Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

shRNA targeting of ferritin heavy chain activates H19/miR-675 axis in K562 cells.

PURPOSE: The heavy subunit of the iron storage protein ferritin (FHC) is essential for the intracellular iron metabolism and, at the same time, it represents a central hub of iron-independent pathways, such as cell proliferation, angiogenesis, p53 regulation, chemokine signalling, stem cell expansion, miRNAs expression. In this work we have explored the ability of FHC to modulate gene expression in K562 cells, through the up-regulation of the lncRNA H19 and its cognate miR-675. MATERIALS AND METHODS: Targeted silencing of FHC was performed by lentiviral-driven shRNA strategy. FHC reconstitution was obtained by full length FHC cDNA transfection with Lipofectamine 2000. ROS amounts were determined with the redox-sensitive probe H2DCFDA. H19, miR-675, miR-107, Twist1, ID3, EPHB6, GNS, ANK1 and SMAD6 mRNA amounts were quantified by Taqman assay and qPCR analysis. RESULTS: FHC silencing in K562 cells modulates gene expression through the up-regulation of the lncRNA H19 and its cognate miR-675. Experimental findings demonstrate that the molecular mechanism underlying this phenomenon is represented by an FHC knock-down-triggered increase in reactive oxygen species (ROS) production. CONCLUSIONS: In this paper we uncover a so far not described function of the ferritin heavy subunit in the control of lncRNA pathways.

Epithelial-to-mesenchymal transition in FHC-silenced cells: the role of CXCR4/CXCL12 axis.

BACKGROUND: Ferritin plays a central role in the intracellular iron metabolism; the molecule is a nanocage of 24 subunits of the heavy and light types. The heavy subunit (FHC) is provided of a ferroxidase activity and thus performs the key transformation of iron in a non-toxic form. Recently, it has been shown that FHC is also involved in additional not iron-related critical pathways including, among the others, p53 regulation, modulation of oncomiRNAs expression and chemokine signalling. Epithelial to mesenchymal transition (EMT) is a cellular mechanism by which the cell acquires a fibroblast-like phenotype along with a decreased adhesion and augmented motility. In this work we have focused our attention on the role of the FHC on EMT induction in the human cell lines MCF-7 and H460 to elucidate the underlying molecular mechanisms. METHODS: Targeted silencing of the FHC was performed by lentiviral-driven shRNA strategy. Reconstitution of the FHC gene product was obtained by full length FHC cDNA transfection with Lipofectamine 2000. MTT and cell count assays were used to evaluate cell viability and proliferation; cell migration capability was assayed by the wound-healing assay and transwell strategy. Quantification of the CXCR4 surface expression was performed by flow cytometry. RESULTS: Experimental data indicated that FHC-silenced MCF-7 and H460 cells (MCF-7shFHC, H460shFHC) acquire a mesenchymal phenotype, accompanied by a significant enhancement of their migratory and proliferative capacity. This shift is coupled to an increase in ROS production and by an activation of the CXCR4/CXCL12 signalling pathway. We present experimental data indicating that the cytosolic increase in ROS levels is responsible for the enhanced proliferation of FHC-silenced cells, while the higher migration rate is attributable to a dysregulation of the CXCR4/CXCL12 axis. CONCLUSIONS: Our findings indicate that induction of EMT, increased migration and survival depend, in MCF-7 and H460 cells, on the release of FHC control on two pathways, namely the iron/ROS metabolism and CXCR4/CXCL12 axis. Besides constituting a further confirmation of the multifunctional nature of FHC, this data also suggest that the analysis of FHC amount/function might be an important additional tool to predict tumor aggressiveness.

Behaviour of dental pulp stem cells on different types of innovative mesoporous and nanoporous silicon scaffolds with different functionalizations of the surfaces.

Dental pulp stem cells (DPSCs) are stem cells found in the dental pulp. The ability of DPSCs to differentiate towards odontoblastic and osteoblastic phenotype was reported first in the literature, then in the following years, numerous studies on odontogenesis were carried out, starting from mesenchymal stem cells isolated from tissues of dental and oral origin. The aim of this research was to evaluate the behaviour of DPSCs grown on silicon nanoporous and mesoporous matrices and differentiated towards the osteogenic phenotype, but also to investigate the use of DPSCs in pilot studies focused on the biological compatibility of innovative dental biomaterials. Twenty-eight silicon samples were created with standardized procedures. These scaffolds were divided into samples made of silicon bulk, nanoporous silicon, mesoporous silicon, nanoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS) and methanol (MeOH), nanoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS)/toluene, mesoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS) and methanol (MeOH) andmesoporous silicon functionalized with (3-Aminopropyl) Trimethoxysilane (APTMS)/toluene. DPSC proliferation on the tested silicon scaffolds was analyzed at 3 and 5 days. The assay showed that DPSCs proliferated better on mesoporous scaffolds functionalized with APTMS/toluene compared to a silicon one. These results show that the functionalization of silicon scaffold with APTMS/toluene supports the growth of DPSCs and could be used for future applications in tissue engineering.

A proteomics-driven assay defines specific plasma protein signatures in different stages of Ménière's disease.

Ménière's disease (MD) is a common disorder of the inner ear whose hallmarks are vertigo, tinnitus, aural fullness, and progressive hearing loss. The degree of severity of the disease is quite heterogeneous, and so is its pathogenesis. A multifactorial inheritance of intrinsic and extrinsic factors has been described, but there is not a common agreement on the molecular basis of MD. In a recent article, we have demonstrated that patients suffering from MD share a common plasma proteomic signature, characterized by the presence of several up- and down-regulated proteins. In this study, we have further extended our analysis and show that the differential expression of plasma proteins can identify specific subsets of MD-affected individuals, depending on their stage. Our findings confirm our plasma proteomics-driven approach as a powerful tool for early diagnosis of MD and uncover a potentially starring role for some proteins in the development and fate of this frustrating disease, whose pathogenesis still remains unclear.

Biomarker discovery by plasma proteomics in familial Brugada Syndrome.

Brugada Syndrome (BS) is a polygenic inherited cardiac disease characterized by life-threatening arrhythmias and high incidence of sudden death. In this study, two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (LC-MS/MS) was used to investigate specific changes in the plasma proteome of BS patients and family members sharing the same gene mutation (SCN5AQ1118X), with the aim to identify novel disease biomarkers. Our data demonstrate that the levels of several proteins were significantly altered in BS patients compared with controls. In particular, apolipoprotein E, prothrombin, vitronectin, complement-factor H, vitamin-D-binding protein, voltage-dependent anion-selective channel protein 3 and clusterin were considerably increased in plasma sample of BS patients, whereas alpha-1-antitrypsin, fibrinogen and angiotensinogen were considerably decreased; moreover, post-translational modifications of antithrombin-III were detected in all affected individuals. On the light of these results, we hypothesize that these proteins might be considered as potential markers for the identification of disease status in BS.

Sgk1 enhances RANBP1 transcript levels and decreases taxol sensitivity in RKO colon carcinoma cells.

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.

Negative transcriptional regulation of the human periostin gene by YingYang-1 transcription factor.

Periostin (POSTN), an osteoblast-specific secreted protein known to be associated with cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. At present, little is known about the mechanisms underlying its transcriptional control either in physiological or neoplastic conditions. In this study we demonstrate that the ability of the human POSTN promoter to drive transcription mostly depends on the activity of YingYang-1 (YY1) zinc finger transcription factor. YY1, whose regulatory role in biology includes, besides transcriptional control, also chromatin remodeling, DNA damage repair and tumorigenesis, acts as a strong negative modulator of the POSTN expression. We retain that the identification of the functional role of YY1 in the transcriptional control of the human POSTN gene adds new insights in the studies focused on gene expression in normal and transformed cells.

Proteomic analysis in canine leishmaniasis.

The aim of this study was to perform a proteomic analysis on serum of dogs naturally infected with Leishmania parasite. Sera from 24 dogs, n. 8 with high IFAT titre of anti-Leishmania antibodies (>or= 1:640), n. 8 with uncertain titre (= 1:40), and n. 8 with IFAT negative were used. Sera of each group were pooled together to form three pools: P (high titre); U (uncertain titre); and N (negative). The P pool was analyzed, using a mass spectrometry-based approach to search for Leishmania proteins (qualitative analysis). In a second experiment, protein signal intensities of U and P pools were compared with the signal intensities of N pool by a quantitative mass spectrometry method based on isotopic dilution. The quantitative analysis detected a total of 70 proteins, of which 17 and 5 resulted over- and under-represented in sample P, respectively.

Modeling and designing a proteomics application on PROTEUS.

OBJECTIVES: Biomedical applications, such as analysis and management of mass spectrometry proteomics experiments, involve heterogeneous platforms and knowledge, massive data sets, and complex algorithms. Main requirements of such applications are semantic modeling of the experiments and data analysis, as well as high performance computational platforms. In this paper we propose a software platform allowing to model and execute biomedical applications on the Grid. METHODS: Computational Grids offer the required computational power, whereas ontologies and workflow help to face the heterogeneity of biomedical applications. In this paper we propose the use of domain ontologies and workflow techniques for modeling biomedical applications, whereas Grid middleware is responsible for high performance execution. As a case study, the modeling of a proteomics experiment is discussed. RESULTS: The main result is the design and first use of PROTEUS, a Grid-based problem-solving environment for biomedical and bioinformatics applications. CONCLUSION: To manage the complexity of biomedical experiments, ontologies help to model applications and to identify appropriate data and algorithms, workflow techniques allow to combine the elements of such applications in a systematic way. Finally, translation of workflow into execution plans allows the exploitation of the computational power of Grids. Along this direction, in this paper we present PROTEUS discussing a real case study in the proteomics domain.

Molecular mechanisms of corticosteroid actions in chronic inflammatory airway diseases.

Although corticosteroids have been used for a long time as a very effective therapy of airway inflammatory diseases such as asthma, only recently the molecular basis of their mechanism of action has begun to be elucidated. These hormones exert their biological and pharmacological actions by binding to cytoplasmic receptors that, upon activation, translocate to the nucleus where they interact with specific genomic sequences thus modulating gene expression. However, many glucocorticoid effects responsible for their anti-inflammatory and anti-asthmatic activity take place irrespectively of receptor binding to DNA. In particular, ligand-bound glucocorticoid receptors can repress several different pro-inflammatory genes by physically associating, via protein-protein interactions, with various transcription factors and with the macromolecular complexes implicated in regulation of chromatin structure and function. In this regard, an important role is played by the influences of corticosteroids on the intrinsic histone acetyltransferase and deacetylase functions of coactivators and corepressors, respectively. Furthermore, the signal transduction pathways mediated by mitogen-activated protein kinases are newly recognized, key targets of glucocorticoids. Indeed, these enzymatic cascades are crucially involved in the regulation of gene expression in that they are essential for the activity of a high number of transcription factors. Therefore, the recent advances made in such a rapidly growing research field are providing new insights into the mode of action of corticosteroids, thereby also unveiling novel promising therapeutic strategies directly targeted to the molecular events underlying the inflammatory, immune, and apoptotic processes implicated in the pathogenesis of asthma and other airway diseases.

Oxidative stress and lung diseases.

Several different lung diseases are characterized by an oxidant/antioxidant imbalance, which is a major cause of cell damage. Oxidative stress activates a complex network of intracellular signal transduction pathways involved in the regulation of transcription factors such as nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1). Within this context, a key role is played by mitogen-activated protein kinases (MAPK), which are highly expressed by pulmonary endothelial and airway epithelial cells. By exposing these cell lines to oxidant agents, our group has shown that oxidative stress leads to a significant MAPK activation, which can be effectively inhibited by corticosteroids. We believe that studies such as ours may contribute to further elucidate the molecular events underlying the therapeutic action of these drugs in many respiratory disorders caused by oxidative/proinflammatory pathogenic mechanisms. In addition, our findings may help to unveil new anti-oxidant treatments based on MAPK modulation.

Transcriptional regulation of the mismatch repair gene hMLH1.

We have characterized the promoter region of the human gene coding for the MLH1 mismatch repair protein. The total transcriptional activity of the hMLH1 promoter is driven by two positive cis-elements included between nucleotides -300 and -220. The upstream element is a canonical CCAAT box, and it is recognized by the heterotrimeric transcription factor NF-Y. On the other hand, the downstream element is recognized by a nuclear factor of about 120 kDa. Variations in hMLH1 intracellular levels may influence the surveillance of the genome integrity. The identification of the two elements may shad some light on the regulation of the transcriptional regulation of hMLH1 expression.

Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer.

Several genes have been involved in the pathogenesis of hereditary breast/ovarian cancer (BOC), but mutations in the BRCA1 gene are by far the most recurrent. In this study, we report the identification of a founder mutation in a geographically and historically homogeneous population from Calabria, a south Italian region. A screening performed on 24 patients from unrelated families highlighted the high prevalence of a 5083del19 alteration in the BRCA1 gene, which accounts for 33% of the overall gene mutations. The same mutation was also detected in 4 patients, all of Calabrian origin, referred to us by research centres from the north of Italy. Allelotype analysis, performed on probands and unaffected family members revealed the presence a common allele, therefore suggesting a founder effect due to a common ancestor. Our findings underscore the importance of ethnic background homogeneity in patients' selection and highlight the usefulness of founder mutations as a potential tool for optimisation of preclinical diagnosis in gene carriers and therapeutic approaches in affected individuals.

Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Effects of glucocorticoids on activation of c-jun N-terminal, extracellular signal-regulated, and p38 MAP kinases in human pulmonary endothelial cells.

Mitogen-activated protein kinases (MAPK) play a central role in signal transduction by regulating many nuclear transcription factors involved in inflammatory, immune, and proliferative responses. The aim of this study was to investigate, in human pulmonary endothelial cells, the effects of synthetic glucocorticosteroids on activation of c-jun N-terminal kinases, extracellular signal-regulated kinases, and p38 subgroups of the MAPK family. Human microvascular endothelial cells from lung were stimulated for 2 h with either H(2)O(2) (2 mM), IL-1beta (10 ng/mL), or tumour necrosis factor-alpha (10 ng/mL). Under these conditions, a remarkable increase in the phosphorylation pattern of c-jun N-terminal kinases, extracellular signal-regulated kinases 1/2, and p38 was detected. Pretreatment for 12 h with dexamethasone (100 nM) was able to prevent phosphorylation-dependent MAPK activation in stimulated cells, without substantially affecting the expression levels of these enzymes. Our results suggest that inhibition of MAPK signaling pathways in human pulmonary endothelial cells may significantly contribute, by interfering with activation of several different transcription factors, to the antiinflammatory and immunosuppressive effects of glucocorticosteroids.

Changes in myocardial cytoskeletal intermediate filaments and myocyte contractile dysfunction in dilated cardiomyopathy: an in vivo study in humans.

AIM: To investigate in vivo the intermediate cytoskeletal filaments desmin and vimentin in myocardial tissues from patients with dilated cardiomyopathy, and to determine whether alterations in these proteins are associated with impaired contractility. METHODS: Endomyocardial biopsies were performed in 12 patients with dilated cardiomyopathy and in 12 controls (six women with breast cancer before anthracycline chemotherapy and six male donors for heart transplantation). Biopsy specimens were analysed by light microscopy and immunochemistry (desmin, vimentin). Myocyte contractile protein function was evaluated by the actin-myosin in vitro motility assay. Left ventricular ejection fraction was assessed by echocardiography and radionuclide ventriculography. RESULTS: Patients with dilated cardiomyopathy had a greater cardiomyocyte diameter than controls (p < 0.01). The increase in cell size was associated with a reduction in contractile function, as assessed by actin-myosin motility (r = -0.643; p < 0.01). Quantitative immunochemistry showed increased desmin and vimentin contents (p < 0.01), and the desmin distribution was disturbed in cardiomyopathy. There was a linear relation between desmin distribution and actin-myosin sliding in vitro (r = 0.853; p < 0.01) and an inverse correlation between desmin content and ejection fraction (r = -0.773; p < 0.02). Negative correlations were also found between myocardial vimentin content and the actin-myosin sliding rate (r = -0.74; p < 0.02) and left ventricular ejection fraction (r = -0.68; p < 0.01). CONCLUSIONS: Compared with normal individuals, the myocardial tissue of patients with dilated cardiomyopathy shows alterations of cytoskeletal intermediate filament distribution and content associated with reduced myocyte contraction.

Calcium antagonist isradipine improves abnormal endothelium-dependent vasodilation in never treated hypertensive patients.

OBJECTIVE: To examine whether middle (two months) and long-term (six months) isradipine sustained-release treatment improves endothelium-dependent vasodilation in never treated hypertensive patients. METHODS: The responses of the forearm vasculature to acetylcholine (7.5, 15 and 30 micrograms/min) and sodium nitroprusside (0.8, 1.6, 3.2 micrograms/min) were evaluated in 12 normotensive controls (seven men and five women, aged 25 to 49 years), and in 12 hypertensives (eight men and four women, aged 20 to 47 years) at baseline and after two and six months of isradipine sustained-release treatment. Drugs were infused into the brachial artery, and forearm blood flow was measured by strain-gauge plethysmography. RESULTS: At baseline, the response to acetylcholine was significantly lower in hypertensives vs controls: at the highest dose (30 micrograms/min), forearm blood flow was 28.6 +/- 2.4 ml/100 ml of tissue per min in the controls vs 8.9 +/- 1.0 ml/100 ml of tissue per min in hypertensive (p < 0.0001). Similarly, vascular resistance was significantly (p < 0.0001) higher in hypertensives: 4.8 +/- 0.5 units (controls) vs 15.1 +/- 1.7 units (hypertensives). After isradipine treatment, the forearm blood flow in hypertensive patients changed from 8.9 +/- 1.0 ml/100 ml of tissue per min to 16.0 +/- 1.2 ml/100 ml of tissue per min (two months; p < 0.0001) and 15.2 +/- 1.4 ml/100 ml of tissue per min (six months; p < 0.0001). Isradipine treatment did not modify the vasodilating effect of sodium nitroprusside. CONCLUSIONS: Our data demonstrate for the first time that the calcium antagonist isradipine improves acetylcholine-induced vasodilation in hypertensives.

Familial hypertrophic cardiomiopathy: molecular basis and genotype-phenotype correlations.

The impact of molecular genetics in the diagnosis and management of various forms of heritable cardiac or vascular disorders is continuously increasing thanks to the newly available laboratory tools. Familial hypertrophic cardiomyopathy (FHC), an autosomal dominant inherited disease characterized by unexplained left ventricular hypertrophy and a wide range of clinical symptoms, is the first cardiac disorder whose genetic bases have been elucidated. Linkage analysis studies have shown a statistically significant association between the disease status and at least seven genetic loci, all coding for sarcomeric proteins, in unrelated kindreds. A major challenge for physicians is to make an accurate and early diagnosis, not only on the basis of the traditional tools (i.e. physical examination and electro-echocardiography) but also to focus on the impact of genotype on clinical manifestations of FHC. In this review we present the more recent findings on the genetic basis of FHC and analyze the genotype-phenotype correlations of this disorder, whose expression may be modulated by additional factors (modifier genes, genetic background, environmental factors) other than mutations in any of the sarcometric proteins.