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This paper describes a coupled experimental and computational fluid dynamics (CFD) campaign conducted on a 1.5 intermediate turbine stage in the full range of operating conditions, from start-up to design point under variable expansion ratio and physical speed. The test maintains engine similitude conditions and allows direct comparison with CFD data to assess the predictions accuracy. The choice of variables to describe the speedlines is also addressed by using both measured and predicted data. A discussion on velocity ratio versus corrected speed illustrates the advantages of the former parameter the adoption of which produces constant shape curves in a very wide range of operating conditions. The comparison between measurements and predictions suggests that CFD, in conjunction with performance correlations, is a viable tool to predict speedlines in a fairly wide range of conditions, provided that geometrical and operational details are carefully matched.

In this work, the effects of turbine center frame (TCF) wakes on the aeromechanical behavior of the downstream low-pressure turbine (LPT) blades are numerically investigated and compared with the experimental data. A small industrial gas turbine has been selected as a test case, composed of a TCF followed by the two low-pressure stages and a turbine rear frame (TRF) before the exhaust plenum. Full annulus unsteady computations of the whole low-pressure module have been performed. Two operating conditions, full (100%) and partial (50%) load, have been investigated with the aim of highlighting the impact of TCF wakes convection and diffusion through the downstream rows. Attention was paid to the harmonic content of rotors’ blades. The results show a slower decay of the wakes through the downstream rows in off-design conditions compared with the design point. The analysis of the rotors’ frequency spectrum reveals that moving from design to off-design conditions, the effect of the TCF does not change significantly. The harmonic contribution of all turbine components has been extracted, highlighting the effect of statoric parts on the last LPT blade. The TCF harmonic content remains the most relevant from an aeromechanic point of view as per experimental evidence, and it is considered for an forced response analysis (FRA) on the last LPT blade itself. Finally, aerodynamic and aeromechanic predictions have been compared with the experimental data to validate the numerical approach. Some general design solutions aimed at mitigating the TCF wakes impact are discussed.

In this work, the effects of Turbine Center Frame (TCF) wakes on the aeromechanical behavior of the downstream Low Pressure Turbine (LPT) blades are numerically investigated and compared with experimental data. A small industrial gas turbine has been selected as a test case, composed of a TCF followed by the two low pressure stages and a Turbine Rear Frame (TRF) before the exhaust plenum. Full annulus unsteady computations of the whole low-pressure module have been performed. Two operating conditions, full (100%) and partial (50%) load, have been investigated with the aim of highlighting the impact of TCF wakes convection and diffusion through the downstream rows. Attention was paid to the harmonic content of rotors’ blades. From an aerodynamic point of view, the results show a slower decay of the wakes through the downstream rows in off-design conditions as compared to the design point. The wakes generated by the struts at partial load persist throughout the domain outlet, while they are chopped and circumferentially transported by the rotors motion. This is due to the strong incidence variation at which the TCF works, which induces the growth of wide regions of separated flow on the rear part of the struts. Nevertheless, the analysis of the rotors’ frequency spectrum reveals that moving from design to off-design conditions, the effect of the TCF does not change significantly, thanks to the filtering action of the first LPT stage movable Nozzle Guide Vane (NGV). From unsteady calculations the harmonic contribution of all turbine components has been extracted, highlighting the effect of statoric parts on the last LPT blade. Anyhow the TCF harmonic content remains the most relevant from an aeromechanic point of view as per experimental evidence, and it is considered for a Forced Response Analysis (FRA) on the last LPT blade itself. Finally, aerodynamic and aeromechanic predictions have been compared with the experimental data to validate the numerical approach. In the last part of this paper some general design solutions, that can help mitigation of the TCF wakes impact, are discussed.

Ataxia telangiectasia (A-T) is a rare incurable neurodegenerative disease caused by biallelic mutations in the gene for ataxia-telangiectasia mutated (ATM). The lack of a functional ATM kinase leads to a pleiotropic phenotype, and oxidative stress is considered to have a crucial role in the complex physiopathology. Recently, steroids have been shown to reduce the neurological symptoms of the disease, although the molecular mechanism of this effect is largely unknown. In the present study, we have demonstrated that dexamethasone treatment of A-T lymphoblastoid cells increases the content of two of the most abundant antioxidants [glutathione (GSH) and NADPH] by up to 30%. Dexamethasone promoted the nuclear accumulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 to drive expression of antioxidant pathways involved in GSH synthesis and NADPH production. The latter effect was via glucose 6-phosphate dehydrogenase activation, as confirmed by increased enzyme activity and enhancement of the pentose phosphate pathway rate. This evidence indicates that glucocorticoids are able to potentiate antioxidant defenses to counteract oxidative stress in ataxia telangiectasia, and also reveals an unexpected role for dexamethasone in redox homeostasis and cellular antioxidant activity.

Oxidative stress has been shown to play a crucial role in the pathophysiology of the neurodegenerative disease Ataxia Telangiectasia. We have recently demonstrated that Dexamethasone treatment is able to counteract the oxidative state by promoting nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation. However, substantial gaps remain in our knowledge of the underlying molecular mechanism(s) according to which Dexamethasone acts as an NRF2 inducer. Herein we investigate the possible effects of the drug on the main NRF2 activation pathways by initially focusing on key kinases known to differently affect NRF2 activation. Neither AKT nor ERK1/2, known to be NRF2-activating kinases, were found to be activated upon Dexamethasone treatment, thus excluding their involvement in the transcription factor nuclear shift. Likewise, GSK3 inactivating kinase was not inhibited, thus ruling out its role in NRF2 activation. On the other hand, p38 MAPK, another NRF2-inhibitory kinase, was indeed switched-off in Ataxia Telangiectasia cells by Dexamethasone-mediated induction of DUSP1 phosphatase, and therefore it appeared that it might account for NRF2 triggering. However, this mechanism was excluded by the use of a selective p38 inhibitor, which failed to cause a significant NRF2 nuclear shift and target gene induction. Finally, dexamethasone effects on the classical oxidative pathway orchestrated by KEAP1 were addressed. Dexamethasone was found to decrease the expression of the inhibitor KEAP1 at both mRNA and protein levels and to induce the shift from the reduced to the oxidized form of KEAP1, thus favouring NRF2 translocation into the nucleus. Furthermore, preliminary data revealed very low levels of the negative regulator Fyn in Ataxia Telangiectasia cells, which might account for the prolonged NRF2-activated gene expression.

Ataxia telangiectasia (AT) is a rare incurable genetic disease caused by biallelic mutations in the Ataxia telangiectasia-mutated gene. Intra-erythrocyte infusion of dexamethasone improves clinical outcomes in AT patients; however, the molecular mechanisms that lead to this improvement remain unknown. Hence, to gain a better understanding of these mechanisms, we assessed the effects of glucocorticoid administration on gene expression in the blood of AT patients. Whole blood was obtained from nine children enrolled in a phase two clinical trial, who were being treated with dexamethasone (AT Dexa), from six untreated AT patients (AT) and from six healthy volunteers (WT). CodeLink Whole Genome Bioarrays were used to assess transcript expression. The reliability of the differentially expressed genes (DEGs) was verified by qRT-PCR analysis. The enriched Gene Ontology (GO) terms and the pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) of DEGs obtained by group comparisons were achieved using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Functional network analyses were computed by Reactome FI. The likely involved transcription factors were revealed by iRegulon. Among the identified DEGs influenced by the pathology and restored by dexamethasone, we detected 522 upregulated probes coding for known proteins, while 22 probes were downregulated, as they were in healthy subjects. These results provide useful information and represent a first step towards gaining a better understanding of the underlying mechanisms of the effects of dexamethasone on AT patients.

Background Ataxia Telangiectasia (AT) is a rare incurable genetic disease, caused by biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this syndrome. Nevertheless, the molecular mechanism underlying the glucocorticoid action in AT patients is not yet understood. Recently, we have demonstrated that Dexamethasone treatment may partly restore ATM activity in AT lymphoblastoid cells by a new ATM transcript, namely ATMdexa1. Results In the present study, the new ATMdexa1 transcript was also identified in vivo, specifically in the PMBCs of AT patients treated with intra-erythrocyte Dexamethasone (EryDex). In these patients it was also possible to isolate new “ATMdexa1 variants” originating from canonical and non-canonical splicing, each containing the coding sequence for the ATM kinase domain. The expression of the ATMdexa1 transcript family was directly related to treatment and higher expression levels of the transcript in patients’ blood correlated with a positive response to Dexamethasone therapy. Neither untreated AT patients nor untreated healthy volunteers possessed detectable levels of the transcripts. ATMdexa1 transcript expression was found to be elevated 8 days after the drug infusion, while it decreased 21 days after treatment. Conclusions For the first time, the expression of ATM splicing variants, similar to those previously observed in vitro, has been found in the PBMCs of patients treated with EryDex. These findings show a correlation between the expression of ATMdexa1 transcripts and the clinical response to low dose dexamethasone administration. Electronic supplementary material The online version of this article (doi:10.1186/s13023-017-0669-2) contains supplementary material, which is available to authorized users.

Ataxia telangiectasia is a rare genetic disease and no therapy is currently available. Glucocorticoid analogues have been shown to improve the neurological symptoms of treated patients. In the present study ataxia telangiectasia and wild type cells were used as a cellular model and treated with dexamethasone. The cells were subsequently investigated for membrane and whole cell mechanical properties by atomic force microscopy. In addition, cytoskeleton protein dynamics and nuclear shapes were assayed by fluorescence microscopy, while western blots were used to assess actin and tubulin content. At the macro level, dexamethasone directly modified the cell shape, Young's modulus and cytoskeleton protein dynamics. At the nano level, the roughness of the cell surface and the local nano-mechanical proprieties were found to be affected by Dexa. Our results show that ataxia telangiectasia and wild type cells are affected by Dexa, although there are dissimilarities in some macro-level and nano-level features between the tested cell lines. The Young's modulus of the cells appears to depend mainly on nuclear shape, with a slight contribution from the tested cytoskeleton proteins. The current study proposes that dexamethasone influences ataxia telangiectasia cell membranes contents, cell components and cell shape.

In the present paper we describe an atomic force microscopy (AFM)-based method for the quantitative analysis of FK506 (Tacrolimus) in whole blood (WB) samples. Current reference methods used to quantify this immunosuppressive drug are based on mass spectrometry. In addition, an immunoenzymatic assay (ELISA) has been developed and is widely used in clinic, even though it shows a small but consistent overestimation of the actual drug concentration when compared with the mass spectrometry method. The AFM biosensor presented herein utilises the endogen drug receptor, FKBP12, to quantify Tacrolimus levels. The biosensor was first assayed to detect the free drug in solution, and subsequently used for the detection of Tacrolimus in blood samples. The sensor was suitable to generate a dose-response curve in the full range of clinical drug monitoring. A comparison with the clinically tested ELISA assay is also reported.

Multidetector computed tomography (MDCT) is one of the best diagnostic tools for the diagnosis of pulmonary embolism (PE). However, differences in MDCT interpretation, depending on the operator personal expertise, is an important factor that could interfere with the right diagnosis and, consequently, with the more adequate and well-timed therapy. The aim of the present study was to evaluate the interobserver agreement in the interpretation of MDCT for the diagnosis of acute PE. On a blind basis, 4 radiologists with different expertise in CT interpretation evaluated 46 different MDCT executed for acute PE. They had to verify the presence or absence of PE and, in the positive case, localize (right-left) and quantify (massive, segmentarian or subsegmentarian) it. The interobserver concordance was expressed using the Cohen K statistic. The mean concordance between the 4 operators was high (0.82; range, 0.68-0.95). Ruling out the massive PE cases, the mean concordance over the other cases was only moderate (0.47; range, 0.16-0.84). We found a very good interobserver agreement in MDCT evaluation for the diagnosis of massive PE, whereas we observed a lower concordance in regard to segmentarian and subsegmentarian PE. In the case of negative or nonmassive PE diagnosis, a second evaluation of the CT performed by an expert CT radiologist would probably be effective to decrease the CT evaluation error.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder caused by biallelic mutations in the Ataxia Telangiectasia-mutated gene. A-T shows a complex phenotype ranging from early-onset progressive neurodegeneration to immunodeficiencies, high incidence of infections, and tumors. Unfortunately, no therapy is up to now available for treating this condition. Recently, the short term treatment of ataxia-telangiectasia patients with glucocorticoids was shown to improve their neurological symptoms and possibly reverse cerebellar atrophy. Thus, corticosteroids represent an attractive approach for the treatment of this neurodegenerative disease. However, the molecular mechanism involved in glucocorticoid action in A-T is yet unknown. The aim of our work is to construct cDNA libraries containing those genes which are transactivated by the glucocorticoid analogue, dexamethasone, in A-T human cells. For this purpose, suppression subtractive hybridization has been performed on ATM-null lymphoblastoid cell transcriptome extracted following drug administration. Annotation of whole genes contained in the libraries has been obtained by coupling subtractive hybridization with microarray analysis. Positive transcripts have been validated by quantitative PCR. Through in silico analyses, identified genes have been classified on the basis of the pathway in which they are involved, being able to address signaling required for dexamethasone action. Most of the induced transcripts are involved in metabolic processes and regulation of cellular processes. Our results can help to unravel the mechanism of glucocorticoid action in the reversion of A-T phenotype. Moreover, the induction of a specific region of the ATM transcript has been identified as putative biomarker predictive of dexamethasone efficacy on ataxic patients.

Aims: The introduction of HBV vaccination in Italy has reduced the incidence and prevalence of HBV infection. However, increasing immigration from countries in which HBV is endemic has led to a wave of new HBsAg-positive subjects and their clinical impact needs evaluation. We compared the serological, clinical and epidemiological data relating to Italian and non-Italian subjects referred to a hospital in Northern Italy. Study Design: We retrospectively analysed the laboratory and clinical records of 488 subjects, including 107 (21.9%) non-Italians, in order to find data concerning clinical diagnoses, possible routes of infection, anti-HCV and anti-HDV antibodies. Results: The differences in gender distribution and mean age between the non-Italian and Italian patients were statistically significant (p

Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of nucleotide residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.

The introduction of HBV vaccination in Italy has led to a decline in new HBV infections. Increasing immigration over recent years suggests a change in short-term epidemiology of HBV. The aim of this study was to assess the prevalence of HBV infection in the general population living in the catchment area of Legnano Hospital (Northern Italy). In the period 2007-2008, 22,758 inpatients and outpatients were examined for Hepatitis B surface antigen (HBsAg), of whom 1,654 (7.3%) were of foreign origin. Of the 488 patients who were positive for HBsAg (2.1%), 381 (1.8%) were Italian and 107 (6.5%) were born in other countries. In terms of age, the prevalence of HBsAg was significantly higher among non- Italians in every age group (other than those aged >60 and

Drug delivery is a growing field of interdisciplinary activities that combine the use of new materials with the biochemical properties of selected drugs, with the aim of improving their therapeutic action and reducing their toxicity. In few cases, proper medical devices have been also realized to implement new drug delivery modalities. In this article, we have summarized available information and our experience on the use of autologous Red Blood Cells as carriers for drugs to be released within the vascular system. This is not a comprehensive review, but it focuses on the mechanisms that are available to distribute drugs in circulation by carrier red blood cells and provide illustrative examples on how this is currently obtained. We have not included a summary of clinical data collected in recent years using this technology but simply provided proper references for the interested readers. Finally, a special attention is devoted to the possibility of entrapping, into autologous red blood cells, recombinant drug-binding proteins. This new strategy is opening the way at a new modality to influence the vascular distribution of drugs by realizing a dynamic circulating container (the engineered red cell) capable of reversible binding and transportation of one or more drugs of interest selected on the bases of the red cell entrapped target proteins. This new modality is not yet fully developed and explored but will certainly provide a technical solution to the problem of stabilizing drug concentration in circulation improving drug efficacy and reducing drug toxicity.

Cyclosporine A (CsA) and tacrolimus (also known as FK506) are natural compounds with immunosuppressive activity that have improved the outcome of organ transplantation. Unfortunately, both drugs are characterised by high pharmacokinetic variability, poor bioavailability and high toxicity. Until now, no optimal method to deliver immunosuppressant drugs into circulation has been developed. Here we propose the use of engineered erythrocytes as a drug delivery system for the release of immunosuppressants in circulation in order to modify their pharmacokinetic and restrain toxic effects. After administration, FK506 and CsA mainly distribute within erythrocytes owing to the presence into these cells of immunophilins that bind the drugs with very high affinity (FKBP12 for FK506 and cyclophilin A for CsA); therefore, a new strategy aimed to increase the amount of FK506/CsA carried by erythrocytes by increasing the intra-erythrocytic concentration of the respective binding proteins has been developed. We manufactured recombinant forms of human FKBP12 and cyclophilin A to be loaded into RBC through a hypotonic dialysis and isotonic resealing procedure. Erythrocytes loaded with 3.5±1.3, 7.5±3.1 and 15.5±0.4nmol FKBP12 were able to bind 3.5±1.5, 6.0±1.9 and 11.4±2.9μg FK506 per millilitre RBC, respectively, while RBC loaded with 4.0±0.6, 5.0±0.8 and 15.9±2.4nmol of cyclophilin A could bind 8.9±3.4, 12.2±3.5 and 17.0±3.2μg CsA. Thus, both engineered RBC were demonstrated able to bind up to an order of magnitude more drug than corresponding native erythrocytes (1.0±0.3μg FK506 and 3.2±0.3μg CsA). Moreover, FK506 released from FKBP12-RBC is able to be up-taken by T lymphocytes and inhibit IL-2 expression in vitro as free administered drug. In summary, our results indicate that diffusible immunosuppressants could be entrapped into red cells (thanks to the loading of the respective target protein) and suggest that immunophilin-loaded RBC could be employed as potential delivery system for immunosuppressive agents.

Cardiac autonomic abnormalities have been described in Parkinson’s disease. Little is known about possible alterations of vascular sympathetic regulatory activity in patients without orthostatic hypotension or symptoms of orthostatic intolerance. Nineteen patients with Parkinson’s disease without orthostatic hypotension (PD), 21 with orthostatic hypotension (PDOH), and 20 healthy controls underwent ECG, beat-to-beat arterial pressure, and respiration recordings while recumbent and during a 75° head-up tilt. Spectrum analysis of RR interval and systolic arterial pressure (SAP) variability provided indices of cardiac sympathovagal interaction (low frequency [LF]/high frequency [HF]) to the sinoatrial node and sympathetic vasomotor control (LF SAP ). Arterial baroreceptor mechanisms were assessed by the spontaneous sequences technique and bivariate spectrum analysis (α index). Plasma catecholamines provided the neurohormonal profile. At rest, hemodynamics and spectral markers of autonomic function were similar in PD and control subjects. Norepinephrine was lower in PD and PDOH than in control subjects. In PDOH, SAP was higher, whereas LF/HF ratio and LF SAP were lower compared with control subjects. During tilt, SAP was unchanged in PD; however, similar to PDOH, the increase of heart rate, LF/HF ratio, and LF SAP was blunted compared with control subjects. Baroreflex indices were unmodified in PD and PDOH compared with control subjects. Initial alterations in both cardiac and vascular sympathetic modulatory activity were found in PD and revealed by a gravitational stimulus. Prompt recognition of sympathetic abnormalities might result in earlier therapeutic intervention, reduced orthostatic intolerance, and increased quality of life.