Natalia Malara

Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung

Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

Simultaneous inhibition of the constitutively activated nuclear factor κB and of the Interleukin-6 pathways is necessary and sufficient to completely overcome apoptosis resistance of human U266 myeloma cells

Elevated Nuclear Factor κB (NF-κB) levels have been reported in multiple myeloma cells derived from patients relapsing after chemotherapy. In the search of an in vitro a model with molecular features similar to relapsing lesions, we focused our attention on an IL-6 autocrine human myeloma cell line (U266), characterized by apoptosis resistance due to up-regulation of two constitutive signaling pathways: NF-κB and STAT-3. NF-κB activity was inhibited with proteasome inhibitory agents, such as PS-341 and Withaferin A, with an IKK inhibitor (Wedelolactone) or with the adenoviral vector HD IκBαmut-IRES-EGFP encoding a mutant IκBα protein, resistant to proteasomal degradation. We observed that the NF-κB intracellular dislocation at the beginning of the treatment affected therapeutic effectiveness of PS-341, Withaferin A and Wedelolactone; interestingly, the adenoviral vector was highly effective in inducing apopotosis even with NF-κB being predominantly nuclear at the time of infection. We also observed that U266 treated with the Interleukin-6 antagonist Sant7 exhibited reduced STAT3 activity and preferential cytoplasmic NF-κB location; moreover they became capable of undergoing apoptosis mainly from the G1 phase. Adenoviral vector treated U266 have NF-κB localized completely in the cytoplasm and also showed down-regulation of nuclear phospho STAT-3. Finally, combined targeting of NF-κB and STAT3 signaling pathways was the most effective treatment in inducing apoptosis. These findings suggest that combined NF-κB κB and STAT3 targeting warrants further investigations in other apoptosis resistant MM cell lines as well as in suitable MM animal models.

The treatment of hyperuricemia

Hyperuricemia has long been established as the major etiologic factor in gout. Alongside with an inflammatory state triggered by urate crystal deposition in the joints, hyperuricemia displayed additional pathophysiological consequences leading to tissue inflammation mainly in the vascular wall. Thus, therapeutic strategies used to treat hyperuricemia in the past decades have often been focused on limiting acute episodes. Recently, evidence has been accumulated suggesting that chronic urate deposition requires a correct treatment not limited to acute episodes based on the modulation of the activity of key enzymes involved in metabolism and excretion of urate including xanthine oxidoreductase (XO) and URAT1. The present review article will try to summarize the most recent evidences on the efficacy of XO inhibitors and uricosuric compounds in lowering uric acid levels in both the bloodstream and peripheral tissues. In particular, we will focus on the effect of novel XO inhibitors in counteracting uric acid overproduction. On the other hand, the effect of lowering uric acid levels via XO inhibition will be correlated with attenuation oxidative stress which leads to endothelial dysfunction thereby contributing to the pathophysiology of diabetes, hypertension, arteriosclerosis, and chronic heart failure. Hence, scavenging and prevention of the XO generated oxygen radical accumulation emerge as an intriguing novel treatment option to counteract uric acid-induced tissue damages.

Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial homeostasis maintenance. Both CEC and EPC are thought to represent potential biomarkers in several clinical conditions involving endothelial turnover/remodeling. Although different flow cytometry methods for CEC and EPC characterization have been published so far, none of them have reached consistent conclusions, therefore consensus guidelines with respect to CEC and EPC identification and quantification need to be established. Here, we have carried out an in depth investigation of CEC and EPC phenotypes in healthy PB, CB and BM samples, by optimizing a reliable polychromatic flow cytometry (PFC) panel. Results showed that the brightness of CD34 expression on healthy PB and CB circulating cells represents a key benchmark for the identification of CEC (CD45neg/CD34bright/CD146pos) respect to the hematopoietic stem cell (HSC) compartment (CD45dim/CD34pos/CD146neg). This approach, combined with a dual‐platform counting technique, allowed a sharp CEC enumeration in healthy PB (n = 38), and resulting in consistent CEC counts with previously reported data (median = 11.7 cells/ml). In parallel, by using rigorous PFC conditions, CD34pos/CD45dim/CD133pos/VEGFR2pos EPC were not found in any healthy PB or CB sample, since VEGFR2 expression was never detectable on the surface of CD34pos/CD45dim/CD133pos cells. Notably, the putative EPC phenotype was observed in all analyzed BM samples (n = 12), and the expression of CD146 and VEGFR2, on BM cells, was not restricted to the CD34bright compartment, but also appeared on the HSC surface. Altogether, our findings suggest that the previously reported EPC antigen profile, defined by the simultaneous expression of VEGFR2 and CD133 on the surface of CD45dim/CD34pos cells, should be carefully re‐evaluated and further studies should be conducted to redefine EPC features in order to translate CEC and EPC characterization into clinical practice.

Electroless Deposition and Nanolithography Can Control the Formation of Materials at the Nano-Scale for Plasmonic Applications

The new revolution in materials science is being driven by our ability to manipulate matter at the molecular level to create structures with novel functions and properties. The aim of this paper is to explore new strategies to obtain plasmonic metal nanostructures through the combination of a top down method, that is electron beam lithography, and a bottom up technique, that is the chemical electroless deposition. This technique allows a tight control over the shape and size of bi- and three-dimensional metal patterns at the nano scale. The resulting nanostructures can be used as constituents of Surface Enhanced Raman Spectroscopy (SERS) substrates, where the electromagnetic field is strongly amplified. Our results indicate that, in electroless growth, high quality metal nanostructures with sizes below 50 nm may be easily obtained. These findings were explained within the framework of a diffusion limited aggregation (DLA) model, that is a simulation model that makes it possible to decipher, at an atomic level, the rules governing the evolution of the growth front; moreover, we give a description of the physical mechanisms of growth at a basic level. In the discussion, we show how these findings can be utilized to fabricate dimers of silver nanospheres where the size and shape of those spheres is controlled with extreme precision and can be used for very large area SERS substrates and nano-optics, for single molecule detection.

Protein–Carbohydrate Complex Reveals Circulating Metastatic Cells in a Microfluidic Assay

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3--a protein that is correlated to the progress of tumor and metastasis--is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interactions strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine.

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.

Animal models of cardiac cachexia

Cachexia is the loss of body weight associated with several chronic diseases including chronic heart failure (CHF). The cachectic condition is mainly due to loss of skeletal muscle mass and adipose tissue depletion. The majority of experimental in vivo studies on cachexia rely on animal models of cancer cachexia while a reliable and appropriate model for cardiac cachexia has not yet been established. A critical issue in generating a cardiac cachexia model is that genetic modifications or pharmacological treatments impairing the heart functionality and used to obtain the heart failure model might likely impair the skeletal muscle, this also being a striated muscle and sharing with the myocardium several molecular and physiological mechanisms. On the other hand, often, the induction of heart damage in the several existing models of heart failure does not necessarily lead to skeletal muscle loss and cachexia. Here we describe the main features of cardiac cachexia and illustrate some animal models proposed for cardiac cachexia studies; they include the genetic calsequestrin and Dahl salt-sensitive models, the monocrotaline model and the surgical models obtained by left anterior descending (LAD) ligation, transverse aortic constriction (TAC) and ascending aortic banding. The availability of a specific animal model for cardiac cachexia is a crucial issue since, besides the common aspects of cachexia in the different syndromes, each disease has some peculiarities in its etiology and pathophysiology leading to cachexia. Such peculiarities need to be unraveled in order to find new targets for effective therapies.

The effect of peroxynitrite decomposition catalyst MnTBAP on aldehyde dehydrogenase-2 nitration by organic nitrates: role in nitrate tolerance.

Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event in GTN tolerance, an effect counteracted both in vitro and in vivo by novel peroxynitrite decomposition catalyst.

From nucleotides to DNA analysis by a SERS substrate of a self similar chain of silver nanospheres

In this work we realized a device of silver nanostructures designed so that they have a great ability to sustain the surface-enhanced Raman scattering effect. The nanostructures were silver self-similar chains of three nanospheres, having constant ratios between their diameters and between their reciprocal distances. They were realized by electron beam lithography, to write the pattern, and by silver electroless deposition technique, to fill it with the metal. The obtained device showed the capability to increase the Raman signal coming from the gap between the two smallest nanospheres (whose size is around 10 nm) and so it allows the detection of biomolecules fallen into this hot spot. In particular, oligonucleotides with 6 DNA bases, deposited on these devices with a drop coating method, gave a Raman spectrum characterized by a clear fingerprint coming from the hot spot and, with the help of a fitting method, also oligonucleotides of 9 bases, which are less than 3 nm long, were resolved. In conclusion the silver nanolens results in a SERS device able to measure all the molecules, or part of them, held into the hot spot of the nanolenses, and thus it could be a future instrument with which to analyze DNA portions.