Changes in actin and tubulin expression in osteogenic cells cultured on bioactive glass‐based surfaces

The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass‐based materials are due to altered mRNA and protein levels. Primary rat‐derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real‐time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals. Microsc. Res. Tech. 78:1046–1053, 2015. © 2015 Wiley Periodicals, Inc.     

Reliability of RF MEMS switches due to charging effects and their circuital modelling

The reliability of RF MEMS switches is typically reduced by charging effects occurring in the dielectrics. The aim of this paper is to discuss these effects, and to propose analytical and equivalent circuit models which account for most of the physical contributions present in the structure.     

Lipid characteristics of RSV-transformed Balb/c 3T3 cell lines with different spontaneous metastatic potentials

To determine whether a metastatic phenotype may be corelated with a characteristic lipid pattern, we compared the lipid composition of low metastasizing Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (B77-3T3 cells) with that of a subclone isolated by growth in 0.6% agar, the B77-AA6 cells, which exhibit a high capacity for spontaneous metastasis. B77-3T3 cells revealed characteristics in their lipid composition common to other systems of transformed cells, i.e., an accumulation of ether-linked lipids, a reduction of the more complex gangliosides, an increase of oleic acid (18∶1) and a decrease of arachidonic (20∶4) and C22 polyunsaturated fatty acids in phospholipids. High metastatic B77-AA6 cells showed: a) an even more marked decrease of complex gangliosides; b) a more pronounced increase of 18∶1 and decrease of 20∶4 and 22 polyunsaturated fatty acids in certain phospholipid classes; and c) a higher percentage of alkyl-acyl subfractions in both phosphatidylcholine and phosphatidylethanolamine than B77-3T3 cells. Comparing the data for other systems of metastatic cells with those of lipid studies of spontaneously metastasizing B77-AA6 cell system leads us to conclude that the metastatic phenotype is characterized by a change in ether-linked lipids, rather than in fatty acids.     

Development of an Expert Model to Assess Falls from Height Hazards in Construction Sites

This paper reports on the current state of an ongoing research project which is aimed at implementing intelligent models for hardly predictable hazard scenarios identification in construction sites. As any programmatic actions cannot deal with the unpredictable nature of many risk dynamics, an attempt to improve the current approach for safety management in the construction industry will be presented in this paper. To this aim, the features offered by Bayesian networks have been exploited. The present research has led to the definition of a probabilistic model using elicitation techniques from subjective knowledge. This model, which might be meant as a reliable knowledge map about accident dynamics, showed that a relevant part of occurrences fall in the ＂hardly predictable hazards＂ category, which cannot be warded off by programmatic safety measures. Hence, more effort turned out to be needed in order to manage those hardly predictable hazardous scenarios. Consequently, further developments of this research project will focus on a real time monitoring system for the identification of unpredictable hazardous events in construction.     

Optical tweezers for synchrotron radiation probing of trapped biological and soft matter objects in aqueous environments

Investigations of single fragile objects manipulated by optical forces with high brilliance X-ray beams may initiate the development of new research fields such as protein crystallography in an aqueous environment. We have developed a dedicated optical tweezers setup with a compact, portable, and versatile geometry for the customary manipulation of objects for synchrotron radiation applications. Objects of a few micrometers up to a few tens of micrometers size can be trapped for extended periods of time. The selection and positioning of single objects out of a batch of many can be performed semi-automatically by software routines. The performance of the setup has been tested by wide-angle and small-angle X-ray scattering experiments on single optically trapped starch granules, using a synchrotron radiation microbeam. We demonstrate here for the first time the feasibility of microdiffraction on optically trapped protein crystals. Starch granules and insulin crystals were repeatedly raster-scanned at about 50 ms exposure/raster-point up to the complete loss of the structural order. Radiation damage in starch granules results in the appearance of low-angle scattering due to the breakdown of the polysaccharide matrix. For insulin crystals, order along the densely packed [110] direction is preferentially maintained until complete loss of long-range order.     

Disease mapping in veterinary epidemiology: a Bayesian geostatistical approach

Model-based geostatistics and Bayesian approaches are useful in the context of veterinary epidemiology when point data have been collected by appropriate study design. We take advantage of an example of Epidemiological Surveillance on urban settings where a two-stage sampling design with first stage transects is applied to study the risk of dog parasite infection in the city of Naples, 2004-2005. We specified Bayesian Gaussian spatial exponential models and Bayesian kriging were performed to predict the continuous risk surface of parasite infection on the study region. We compared the results with those obtained by the application of hierarchical Bayesian models on areal data (proportion of positive specimens by transect). The models results were consistent with each other and the Bayesian geostatistical approach proved to be more accurate in identifying areas at risk of zoonotic parasitic diseases. In general, larger risk areas were identified at the city border where wild dogs mixed with domestic dogs and human or urban barriers were less present.     

A Beckwith–Wiedemann-Associated CDKN1C Mutation Allows the Identification of a Novel Nuclear Localization Signal in Human p57Kip2

p57^Kip2 protein is a member of the CIP/Kip family, mainly localized in the nucleus where it exerts its Cyclin/CDKs inhibitory function. In addition, the protein plays key roles in embryogenesis, differentiation, and carcinogenesis depending on its cellular localization and interactors. Mutations of CDKN1C, the gene encoding human p57^Kip2, result in the development of different genetic diseases, including Beckwith–Wiedemann, IMAGe and Silver–Russell syndromes. We investigated a specific Beckwith–Wiedemann associated CDKN1C change (c.946 C>T) that results in the substitution of the C-terminal amino acid (arginine 316) with a tryptophan (R316W-p57^Kip2). We found a clear redistribution of R316W-p57^Kip2, in that while the wild-type p57^Kip2 mostly occurs in the nucleus, the mutant form is also distributed in the cytoplasm. Transfection of two expression constructs encoding the p57^Kip2 N- and C-terminal domain, respectively, allows the mapping of the nuclear localization signal(s) (NLSs) between residues 220–316. Moreover, by removing the basic RKRLR sequence at the protein C-terminus (from 312 to 316 residue), p57^Kip2 was confined in the cytosol, implying that this sequence is absolutely required for nuclear entry. In conclusion, we identified an unreported p57^Kip2 NLS and suggest that its absence or mutation might be of relevance in CDKN1C-associated human diseases determining significant changes of p57^Kip2 localization/regulatory roles.     

Extension of Collagen Deposition in COVID-19 Post Mortem Lung Samples and Computed Tomography Analysis Findings

Lung fibrosis has specific computed tomography (CT) findings and represents a common finding in advanced COVID-19 pneumonia whose reversibility has been poorly investigated. The aim of this study was to quantify the extension of collagen deposition and aeration in postmortem cryobiopsies of critically ill COVID-19 patients and to describe the correlations with qualitative and quantitative analyses of lung CT. Postmortem transbronchial cryobiopsy samples were obtained, formalin fixed, paraffin embedded and stained with Sirius red to quantify collagen deposition, defining fibrotic samples as those with collagen deposition above 10%. Lung CT images were analyzed qualitatively with a radiographic score and quantitatively with computer-based analysis at the lobe level. Thirty samples from 10 patients with COVID-19 pneumonia deceased during invasive mechanical ventilation were included in this study. The median [interquartile range] percent collagen extension was 6.8% (4.6–16.2%). In fibrotic compared to nonfibrotic samples, the qualitative score was higher (260 (250–290) vs. 190 (120–270), p = 0.036) while the gas fraction was lower (0.46 (0.32–0.47) vs. 0.59 (0.37–0.68), p = 0.047). A radiographic score above 230 had 100% sensitivity (95% confidence interval, CI: 66.4% to 100%) and 66.7% specificity (95% CI: 41.0% to 92.3%) to detect fibrotic samples, while a gas fraction below 0.57 had 100% sensitivity (95% CI: 66.4% to 100%) and 57.1% specificity (95% CI: 26.3% to 88.0%). In COVID-19 pneumonia, qualitative and quantitative analyses of lung CT images have high sensitivity but moderate to low specificity to detect histopathological fibrosis. Pseudofibrotic CT findings do not always correspond to increased collagen deposition.     

High SPARC Expression Starting from Dysplasia,Associated with Breast Carcinoma,Is Predictive for Bone Metastasis without Enhancement of Plasma Levels

In order to become established in the skeleton, metastatic cells disseminating from the breast carcinoma need to acquire organ-specific traits. There are no effective predictors for who will develop bone metastasis to guide long-term predictive therapy. Our purpose was to individuate events critical for bone colonization to make a molecular classification of breast carcinoma useful for bone-metastasis outcome. In dysplasia adjacent to carcinoma and in pair-matched specimens of bone metastasis we examined SPARC expression and localization as well as Endothelin 1/ET_AR signals by immunohistochemistry, and the evaluation of plasma levels of SPARC by ELISA was also performed. In patients with breast carcinoma metastasizing to bone, SPARC and Endothelin 1/ET_AR axis were highly expressed from dysplasia until bone metastasis, but the SPARC plasma level was as low as that of normal women, in contrast to patients that never develop bone metastasis, suggesting that circulating SPARC was counter adhesive. Altogether, the early identification of SPARC/Endothelin 1/ET_AR in dysplastic lesions would be important to devise therapies preventing metastasis engraftment, since often carcinoma cells spread to distant organs at the time or even before patients present with cancer.     

A theoretically-based novel protocol for the analytic treatment of the glass failure stresses associated with coaxial double ring test method

With the aim of validating a new standardized Coaxial Double Ring testing procedure, without overpressure and with fixed geometry, an ad hoc theoretical approach has been proposed here to rearrange the laboratory outcomes accounting for the effects of the geometric non-linearities associated with such a testing configuration. By borrowing this idea, once the experimental values of the failure load have been determined, it has been possible to obtain an expression in closed form (fully defined by only two coefficients) of the maximum tensile stress (σ_max) in the core of the specimen. Following this, in order to make the laboratory outcomes comparable and homogeneous, the σ_max-values have been then re-scaled to a common reference condition (equibiaxial stress on a reference area, σ_eqbx), by means of the use of a correction coefficient (K) able to determine, under a condition of equal probability of failure, the effective area (A_eff) of the tested specimens. After being corrected to account for the effects of the stress corrosion cracking (static fatigue effect), all re-scaled data have been finally interpreted using a Weibull-type statistical distribution to determine the main fractile values of the glass strength. Doing so, despite some unavoidable approximations, this procedure furnished a highly effective means of determining the bending strength of float glass. Unlike the pure numerical approach proposed in codes and literature, which requires to correct the experimental data via FEM simulation, the rationale behind the proposed approach is in fact to elaborate the experimental data through an analytic treatment of the problem, which would greatly facilitate the interpretation of the data as well as the standardization of the testing procedure.