Publications (28)98.06 Total impact
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Article: The Isolectin IB4 binds RET Receptor Tyrosine Kinase in microglia.
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ABSTRACT: Ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (GDNF). Ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. Ret expression has been as well demonstrated on microglia and several evidence indicate that GDNF regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. Here we demonstrated that plant lectin Griffonia (Bandeiraea) simplicifolia lectin I, isolectin B4 (IB4), commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of Ret on the surface of living NIH3T3 fibroblasts cells stably transfected with Ret as well as in adult rat brain as revealed by immunoblotting. Further, confocal immunofluorescence analysis demonstrated a clear overlap in staining between pRet and IB4 in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pMCAO). Interestingly, IB4 staining identified activated or amoeboid Ret-expressing microglia under ischemic conditions. Collectively, our data indicate Ret receptor as one of the IB4-reactive glycoconjugate accounting for the IB4 stain in microglia under physiological and ischemic conditions. © 2013 International Society for Neurochemistry, J. Neurochem. (2013) 10.1111/jnc.12209.Journal of Neurochemistry 02/2013; · 4.06 Impact Factor -
Article: Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay.
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ABSTRACT: MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin-alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L(-1) and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.Analytical and Bioanalytical Chemistry 10/2012; · 3.78 Impact Factor -
Article: Targeting Axl With an High-affinity Inhibitory Aptamer.
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ABSTRACT: Axl is a tyrosine kinase receptor that was first identified as a transforming gene in human myeloid leukemia. Recent converging evidence suggests its implication in cancer progression and invasion for several solid tumors, including lung, breast, brain, thyroid, and pancreas. In the last decade, Axl has thus become an attractive target for therapeutic development of more aggressive cancers. An emerging class of therapeutic inhibitors is now represented by short nucleic acid aptamers. These molecules act as high affinity ligands with several advantages over conventional antibodies for their use in vivo, including their small size and negligible immunogenicity. Furthermore, these molecules can easily form conjugates able to drive the specific delivery of interfering RNAs, nanoparticles, or chemotherapeutics. We have thus generated and characterized a selective RNA-based aptamer, GL21.T that binds the extracellular domain of Axl at high affinity (12 nmol/l) and inhibits its catalytic activity. GL21.T blocked Axl-dependent transducing events in vitro, including Erk and Akt phosphorylation, cell migration and invasion, as well as in vivo lung tumor formation in mice xenografts. In this respect, the GL21.T aptamer represents a promising therapeutic molecule for Axl-dependent cancers whose importance is highlighted by the paucity of available Axl-specific inhibitory molecules.Molecular Therapy 08/2012; · 6.87 Impact Factor -
Chapter: Nucleic Acid Aptamers for In Vivo Molecular Imaging
03/2012; , ISBN: 978-953-51-0359-2 -
Article: New insight into clinical development of nucleic acid aptamers.
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ABSTRACT: Nucleic acid-based aptamers have been shown as high-affinity ligands and potential antagonists of disease-associated proteins. Aptamers, isolated from combinatorial libraries by an iterative in vitro selection process, discriminate between closely related targets and are characterized by high specificity and low toxicity thus representing a valid alternative to antibodies to target specific proteins of biomedical interest. Moreover, they are non-immunogenic and can be easily stabilized by chemical modifications thus expanding their therapeutic potential. Here, we will focus on the structural and functional features of aptamers that have entered the clinical development pipeline together with those aptamers holding great potential as therapeutics in preclinical studies. The future perspectives of aptamers as therapeutics will be discussed as well.Discovery medicine 06/2011; 11(61):487-96. -
Article: A regulatory mechanism involving TBP-1/Tat-Binding Protein 1 and Akt/PKB in the control of cell proliferation.
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ABSTRACT: TBP-1 /Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a multifunctional protein, originally identified as a regulator of HIV-1-Tat mediated transcription. It is an AAA-ATPase component of the 19S regulative subunit of the proteasome and, as other members of this protein family, fulfils different cellular functions including proteolysis and transcriptional regulation. We and others reported that over expression of TBP-1 diminishes cell proliferation in different cellular contexts with mechanisms yet to be defined. Accordingly, we demonstrated that TBP-1 binds to and stabilizes the p14ARF oncosuppressor increasing its anti-oncogenic functions. However, TBP-1 restrains cell proliferation also in the absence of ARF, raising the question of what are the molecular pathways involved. Herein we demonstrate that stable knock-down of TBP-1 in human immortalized fibroblasts increases cell proliferation, migration and resistance to apoptosis induced by serum deprivation. We observe that TBP-1 silencing causes activation of the Akt/PKB kinase and that in turn TBP-1, itself, is a downstream target of Akt/PKB. Moreover, MDM2, a known Akt target, plays a major role in this regulation. Altogether, our data suggest the existence of a negative feedback loop involving Akt/PKB that might act as a sensor to modulate TBP-1 levels in proliferating cells.PLoS ONE 01/2011; 6(10):e22800. · 4.09 Impact Factor -
Article: A neutralizing RNA aptamer against EGFR causes selective apoptotic cell death.
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ABSTRACT: Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity. Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA-aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors.PLoS ONE 01/2011; 6(9):e24071. · 4.09 Impact Factor -
Article: Recent Advance in Biosensors for microRNAs Detection in Cancer
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ABSTRACT: MicroRNAs (miRNAs) are short non-protein-coding RNA molecules that regulate the expression of a wide variety of genes. They act by sequence-specific base pairing in the 3" untranslated region (3"UTR) of the target mRNA leading to mRNA degradation or translation inhibition. Recent studies have implicated miRNAs in a wide range of biological processes and diseases including development, metabolism and cancer, and revealed that expression levels of individual miRNAs may serve as reliable molecular biomarkers for cancer diagnosis and prognosis. Therefore, a major challenge is to develop innovative tools able to couple high sensitivity and specificity for rapid detection of miRNAs in a given cell or tissue. In this review, we focus on the latest innovative approaches proposed for miRNA profiling in cancer and discuss their advantages and disadvantages.Cancers. 01/2011; 3:1877-1898. -
Article: Targeting cancer cells with nucleic acid aptamers.
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ABSTRACT: Aptamers are short, structured, single-stranded RNA or DNA ligands that bind with high affinity to their target molecules, which range from small chemicals to large cell-surface and transmembrane proteins. Aptamers are now emerging as promising molecules to target specific cancer epitopes in clinical diagnosis and therapy. Furthermore, because of their high specificity and low toxicity, aptamers might be considered as the compounds-of-choice for in vivo cell recognition. Specific cancer cell recognition could be capitalized upon for delivering therapeutic nanoparticles, small interfering RNA bioconjugates, chemotherapeutic cargos or molecular imaging probes. In this article, we review recent advances in the use of aptamers for in vivo cancer cell recognition, with a particular focus on novel applications of aptamers for targeting the cell surface.Trends in Biotechnology 10/2010; 28(10):517-25. · 9.15 Impact Factor -
Article: Aptamers as innovative diagnostic and therapeutic agents in the central nervous system.
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ABSTRACT: Aptamers are short non-naturally occurring single stranded DNA or RNA able to bind tightly, due to their specific three-dimensional shapes, to a multitude of targets ranging from small chemical compounds to cells and tissues. Since their first discovery, aptamers became a valuable research tool and show great application to fundamental research, drug selection and clinical diagnosis and therapy. Thanks to their unique characteristics (low size, good affinity for the target, no immunogenicity, chemical structures that can be easily modified to improve their in vivo applications), aptamers may represent a valid alternative to antibodies particularly for the treatment of neurological disorders that urgently needs modalities for drug delivery through the blood brain barrier. Aptamers have excellent potential as reagents for the targeted delivery of active drug substances, either through direct conjugation to the aptamer, or through their encapsulation in aptamer-coated vesicles. We will review here the recent and innovative methods that have been developed and the possible applications of aptamers as inhibitors or tracers in neurological disorders and brain cancer.CNS & neurological disorders drug targets 12/2009; 8(5):393-401. · 3.57 Impact Factor -
Article: GDNF selectively induces microglial activation and neuronal survival in CA1/CA3 hippocampal regions exposed to NMDA insult through Ret/ERK signalling.
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ABSTRACT: The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity.PLoS ONE 02/2009; 4(8):e6486. · 4.09 Impact Factor -
Article: Cell-specific aptamers for targeted therapies.
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ABSTRACT: Many signalling proteins involved in diverse functions such as cell growth and differentiation can act as oncogenes and cause cellular transformation. These molecules represent attractive targets for cancer diagnosis or therapy and therefore are subject to intensive investigation. Aptamers are small, highly structured nucleic acid molecules, isolated from combinatorial libraries by a procedure termed SELEX. Aptamers bind to a target molecule by providing a limited number of specific contact points imbedded in a larger, defined three-dimensional structure. Recently, aptamers have been selected against whole living cells, opening a new path which presents three major advantages: (1) direct selection without prior purification of membrane-bound targets, (2) access to membrane proteins in their native conformation similar to the in vivo conditions and (3) identification of (new) targets related to a specific phenotype. The ability to raise aptamers against living cells opens some attractive possibilities for new therapeutic and delivery approaches. In this chapter, the most recent advances in the field will be reviewed together with detailed descriptions of the relevant experimental approaches.Methods in molecular biology (Clifton, N.J.) 02/2009; 535:59-78. -
Article: Elevated expression of the tyrosine phosphatase SHP-1 defines a subset of high-grade breast tumors.
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ABSTRACT: Protein tyrosine phosphatases are key regulators of intracellular signaling that contribute to determining cancer cell growth, which thus makes them attractive targets for therapeutic and diagnostic agents. SHP-1 phosphotyrosine phosphatase is rarely expressed in epithelial tumor cells, but expression has been found in several breast cancer cell lines and tumors. To determine the potential significance of SHP-1 as a prognostic marker in the clinical setting, we examined SHP-1 protein expression in breast tumors. We analyzed SHP-1 expression by immunohistochemistry in a breast tissue microarray composed of 2,081 cores, either alone or in combination with known prognostic markers. Our data showed that SHP-1 expression was confined to a well-defined subset of high-grade tumors characterized by unique biological parameters. SHP-1 expression correlated directly with expression of the tyrosine kinase receptor HER-2 and inversely with expression of the estrogen receptor, while it was weakly associated with Bcl-2 expression. Levels of SHP-1 were correlated with conventional pathologic parameters of tumor aggressiveness and were associated with reduced patient survival, suggesting that elevated expression of SHP-1 is a common molecular abnormality in a defined subset of breast tumors and might be used in routine diagnosis to identify patients with high-risk tumors.Oncology 01/2009; 77(6):378-84. · 2.27 Impact Factor -
Article: Differential SELEX in human glioma cell lines.
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ABSTRACT: The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers.PLoS ONE 01/2009; 4(11):e7971. · 4.09 Impact Factor -
Article: A cross-talk between TrkB and Ret tyrosine kinases receptors mediates neuroblastoma cells differentiation.
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ABSTRACT: Understanding the interplay between intracellular signals initiated by multiple receptor tyrosine kinases (RTKs) to give the final cell phenotype is a major pharmacological challenge. Retinoic acid (RA)-treatment of neuroblastoma (NB) cells implicates activation of Ret and TrkB RTKs as critical step to induce cell differentiation. By studying the signaling interplay between TrkB and Ret as paradigmatic example, here we demonstrate the existence of a cross-talk mechanism between the two unrelated receptors that is needed to induce the cell differentiation. Indeed, we show that TrkB receptor promotes Ret phosphorylation by a mechanism that does not require GDNF. This reveals to be a key mechanism, since blocking either TrkB or Ret by small interfering RNA causes a failure in NB biochemical and morphological differentiation. Our results provide the first evidence that a functional transactivation between distinct tyrosine kinases receptors is required for an important physiological process.PLoS ONE 02/2008; 3(2):e1643. · 4.09 Impact Factor -
Article: Shp2 in PC12 cells: NGF versus EGF signalling.
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ABSTRACT: The balance between specific signals from different growth factors dictates the biological response of mammalian cells including cell proliferation, differentiation and survival. PC12 cells represent a model of choice to compare the signalling of differentiative growth factors, as NGF, and of mitogenic growth factors, as EGF. In these cells the prolonged activity of the ERK kinase dictates the decision of cells to differentiate. Here we focused on the cytosolic tyrosine phosphatase Shp2 as an established regulator of the Ras-ERK cascade, to elucidate its involvement in determining the stimulation-dependent PC12 cell fate. To this end, we generated PC12 derived cell lines that express the interfering mutant of Shp2 under a tetracycline-inducible promoter. Our findings show that Shp2 participates to the opposite effects induced in PC12 cells by EGF and NGF and that the interactions with the multidocking Gab2 protein mediate such effects.Cellular Signalling 07/2007; 19(6):1193-200. · 4.06 Impact Factor -
Article: Nucleic acid-based aptamers as promising therapeutics in neoplastic diseases.
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ABSTRACT: Isolated through combinatorial libraries by an iterative in vitro selection process, small single-stranded nucleic acid compounds, named aptamers, have been developed as high-affinity ligands for a variety of targets, ranging from small chemical compounds to large proteins. In the last years, an increasing number of aptamers has been generated that represent potential antagonists of the disease-associated target proteins. These molecules have been shown to discriminate between even closely related targets, thus representing a valid alternative to antibodies or other biomimetic receptors for the development of biosensors and other bioanalytical methods. Moreover, they can be easily stabilized by chemical modifications for in vivo applications and numerous examples have shown that stabilized aptamers against extracellular targets such as growth factors, receptors, hormones, or coagulation factors are very effective inhibitors of the corresponding protein function, thus resulting as useful reagents for target validation in a variety of diseases, including cancer. Indeed, many signaling proteins involved in diverse functions such as cell growth and differentiation can act as oncogenes and cause cellular transformation, thus making these high affinity ligands promising tools for cancer diagnosis or therapy.Methods in molecular biology (Clifton, N.J.) 02/2007; 361:187-200. -
Article: An autocrine loop involving ret and glial cell-derived neurotrophic factor mediates retinoic acid-induced neuroblastoma cell differentiation.
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ABSTRACT: In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.Molecular Cancer Research 08/2006; 4(7):481-8. · 4.29 Impact Factor -
Article: Aptamer-Based Technologies as New Tools for Proteomics in Diagnosis and Therapy
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ABSTRACT: Proteomics has provided a tool to define protein profile of a specific cell or tissue and to associate protein expression levels and post-translational modifications with disease states therefore developing innovative technologies for measurement of protein levels has become a major challenge of the last few years. Specific nucleic acid-based compounds, named aptamers, have been shown as high-affinity ligands and potential antagonists of disease-associated proteins. Aptamers, isolated from combinatorial libraries by an iterative in vitro selection process, discriminate between closely related targets thus representing a valid alternative to antibodies or other bio-mimetic receptors, for the development of biosensors and other bio-analytical methods. Moreover they can be easily stabilized by chemical modifications for in vivo applications and numerous examples have shown that stabilized aptamers against extracellular targets such as growth factors, receptors, hormones or coagulation factors are very effective inhibitors of the corresponding protein function. By integrating the aptamer-based biosensor development with the maturing technology for in vitro selection of anti-protein aptamers results in the highthroughput production of proteome chips. Furthermore, aptamer arrays and biosensors will reveal the most effective tools for the detection of biomolecular interactions and the identification of protein targets, particularly with regard to those not detectable by known receptors like enzymes or antibodies. We will review here the main and innovative methods based on the use of aptamers as biosensors for protein detection that, in alternative or combined to the classical proteomic approaches, could reveal suitable for both diagnostic and therapeutic purposes.Frontiers in Drug Design & Discovery 02/2006; 2(1):103-119. -
Article: Comparison of different strategies to select aptamers against a transmembrane protein target.
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ABSTRACT: Binding of aptamers is dependent on their target conformation, which in turn is conditioned by the target's environment. Therefore, selection of aptamers against the active forms of membrane proteins could require their correct membrane insertion in order to maintain their native conformation. Here, we compare different SELEX strategies to identify aptamers against the mutated form of the membrane receptor tyrosine kinase RET(C634Y). (1) selections S1 and S2 against living cells transformed to express the protein yielded a minority of RET-targeted aptamers while the bulk of aptamers recognized more abundant membrane proteins, suggesting that a high level of expression of the target protein is crucial to allow the isolation of aptamers at cell surface; (2) selection S3 against the purified extracellular moiety of RET yielded aptamers unable to recognize RET expressed at the cell membrane; (3) crossover selections S4 and S5 alternating cells and recombinant RET enhanced the enrichment of the aptamers directed against RET; however, these aptamers displayed a weaker affinity for Ret than those obtained with S1 and S2. In our case, using transformed cell lines as the partitioning matrix during SELEX appears to be essential in order to obtain aptamers able to recognize the RET receptor tyrosine kinase in its physiologic environment.Oligonucleotides 02/2006; 16(4):323-35. · 2.80 Impact Factor
Top co-authors
Top Journals
- PLoS ONE (5)
- Cellular Signalling (4)
- Methods in molecular biology (Clifton, N.J.) (2)
- FEBS Letters (1)
- Oncology (1)
Institutions
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2004–2013
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Università degli Studi di Napoli Federico II
- • Department of Neuroscience
- • Department of Molecular Medicine and Health Biotechnology
- • Department of Organic and Biological Chemistry
Portici, Campania, Italy
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2006–2012
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Istituto di Endocrinologia ed Oncologia Sperimentale "G. Salvatore"
Napoli, Campania, Italy -
National Research Council
Roma, Latium, Italy
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2008
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CNR NANO - Istituto Nanoscienze Consiglio Nazionale delle Ricerche
Napoli, Campania, Italy
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