SOCS3 deregulation contributes to aberrant activation of the JAK/STAT pathway in precursor T-cell neoplasms

Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T-ALL/LBL, no specific therapy has been approved for T-ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T-ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T-ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T-ALL/LBLComunidad de Madrid, Grant/Award Number: B2017/BMD-3778; LINFOMAS-CM; Fundación Científica Asociación Española Contra el Cáncer, Grant/Award Number: PROYE18054PIRI; Fundación Ramón Areces, Grant/Award Number: CIVP19S7917; Instituto de Investigación Sanitaria Fundación Jiménez Díaz; Ministerio de Ciencia, Innovación y Universidades, Grant/ Award Number: RTI2018- 093330-B-I00 and MCIU/FEDER; Ministerio de Economía y Competitividad, Grant/Award Number: SAF2015-70561-R and MINECO/FEDE

Generación de un sistema lentiviral para la expresión de prolactinas recombinantes y su uso para la transducción de células hepáticas

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 10-09-2019Esta tesis tiene embargado el acceso al texto completo hasta el 10-03-2021La participación de la prolactina (PRL) en más de 300 procesos biológicos ha conllevado a que se considere a esta molécula como una hormona pleiotrópica que actúa en diversos procesos fisiológicos y patológicos. No obstante, la propia naturaleza de la PRL y el limitado número de herramientas biotecnológicas de las que se dispone para el estudio de su función, hacen complicado delimitar su contribución en muchos de estos procesos. Por lo tanto, es necesario la generación de nuevas herramientas que permitan modular la acción de la PRL y ayuden a esclarecer su participación en varios de estos escenarios biológicos, entre los que destaca la homeostasis hepática. El objetivo de este trabajo de investigación fue generar un sistema lentiviral que permitiera la modulación de la acción de la PRL y fuera útil para el estudio de su función en diferentes procesos biológicos. Así, clonamos la secuencia codificante de la PRL humana (hPRL), generamos dos de sus mutantes antagonistas y expresamos estas hormonas recombinantes en células eucariotas. Tras comprobar su correcta expresión y respectivas bioactividades, transferimos estas secuencias a vectores lentivirales (LVs) para co-expresarlas junto con la proteína verde fluorescente (GFP) a partir de un único transcrito gracias a la secuencia P2A (hPRL-P2A-GFP). Nuestros resultados mostraron que estos LVs fueron capaces de transducir y expresar de forma eficiente las hPRL recombinantes en distintos tipos celulares. Las hPRL fusionadas al P2A se secuenciaron mediante espectrometría de masas y se comprobó la bioactividad de cada una de ellas en experimentos in vitro. La expresión de los transgenes hPRL-P2A-GFP permitió identificar tanto a las células productoras de hormonas (células GFP positivas), como a las hormonas recombinantes que contienen el epítopo P2A. Además, estas proteínas recombinantes secretadas por las células transducidas fueron capaces de reproducir las acciones autocrinas y paracrinas esperables en cultivos celulares. Tras la caracterización del sistema lentiviral, nuestro siguiente objetivo fue evaluar su capacidad para transducir diferentes líneas celulares establecidas de origen hepático y hepatocitos primarios humanos ex vivo. Nuestros resultados demostraron que los hepatocitos transducidos con nuestro sistema lentiviral expresaban y procesaban correctamente las construcciones hPRL-P2A-GFP in vitro. En resumen, el sistema lentiviral descrito en este trabajo muestra unas características apropiadas para el estudio de las acciones de hPRL en diferentes procesos biológicos, siendo particularmente útil para el estudio de las acciones de la PRL en la homeostasis hepática

Multi-Channeled Polymeric Microsystem for Studying the Impact of Surface Topography on Cell Adhesion and Motility

This paper presents the complete development and experimental validation of a microsystem designed to systematically assess the impact of surface topography on cell adhesion and dynamics. The microsystem includes two pools for culturing cells and for including chemicals. These pools are connected by several channels that have different microtextures, along which the cells crawl from one well to another. The impact of channel surface topography on cell performance, as well as the influence of other relevant factors, can therefore be assessed. The microsystem stands out for its being able to precisely define the surface topographies from the design stage and also has the advantage of including the different textures under study in a single device. Validation has been carried out by culturing human mesenchymal stem cells (hMSCs) on the microsystem pre-treated with a coating of hMSC conditioned medium (CM) produced by these cells. The impact of surface topography on cell adhesion, motility, and velocity has been quantified, and the relevance of using a coating of hMSC-CM for these kinds of studies has been analyzed. Main results, current challenges, and future proposals based on the use of the proposed microsystem as an experimental resource for studying cell mechanobiology are also presented

Composite scaffolds for osteochondral repair obtained by combination of additive manufacturing, leaching processes and hMSC-CM functionalization

Articular repair is a relevant and challenging area for the emerging fields of tissue engineering and biofabrication. The need of significant gradients of properties, for the promotion of osteochondral repair, has led to the development of several families of composite biomaterials and scaffolds, using different effective approaches, although a perfect solution has not yet been found. In this study we present the design, modeling, rapid manufacturing and in vitro testing of a composite scaffold aimed at osteochondral repair. The presented composite scaffold stands out for having a functional gradient of density and stiffness in the bony phase, obtained in titanium by means of computer-aided design combined with additive manufacture using selective laser sintering. The chondral phase is obtained by sugar leaching, using a PDMS matrix and sugar as porogen, and is joined to the bony phase during the polymerization of PDMS, therefore avoiding the use of supporting adhesives or additional intermediate layers. The mechanical performance of the construct is biomimetic and the stiffness values of the bony and chondral phases can be tuned to the desired applications, by means of controlled modifications of different parameters. A humanmesenchymal stemcell (h-MSC) conditionedmedium(CM) is used for improving scaffold response. Cell culture results provide relevant information regarding the viability of the composite scaffolds used

Smart Modification on Magnetic Nanoparticles Dramatically Enhances Their Therapeutic Properties

Magnetic nanoparticles (MNP) are employed as nanocarriers and in magnetic hyperthermia (MH) for the treatment of cancers. Herein, a smart drug delivery system composed of MNP functionalized with the cytotoxic drug gemcitabine (MNP-GEM) has been thoroughly evaluated. The linker employed is based on a disulfide bond and allows the controlled release of GEM under a highly reducing environment, which is frequently present in the cytoplasm of tumor cells. The stability, MH, and the interaction with plasma proteins of the nanoparticles are evaluated, highlighting their great potential for biological applications. Their cytotoxicity is assessed in three pancreatic cancer cell lines with different sensitivity to GEM, including the generation of reactive oxygen species (ROS), the effects on the cell cycle, and the mechanisms of cell death involved. Remarkably, the proposed nanocarrier is better internalized than unmodified nanoparticles, and it is particularly effective in PANC-1 cells, resistant to GEM, but not in non-tumoral keratinocytes. Additionally, its combination with MH produces a synergistic cytotoxic effect in all cancer cell lines tested. In conclusion, MNP-GEM presents a promising potential for treating pancreatic cancer, due to multiple parameters, such as reduced binding to plasma proteins, increased internalization, and synergistic activity when combined with MH

Amplification-free detection of SARS-CoV-2 using gold nanotriangles functionalized with oligonucleotides

Gold nanotriangles (AuNTs) functionalized with dithiolated oligonucleotides have been employed to develop an amplification-free electrochemical biosensor for SARS-CoV-2 in patient samples. Gold nanotriangles, prepared through a seed-mediated growth method and exhaustively characterized by different techniques, serve as an improved electrochemical platform and for DNA probe immobilization. Azure A is used as an electrochemical indicator of the hybridization event. The biosensor detects either single stranded DNA or RNA sequences of SARS-CoV-2 of different lengths, with a low detection limit of 22.2 fM. In addition, it allows to detect point mutations in SARS-CoV-2 genome with the aim to detect more infective SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron. Results obtained with the biosensor in nasopharyngeal swab samples from COVID-19 patients show the possibility to clearly discriminate between non-infected and infected patient samples as well as patient samples with different viral load. Furthermore, the results correlate well with those obtained by the gold standard technique RT-qPCR, with the advantage of avoiding the amplification process and the need of sophisticated equipmentOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work is funded by the Community of Madrid (TRANSNANOAVANSENS S2018/NMT-4349), Spanish Ministry of Economy and Competitiveness (PID2020-116728RBI00, CTQ2015-71955-RED ELECTROBIONET), and the Community of Madrid (project S2018/NMT-4291 TEC2SPACE), Spanish Ministry of Economy and Competitiveness (project CSIC13-4E-1794) and EU (FEDER, FSE). RdC received grants from UAM, Banco Santander, Fundación IMDEA (convocatoria CRUE-CSIC-SANTANDER, fondo supera 2020, project with reference 10.01.03.02.41). JCG received grants from Ramón y Cajal Institute for Health Research (IRYCIS), Madrid, Spain (COVID-19 Grant, 2020/0154). This research was also funded by the Spanish Ministry of Economy and Competitiveness (SAF2017-87305-R, PID2020-119352RB-I00), Instituto de Salud Carlos III (FONDO-COVID19: COV20/00144 and COV20/00122). P.M.R received grants from the Ministry of Economy, Industry and competitiveness of Spain (FPI grant (BES-2017.082521)). IMDEA Nanociencia received grants from the “Severo Ochoa” Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016–0686, CEX2020-001039-S