investigated very little is known about the second class of TKs, Non-Receptor Transmembrane Tyrosine Kinases. Recently, we began to investigate the roles played by these peculiar molecules in hematological tissues and preliminary data are herein presented and discussed. Material & Methods and Results STYK1 is a Non-Receptor Transmembrane TK characterized by a very short extracellular domain, of approximately 25 aminoacid residues followed by a single transmembrane alpha-helix sequence and a conserved cytoplasmic region harboring the catalytic domain, splitted in two halves separated by a short kinase insert sequence, peculiar of class III RTKs, VEGFR and FGFR family members. Indeed, sequence analysis alignment reveals the higher homology with PDGFR and FGFR members. Surprisingly, data mining searches, performed with the Ensembl tools (http://www.ensembl.org) and using blast algorithm with human STYK1 protein as query, revealed that this protein possesses orthologues either in vertebrate model organisms, both mammalian and non-mammalian or in invertebrate metazoan, such as Ciona. Furthermore, a bioinformatic analysis carried-out at CBS (http://www.cbs.dtu.dk/services/NetPhos/) revealed 4 tyrosine residues putatively phosphorylated. Interestingly one of the tyrosine residue displays high homology towards a consensus sequence for the p85 regulatory subunit of the PI3K. Since our interest aims to understand the roles played by STYK1 in hemopoietic cells, we first investigated its expression in different blood cells populations. Therefore the different blood cellular components were separated by immunoaffinity sorting and total mRNA was extracted. Afterwards c-DNA was synthesized and STYK1 mRNA expression studied by RT-PCR. The results reveal that STYK1 mRNA is expressed predominantly in mature lymphocytes. To study more in details its expression within lymphoid population we attempted to separate B-from T-lymphocytes and STYK1 mRNA expression evaluated in the two populations. Furthermore, we also investigated STYK1 expression in leukemic hematological disorders. Nevertheless STYK1 mRNA expression is typical of mature hemopoietic cells populations, such as lymphocytes, its expression resulted deregulated in patients affected by different kind of leukemia (i.e. CML, ALL, AML). Currently a constitutively active form of STYK1 has been generated and its signaling is under investigation. Conclusion The non-receptor transmembrane tyrosine kinase STYK1 represents a novel lymphocyte molecular marker whose expression results deregulated in patients affected by leukemia. Interestingly, due to its homology with class III RTKs like PDGFR, which are easily targeted by small molecules such as Imatinib mesylate, it may represents a novel druggable molecular target for leukemic cells.