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Erratum to: Chapter 13 in: Giampaolo Zuccheri (ed.), DNA Nanotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1811, https://doi.org/10.1007/978-1-4939-8582-1_13.
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The use of DNA aptamers in biosensors for the quantification of pharmaceuticals in the clinics would help to overcome the limitations of antibody-based detection for small molecules. The interest for such systems is proven by the ever-increasing number of aptamer-based solutions for analytics proposed in the literature as proof-of-concept demonstrators. Despite such diversity, these platforms often lack a comparative assessment of their performances against the current standard of practice in the clinics when using real samples. We employed an aptamer against tobramycin discovered in our laboratory to quantify through surface plasmon resonance the concentration of the antibiotic in clinical samples obtained from patients treated with tobramycin and undergoing therapeutic drug monitoring. We then compared the performances of our detection strategy against the current standard of practice. Our results show how, using adequate calibration and matrix complexity reduction, DNA aptamer-based direct assays can assess clinically relevant concentrations of small molecules in patient serum and with good correlation to current standards used in the clinics.
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Aptámeros de Nucleótidos/sangre , Monitoreo de Drogas/normas , Tobramicina/sangre , Antibacterianos/sangre , Monitoreo de Drogas/métodos , Humanos , Resonancia por Plasmón de SuperficieRESUMEN
Capture-SELEX is an effective molecular strategy enabling the discovery of structure-switching aptamers, which might find useful application in molecular detection or separation. We here provide a protocol to perform capture-SELEX for DNA aptamers binding soluble small molecules, which includes a straightforward functional validation by SPR. The SELEX strategy here described is adaptable to any water-soluble molecular target and might foster the development of DNA aptamers binding therapeutic small molecules, at the great advantage of clinical bioanalytics.
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Aptámeros de Nucleótidos/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Aptámeros de Nucleótidos/metabolismo , Técnica SELEX de Producción de Aptámeros/métodos , Resonancia por Plasmón de SuperficieRESUMEN
The isothermal amplification of DNA in minimally buffered conditions allows to perform and monitor nucleic acid amplification with minimal technological and operative requirements. We show in this work how phi29 can operate multiple displacement amplification in minimally buffered conditions producing, as a readout, pH shifts attaining subunits of pH.
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Fagos de Bacillus/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Tampones (Química) , ADN Viral/genética , Genoma Viral , Papillomavirus Humano 16/genética , Humanos , Concentración de Iones de HidrógenoRESUMEN
Label-free approaches to assess cell properties ideally suit the requirements of cell-based therapeutics, since they permit to characterize cells with minimal perturbation and manipulation, at the benefit of sample recovery and re-employment for treatment. For this reason, label-free techniques would find sensible application in adoptive T cell-based immunotherapy. In this work, we describe the label-free and single-cell detection of in vitro activated T lymphocytes in flow through an electrical impedance-based setup. We describe a novel platform featuring 3D free-standing microelectrodes presenting passive upstream and downstream extensions and integrated into microfluidic channels. We employ such device to measure the impedance change associated with T cell activation at electrical frequencies maximizing the difference between non-activated and activated T cells. Finally, we harness the impedance signature of unstimulated T cells to set a boundary separating activated and non-activated clones, so to characterize the selectivity and specificity of the system. In conclusion, the strategy here proposed highlights the possible employment of impedance to assess T cell activation in label-free.
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Técnicas Biosensibles/métodos , Técnicas Analíticas Microfluídicas/métodos , Análisis de la Célula Individual/métodos , Linfocitos T/inmunología , Impedancia Eléctrica , Humanos , Activación de Linfocitos/inmunología , Linfocitos T/químicaRESUMEN
The SMC5/6 complex is the least understood of SMC complexes. In yeast, smc5/6 mutants phenocopy mutations in sgs1, the BLM ortholog that is deficient in Bloom's syndrome (BS). We here show that NSMCE2 (Mms21, in Saccharomyces cerevisiae), an essential SUMO ligase of the SMC5/6 complex, suppresses cancer and aging in mice. Surprisingly, a mutation that compromises NSMCE2-dependent SUMOylation does not have a detectable impact on murine lifespan. In contrast, NSMCE2 deletion in adult mice leads to pathologies resembling those found in patients of BS. Moreover, and whereas NSMCE2 deletion does not have a detectable impact on DNA replication, NSMCE2-deficient cells also present the cellular hallmarks of BS such as increased recombination rates and an accumulation of micronuclei. Despite the similarities, NSMCE2 and BLM foci do not colocalize and concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis-segregation. Our work reveals that SUMO- and BLM-independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice.
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Envejecimiento , Neoplasias/enzimología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Linfocitos B/enzimología , Secuencia de Bases , Células Cultivadas , Segregación Cromosómica , Roturas del ADN de Doble Cadena , Análisis Mutacional de ADN , Replicación del ADN , Femenino , Haploinsuficiencia , Humanos , Ligasas , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas , RecQ Helicasas/metabolismo , Sumoilación , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.
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Regulación de la Expresión Génica , Melanoma/patología , Melanoma/secundario , MicroARNs/metabolismo , Metástasis de la Neoplasia/patología , Factor de Transcripción AP-2/biosíntesis , Animales , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Humanos , Integrinas/metabolismo , Pulmón/patología , Neoplasias Pulmonares/patología , Ratones , MicroARNs/genéticaRESUMEN
CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cell-substrate adhesion and cell migration through physical and functional interactions with integrin receptors. In contrast, little is known about the potential role of CD151 in controlling cell proliferation and survival. We have previously shown that ß4 integrin, a major CD151 partner, not only acts as an adhesive receptor for laminins but also as an intracellular signaling platform promoting cell proliferation and invasive growth upon interaction with Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF). Here we show that RNAi-mediated silencing of CD151 expression in cancer cells impairs HGF-driven proliferation, anchorage-independent growth, protection from anoikis, and tumor progression in xenograft models in vivo. Mechanistically, we found that CD151 is crucially implicated in the formation of signaling complexes between Met and ß4 integrin, a known amplifier of HGF-induced tumor cell growth and survival. CD151 depletion hampered HGF-induced phosphorylation of ß4 integrin and the ensuing Grb2-Gab1 association, a signaling pathway leading to MAPK stimulation and cell growth. Accordingly, CD151 knockdown reduced HGF-triggered activation of MAPK but not AKT signaling cascade. These results indicate that CD151 controls Met-dependent neoplastic growth by enhancing receptor signaling through ß4 integrin-mediated pathways, independent of cell-substrate adhesion.
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Antígenos CD/fisiología , Regulación Neoplásica de la Expresión Génica , Integrina beta4/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Femenino , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Laminina/metabolismo , Ratones , Trasplante de Neoplasias , Interferencia de ARN , Transducción de Señal , Tetraspanina 24RESUMEN
Tudor domains are protein modules that mediate protein-protein interactions, potentially by binding to methylated ligands. A group of germline specific single and multiTudor domain containing proteins (TDRDs) represented by drosophila Tudor and its mammalian orthologs Tdrd1, Tdrd4/RNF17, and Tdrd6 play evolutionarily conserved roles in germinal granule/nuage formation and germ cell specification and differentiation. However, their physiological ligands, and the biochemical and structural basis for ligand recognition, are largely unclear. Here, by immunoprecipitation of endogenous murine Piwi proteins (Miwi and Mili) and proteomic analysis of complexes related to the piRNA pathway, we show that the TDRD group of Tudor proteins are physiological binding partners of Piwi family proteins. In addition, mass spectrometry indicates that arginine residues in RG repeats at the N-termini of Miwi and Mili are methylated in vivo. Notably, we found that Tdrkh/Tdrd2, a novel single Tudor domain containing protein identified in the Miwi complex, is expressed in the cytoplasm of male germ cells and directly associates with Miwi. Mutagenesis studies mapped the Miwi-Tdrkh interaction to the very N-terminal RG/RA repeats of Miwi and showed that the Tdrkh Tudor domain is critical for binding. Furthermore, we have solved the crystal structure of the Tdrkh Tudor domain, which revealed an aromatic binding pocket and negatively charged binding surface appropriate for accommodating methylated arginine. Our findings identify a methylation-directed protein interaction mechanism in germ cells mediated by germline Tudor domains and methylated Piwi family proteins, and suggest a complex mode of regulating the organization and function of Piwi proteins in piRNA silencing pathways.