Sos1 Regulates Macrophage Podosome Assembly and Macrophage Invasive Capacity.

Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.

Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein.

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

Uncertainty evaluation for the quantification of low masses of benzo[a]pyrene: Comparison between the Law of Propagation of Uncertainty and the Monte Carlo method.

A proper evaluation of the uncertainty associated to the quantification of micropollutants in the environment, like Polycyclic Aromatic Hydrocarbons (PAHs), is crucial for the reliability of the measurement results. The present work describes a comparison between the uncertainty evaluation carried out according to the GUM uncertainty framework and the Monte Carlo (MC) method. This comparison was carried out starting from real data sets obtained from the quantification of benzo[a]pyrene (BaP), spiked on filters commonly used for airborne particulate matter sampling. BaP was chosen as target analyte as it is listed in the current European legislation as marker of the carcinogenic risk for the whole class of PAHs. MC method, being useful for nonlinear models and when the resulting output distribution for the measurand is non-symmetric, can particularly fit the cases in which the results of intrinsically positive quantities are very small and the lower limit of a desired coverage interval, obtained according to the GUM uncertainty framework, can be dramatically close to zero, if not even negative. In the case under study, it was observed that the two approaches for the uncertainty evaluation provide different results for BaP masses in samples containing different masses of the analyte, MC method giving larger coverage intervals. In addition, in cases of analyte masses close to zero, the GUM uncertainty framework would give even negative lower limit of uncertainty coverage interval for the measurand, an unphysical result which is avoided when using MC method. MC simulations, indeed, can be configured in a way that only positive values are generated thus obtaining a coverage interval for the measurand that is always positive.

Uncertainty estimation in chemical measurements: carbon dioxide determination at atmospheric concentration.

The present work deals with the evaluation of measurement uncertainty in the determination of carbon dioxide (CO2) concentration in atmosphere, given the high relevance of this greenhouse gas that influences earth climate. In order to carry out CO2 measurements, non dispersive infrared (NDIR) analysers are usually employed as they are stable and scarcely affected by interferences from other air components or pollutants. Typical uncertainty sources are the resolution of the analyser, its time drift and the contributions due to instrument calibration, which is required in order to produce traceable measurement results. The calibration uncertainty takes into account the uncertainty of the composition of the calibration gas mixtures, the instrument repeatability and the possible or residual lack of fit of the adopted mathematical model.

Stability of a NDIR analyser for CO2 at atmospheric concentration.

Carbon dioxide monitoring is significant in the environmental field since this gas plays an important role in the greenhouse effect. In order to determine CO2 concentration and to develop simulation models, it is necessary to carry out measurements which are accurate and comparable in time and space, i.e. SI-traceable. Non-dispersive infrared (NDIR) analysers are employed for CO2 measurements, as they are precise and stable. In order to achieve traceability, such instruments have to be characterized and calibrated. At the Istituto di Metrologia "G. Colonnetti"--CNR, a procedure for calibrating NDIR analysers for CO2 at atmospheric level was developed, which enables to calculate a correction for the analyser output. In addition, a complete uncertainty analysis was carried out and a correct traceability chain was established. The goal of the present work is the study of the stability of a NDIR analyser by repeating calibrations during three years and comparing the correction curves obtained to identify a proper re-calibration interval for such analysers. The investigated instrument has good repeatability and reproducibility, hence satisfactory stability during time, as shown by the short-term and long-term compatibility of calibration curves.

Rapid and sensitive detection of melamine in milk with gold nanoparticles by Surface Enhanced Raman Scattering.

A rapid and sensitive method to detect melamine in liquid milk based on Surface Enhanced Raman Scattering (SERS) spectroscopy is presented, exploiting the selective binding of gold nanoparticles (AuNPs) with this analyte. This interaction promotes the aggregation of the AuNPs inducing a huge enhancement of the melamine signals in the Raman spectrum due to the formation of SERS "hot spots". An external standard calibration method was employed for quantitative analysis and the method was validated for linearity, sensitivity, repeatability and recovery. A good linearity (R(2)=0.99) was found in the concentration range of 0.31-5.0 mg l(-1) in milk with a limit of detection of 0.17 mg l(-1). This method does not require a long extraction procedure (total analysis time can be lower than 30 min) and can be reliably used for melamine detection in milk matrix in accordance with the European law limits.

Computer-aided biophysical modeling: a quantitative approach to complex biological systems.

When dealing with the biophysics of tumors, analytical and numerical modeling tools have long been regarded as potentially useful but practically immature tools. Further developments could not just overturn this predicament, but lead to completely new perspectives in biology. Here, we give an account of our own computational tool and how we have put it to good use, and we discuss a paradigmatic example to outline a path to making cell biology more quantitative and predictive.

Interplay between distribution of live cells and growth dynamics of solid tumours.

Experiments show that simple diffusion of nutrients and waste molecules is not sufficient to explain the typical multilayered structure of solid tumours, where an outer rim of proliferating cells surrounds a layer of quiescent but viable cells and a central necrotic region. These experiments challenge models of tumour growth based exclusively on diffusion. Here we propose a model of tumour growth that incorporates the volume dynamics and the distribution of cells within the viable cell rim. The model is suggested by in silico experiments and is validated using in vitro data. The results correlate with in vivo data as well, and the model can be used to support experimental and clinical oncology.

Expression of α-amylase inhibitors in diploid Triticum species.

The aim of the work was to characterize the expression of various α-amylase inhibitors (αAIs), well known anti-nutritional compounds, for the development of healthier diploid wheat-based functional foods. The salt-soluble protein fractions from the seeds of 53 accessions among Triticum monococcum subsp. monococcum (T.m.), T. monococcum subsp. boeoticum (T.b.) and Triticum urartu (T.u.) were analyzed by immunoblotting after SDS-PAGE and Urea-PAGE using polyclonal antibodies (PABs) raised against 0.19 and 0.28 αAIs expressed in bread-wheat. Reverse zymography with human saliva and Tenebrio molitor α-amylases was used to assay inhibition activity. A great variability of the expression of αAI-related proteins was observed among T.b. and T.u. PABs, and reverse zymography revealed different bands, often not correlating with those present in bread-wheat. Two-dimensional electrophoresis followed by immunoblotting and mass spectrometric analysis identified these proteins as αAIs. Interestingly, no signal was observed within T.m. accessions. This makes T.m. an important candidate for the production of novel functional foods.