Molecular determinants of response to PI3K/akt/mTOR and KRAS pathways inhibitors in NSCLC cell lines

Despite the impressive results obtained in the preclinical setting, all the inhibitors targeting two central cascades in cancer, the PI3K/akt/mTOR and the KRAS/MEK/ERK pathways, have shown, apart from very few exceptions, disappointing efficacy when translated to the clinic. One of the main reasons of their clinical failure seems to be the lack of a clear molecular determinant of response to these drugs. In this study, we tried to address this point by evaluating the cytotoxic activity of different inhibitors targeting the two pathways at different levels in a panel of ten NSCLC cell lines harboring alterations in PI3K, KRAS or both. We were not able to highlight a correlation between the presence of KRAS and PI3K mutations and a specific sensitivity to the different drugs used. Molecular analyses performed after equimolar treatments showed that, independently from the entity of the response, the drugs are able to modulate the activation of their targets. Interestingly, we found that p53 mutational status separates the cell lines according to their sensitivity to PI3K pathway inhibitors treatments. The alterations considered in the PI3K/akt/mTOR and in the KRAS/MEK/ERK pathways in the different NSCLC cell lines are not sufficient to drive treatment choice but rather p53 status is a potential biomarker for the activity of this class of drugs.     

The interleukin-1 receptor in normal and osteoarthritic human articular chondrocytes. Identification as the type I receptor and analysis of binding kinetics and biologic function

Objective. To identify and investigate the kinetic binding properties of interleukin-1 receptors (IL-1R), and examine the abilities of the 2 IL-1 isoforms to stimulate metalloprotease synthesis, in normal and osteoarthritic (OA) chondrocytes. Methods. Receptor affinity and density were determined using radioligand binding experiments and flow cytometry. Immunocytochemical analysis and affinity cross-linking studies were performed for characterization of IL-1R. Results. While no difference in receptor affinity between normal and OA chondrocytes was noted in binding studies (k_d ˜30 pM), a 2-fold increase in receptor density was found in OA chondrocytes as compared with normal chondrocytes (mean 4,069 sites/cell versus 2,315 sites/cell). Flow cytometry experiments also showed a significant increase in receptor density in OA cells, as well as an enhancement in the percentage of positive cells in diseased cartilage compared with normal. Binding data for both IL-1 isoforms revealed a single class of binding sites and receptor specificity. Factors such as IL-2, interferon-sγ, tumor necrosis factor α, and bovine insulin did not compete with IL-1β. By covalent ligand cross-linking and electrophoretic analysis, only type I IL-1R, a protein of 80 kd, was detected on chondrocytes. By immunocytochemical analysis, IL-1R was identified at the cell membrane level, in both normal and OA chondrocytes. The presence of nuclear staining was also observed, but only in OA chondrocytes. Recombinant human IL-1 (α and β) induced the secretion of stromelysin and collagenase in a dose-dependent manner. The IL-1 concentration required for half-maximal metalloprotease stimulation was 3–4 times lower in OA chondrocytes than in normal cells. Conclusion. These results indicate that OA chondrocytes have a higher sensitivity to the stimulation of metalloprotease synthesis by IL-1 than do normal cells. This could be related to the increased levels of IL-1R expressed in the OA cells. The implications of these findings with regard to the possible roles of IL-1 and IL-1R in the pathogenesis of OA are discussed.     

Application of Granular Biocomposites Based on Homogenised Peat for Absorption of Oil Products

Among the various methods for collecting oil spills and oil products, including from the water surface, one of the most effective is the use of sorbents. In this work, three-component bio-based composite granular adsorbents were produced and studied for oil products’ pollution collection. A bio-based binder made of peat, devulcanised crumb rubber from used tyres, and part fly ash as cenospheres were used for absorbent production. The structure, surface morphology, porosity, mechanical properties, and sorption kinetics of the obtained samples were studied. Composite hydrophobicity and sorption capacity to oil products, such as diesel fuel (DF) and motor oil (MO), were determined. The obtained pellets are characterised by a sufficiently pronounced ability to absorb oil products such as DF. As the amount of CR in the granules increases, the diesel absorption capacity increases significantly. The case of 30-70-0 is almost three times higher than the granules from homogenised peat. The increase in q is due to two factors: the pronounced surface hydrophobicity of the samples (Θ = 152°) and a heterogeneous porous granule structure. The presence of the cenosphere in the biocomposite reduces its surface hydrophobicity while increasing the diesel absorption capacity. Relatively rapid realisation of the maximum saturation by the MO was noted. In common, the designed absorbent shows up to 0.7 g·g⁻¹ sorption capacity for MO and up to 1.55 g·g⁻¹ sorption capacity for diesel. A possible mechanism of DF absorption and the limiting stages of the process approximated for different kinetic models are discussed. The Weber–Morris diffusion model is used to primarily distinguish the limiting effect of the external and internal diffusion of the adsorbate on the absorption process.     

Old measures and new scores in spinal muscular atrophy patients

Introduction: A recent Rasch analysis performed on the Hammersmith Functional Motor Scale—Expanded (HFMSE) in patients with spinal muscular atrophy (SMA) identified issues impacting scale validity, redundant items, and disordered thresholds on some items. Methods: We modified the HMFSE scoring based on the Rasch analysis and on expert consensus to establish whether the traditional scoring overestimated the number of patients with changes within 2 points from baseline. Data were collected retrospectively from multicenter data sets in 255 type 2 and 3 SMA patients. Results: The mean 12‐month changes using the new and the traditional scoring system did not differ significantly (P > 0.05). The numbers of patients who improved or decreased by >2 points were also similar. Conclusions: The presence of outliers using the traditional scoring system was not due to overestimation of changes in activities that were tested bilaterally or to discrepancies in the scoring hierarchy of individual items. Muscle Nerve 52:435–437, 2015     

Comparison of three methods for genotyping of prothrombotic polymorphisms

Several methods have been developed to detect common prothrombotic mutations, including factor V Leiden (G1691), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677C. In this study, we compared the accuracy of three different molecular techniques, i.e.: (1) restriction enzyme digestion (RFLP), (2) real time with hybridization probes and final melting curve (Fluorescence Resonance Energy Transfer, FRET), and (3) real time with hydrolysis probes (TaqMan^®). Sequencing was used as the reference standard. Our data showed that RFLPs analysis for the detection of prothrombotic mutations, albeit easy-to-perform, had a limited reliability for assessing correct genotypes. FRET analysis displayed higher resolution than RFLPs. Additionally, FRET analysis was faster and less tedious than sequencing.