Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture

[EN] The use of piezoelectric materials in tissue engineering has grown considerably since inherent bone piezoelectricity was discovered. Combinations of piezoelectric polymers with magnetostrictive nanoparticles (MNP) can be used to magnetoelectrically stimulate cells by applying an external magnetic field which deforms the magnetostrictive nanoparticles in the polymer matrix, deforming the polymer itself, which varies the surface charge due to the piezoelectric effect. Poly(vinylidene) fluoride (PVDF) is the piezoelectric polymer with the largest piezoelectric coefficients, being a perfect candidate for osteogenic differentiation. As a first approach, in this paper, we propose PVDF membranes containing magnetostrictive nanoparticles and a biomimetic heparin/ collagen layer-by-layer (LbL) coating for mesenchymal stem cell culture. PVDF membranes 20% (w/v) with and without cobalt ferrite oxide (PVDF-CFO) 10% (w/w) were produced by non-solvent induced phase separation (NIPS). These membranes were found to be asymmetric, with a smooth surface, crystallinity ranging from 65% to 61%, and an electroactive beta-phase content of 51.8% and 55.6% for PVDF and PVDF-CFO, respectively. Amine groups were grafted onto the membrane surface by an alkali treatment, confirmed by ninhydrin test and X-ray photoelectron spectroscopy (XPS), providing positive charges for the assembly of heparin/collagen layers by the LbL technique. Five layers of each polyelectrolyte were deposited, ending with collagen. Human mesenchymal stem cells (hMSC) were used to test cell response in a short-term culture (1, 3 and 7 days). Nucleus cell counting showed that LbL favored cell proliferation in PVDF-CFO over non-coated membranes.This work has been funded by the Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the PID2019-106099RB-C41/AEI/10.13039/501100011033 and PID2019-106099RB-C43/AEI/10.13039/501100011033 projects and the Associate Laboratory for Green Chemistry-LAQV financed by national funds from FCT/MCTES (UIDB/50006/2020). Maria GuillotFerriols acknowledges the Spanish Government funding of her doctoral thesis through a BES-2017-080398 FPI Grant. The CIBER-BBN (CB06/01/1026) initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. D.M.C is also grateful to the FCT-Fundacao para a Ciencia e Tecnologia for grant SFRH/BPD/121526/2016. Finally, the authors acknowledge funding from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively, also Dr. Carlos Sa (CEMUP) for assistance with the XPS analyses.Guillot-Ferriols, MT.; Rodriguez-Hernandez, J.; Correia, D.; Carabineiro, S.; Lanceros-Méndez, S.; Gómez Ribelles, JL.; Gallego Ferrer, G. (2020). Poly(vinylidene) fluoride membranes coated by heparin/collagen layer-by-layer, smart biomimetic approaches for mesenchymal stem cell culture. Materials Science and Engineering C: Materials for Biological Applications (Online). 117:1-12. https://doi.org/10.1016/j.msec.2020.111281112117Jacob, J., More, N., Kalia, K., & Kapusetti, G. (2018). Piezoelectric smart biomaterials for bone and cartilage tissue engineering. Inflammation and Regeneration, 38(1). doi:10.1186/s41232-018-0059-8Fukada, E., & Yasuda, I. (1957). On the Piezoelectric Effect of Bone. Journal of the Physical Society of Japan, 12(10), 1158-1162. doi:10.1143/jpsj.12.1158Martins, P., Lopes, A. C., & Lanceros-Mendez, S. (2014). Electroactive phases of poly(vinylidene fluoride): Determination, processing and applications. Progress in Polymer Science, 39(4), 683-706. doi:10.1016/j.progpolymsci.2013.07.006Gregorio, R. (2006). Determination of the α, β, and γ crystalline phases of poly(vinylidene fluoride) films prepared at different conditions. Journal of Applied Polymer Science, 100(4), 3272-3279. doi:10.1002/app.23137Sencadas, V., Gregorio, R., & Lanceros-Méndez, S. (2009). α to β Phase Transformation and Microestructural Changes of PVDF Films Induced by Uniaxial Stretch. Journal of Macromolecular Science, Part B, 48(3), 514-525. doi:10.1080/00222340902837527Gregorio, R., & Borges, D. S. (2008). Effect of crystallization rate on the formation of the polymorphs of solution cast poly(vinylidene fluoride). Polymer, 49(18), 4009-4016. doi:10.1016/j.polymer.2008.07.010Sencadas, V., Gregorio Filho, R., & Lanceros-Mendez, S. (2006). Processing and characterization of a novel nonporous poly(vinilidene fluoride) films in the β phase. Journal of Non-Crystalline Solids, 352(21-22), 2226-2229. doi:10.1016/j.jnoncrysol.2006.02.052Buonomenna, M. G., Macchi, P., Davoli, M., & Drioli, E. (2007). Poly(vinylidene fluoride) membranes by phase inversion: the role the casting and coagulation conditions play in their morphology, crystalline structure and properties. European Polymer Journal, 43(4), 1557-1572. doi:10.1016/j.eurpolymj.2006.12.033Ribeiro, C., Costa, C. M., Correia, D. M., Nunes-Pereira, J., Oliveira, J., Martins, P., … Lanceros-Méndez, S. (2018). Electroactive poly(vinylidene fluoride)-based structures for advanced applications. Nature Protocols, 13(4), 681-704. doi:10.1038/nprot.2017.157Liu, F., Hashim, N. A., Liu, Y., Abed, M. R. M., & Li, K. (2011). Progress in the production and modification of PVDF membranes. Journal of Membrane Science, 375(1-2), 1-27. doi:10.1016/j.memsci.2011.03.014Abzan, N., Kharaziha, M., & Labbaf, S. (2019). Development of three-dimensional piezoelectric polyvinylidene fluoride-graphene oxide scaffold by non-solvent induced phase separation method for nerve tissue engineering. Materials & Design, 167, 107636. doi:10.1016/j.matdes.2019.107636Young, T.-H., Chang, H.-H., Lin, D.-J., & Cheng, L.-P. (2010). Surface modification of microporous PVDF membranes for neuron culture. Journal of Membrane Science, 350(1-2), 32-41. doi:10.1016/j.memsci.2009.12.009Gonçalves, R., Martins, P., Correia, D. M., Sencadas, V., Vilas, J. L., León, L. M., … Lanceros-Méndez, S. (2015). Development of magnetoelectric CoFe2O4 /poly(vinylidene fluoride) microspheres. RSC Advances, 5(45), 35852-35857. doi:10.1039/c5ra04409jFernandes, M. M., Correia, D. M., Ribeiro, C., Castro, N., Correia, V., & Lanceros-Mendez, S. (2019). Bioinspired Three-Dimensional Magnetoactive Scaffolds for Bone Tissue Engineering. ACS Applied Materials & Interfaces, 11(48), 45265-45275. doi:10.1021/acsami.9b14001Hermenegildo, B., Ribeiro, C., Pérez-Álvarez, L., Vilas, J. L., Learmonth, D. A., Sousa, R. A., … Lanceros-Méndez, S. (2019). Hydrogel-based magnetoelectric microenvironments for tissue stimulation. Colloids and Surfaces B: Biointerfaces, 181, 1041-1047. doi:10.1016/j.colsurfb.2019.06.023Gonçalves, R., Martins, P., Moya, X., Ghidini, M., Sencadas, V., Botelho, G., … Lanceros-Mendez, S. (2015). Magnetoelectric CoFe2O4/polyvinylidene fluoride electrospun nanofibres. Nanoscale, 7(17), 8058-8061. doi:10.1039/c5nr00453eSilva, J. M., Reis, R. L., & Mano, J. F. (2016). Biomimetic Extracellular Environment Based on Natural Origin Polyelectrolyte Multilayers. Small, 12(32), 4308-4342. doi:10.1002/smll.201601355Costa, R. R., & Mano, J. F. (2014). Polyelectrolyte multilayered assemblies in biomedical technologies. Chemical Society Reviews, 43(10), 3453. doi:10.1039/c3cs60393hCastilla-Casadiego, D. A., Pinzon-Herrera, L., Perez-Perez, M., Quiñones-Colón, B. A., Suleiman, D., & Almodovar, J. (2018). Simultaneous characterization of physical, chemical, and thermal properties of polymeric multilayers using infrared spectroscopic ellipsometry. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 553, 155-168. doi:10.1016/j.colsurfa.2018.05.052Mhanna, R. F., Vörös, J., & Zenobi-Wong, M. (2011). Layer-by-Layer Films Made from Extracellular Matrix Macromolecules on Silicone Substrates. Biomacromolecules, 12(3), 609-616. doi:10.1021/bm1012772Billings, P. C., & Pacifici, M. (2015). Interactions of signaling proteins, growth factors and other proteins with heparan sulfate: mechanisms and mysteries. Connective Tissue Research, 56(4), 272-280. doi:10.3109/03008207.2015.1045066Chen, J., Huang, N., Li, Q., Chu, C. H., Li, J., & Maitz, M. F. (2016). The effect of electrostatic heparin/collagen layer-by-layer coating degradation on the biocompatibility. Applied Surface Science, 362, 281-289. doi:10.1016/j.apsusc.2015.11.227Zhang, K., Huang, D., Yan, Z., & Wang, C. (2017). Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering. Journal of Biomedical Materials Research Part A, 105(7), 1900-1910. doi:10.1002/jbm.a.36053Ferreira, A. M., Gentile, P., Toumpaniari, S., Ciardelli, G., & Birch, M. A. (2016). Impact of Collagen/Heparin Multilayers for Regulating Bone Cellular Functions. ACS Applied Materials & Interfaces, 8(44), 29923-29932. doi:10.1021/acsami.6b09241Castilla-Casadiego, D. A., García, J. R., García, A. J., & Almodovar, J. (2019). Heparin/Collagen Coatings Improve Human Mesenchymal Stromal Cell Response to Interferon Gamma. ACS Biomaterials Science & Engineering, 5(6), 2793-2803. doi:10.1021/acsbiomaterials.9b00008Martins, P., Gonçalves, R., Lanceros-Mendez, S., Lasheras, A., Gutiérrez, J., & Barandiarán, J. M. (2014). Effect of filler dispersion and dispersion method on the piezoelectric and magnetoelectric response of CoFe2O4/P(VDF-TrFE) nanocomposites. Applied Surface Science, 313, 215-219. doi:10.1016/j.apsusc.2014.05.187Gamboa-Martínez, T. C., Luque-Guillén, V., González-García, C., Gómez Ribelles, J. L., & Gallego-Ferrer, G. (2014). Crosslinked fibrin gels for tissue engineering: Two approaches to improve their properties. Journal of Biomedical Materials Research Part A, 103(2), 614-621. doi:10.1002/jbm.a.35210Gregorio, Jr., R., & Cestari, M. (1994). Effect of crystallization temperature on the crystalline phase content and morphology of poly(vinylidene fluoride). Journal of Polymer Science Part B: Polymer Physics, 32(5), 859-870. doi:10.1002/polb.1994.090320509Martins, P., Costa, C. M., & Lanceros-Mendez, S. (2010). Nucleation of electroactive β-phase poly(vinilidene fluoride) with CoFe2O4 and NiFe2O4 nanofillers: a new method for the preparation of multiferroic nanocomposites. Applied Physics A, 103(1), 233-237. doi:10.1007/s00339-010-6003-7Qi, L., Knapton, E. K., Zhang, X., Zhang, T., Gu, C., & Zhao, Y. (2017). Pre-culture Sudan Black B treatment suppresses autofluorescence signals emitted from polymer tissue scaffolds. Scientific Reports, 7(1). doi:10.1038/s41598-017-08723-2Young, T.-H., Cheng, L.-P., Lin, D.-J., Fane, L., & Chuang, W.-Y. (1999). Mechanisms of PVDF membrane formation by immersion-precipitation in soft (1-octanol) and harsh (water) nonsolvents. Polymer, 40(19), 5315-5323. doi:10.1016/s0032-3861(98)00747-2Cheng, L.-P. (1999). Effect of Temperature on the Formation of Microporous PVDF Membranes by Precipitation from 1-Octanol/DMF/PVDF and Water/DMF/PVDF Systems. Macromolecules, 32(20), 6668-6674. doi:10.1021/ma990418lSupriya, S., Kumar, L., & Kar, M. (2018). Optimization of dielectric properties of PVDF-CFO nanocomposites. Polymer Composites, 40(3), 1239-1250. doi:10.1002/pc.24840Lin, D.-J., Beltsios, K., Young, T.-H., Jeng, Y.-S., & Cheng, L.-P. (2006). Strong effect of precursor preparation on the morphology of semicrystalline phase inversion poly(vinylidene fluoride) membranes. Journal of Membrane Science, 274(1-2), 64-72. doi:10.1016/j.memsci.2005.07.043Cai, X., Lei, T., Sun, D., & Lin, L. (2017). A critical analysis of the α, β and γ phases in poly(vinylidene fluoride) using FTIR. RSC Advances, 7(25), 15382-15389. doi:10.1039/c7ra01267eBoccaccio, T., Bottino, A., Capannelli, G., & Piaggio, P. (2002). Characterization of PVDF membranes by vibrational spectroscopy. Journal of Membrane Science, 210(2), 315-329. doi:10.1016/s0376-7388(02)00407-6Liu, J., Lu, X., & Wu, C. (2013). Effect of Preparation Methods on Crystallization Behavior and Tensile Strength of Poly(vinylidene fluoride) Membranes. Membranes, 3(4), 389-405. doi:10.3390/membranes3040389ZHANG, M., ZHANG, A., ZHU, B., DU, C., & XU, Y. (2008). Polymorphism in porous poly(vinylidene fluoride) membranes formed via immersion precipitation process. Journal of Membrane Science, 319(1-2), 169-175. doi:10.1016/j.memsci.2008.03.029Xiao, L., Davenport, D. M., Ormsbee, L., & Bhattacharyya, D. (2015). Polymerization and Functionalization of Membrane Pores for Water Related Applications. Industrial & Engineering Chemistry Research, 54(16), 4174-4182. doi:10.1021/ie504149tDuca, M. D., Plosceanu, C. L., & Pop, T. (1998). Effect of X-rays on poly(vinylidene fluoride) in X-ray photoelectron spectroscopy. Journal of Applied Polymer Science, 67(13), 2125-2129. doi:10.1002/(sici)1097-4628(19980328)67:133.0.co;2-gCorreia, D. M., Ribeiro, C., Sencadas, V., Botelho, G., Carabineiro, S. A. C., Ribelles, J. L. G., & Lanceros-Méndez, S. (2015). Influence of oxygen plasma treatment parameters on poly(vinylidene fluoride) electrospun fiber mats wettability. Progress in Organic Coatings, 85, 151-158. doi:10.1016/j.porgcoat.2015.03.019Kehrer, M., Duchoslav, J., Hinterreiter, A., Cobet, M., Mehic, A., Stehrer, T., & Stifter, D. (2019). XPS investigation on the reactivity of surface imine groups with TFAA. Plasma Processes and Polymers, 16(4), 1800160. doi:10.1002/ppap.201800160Morales-Román, R. M., Guillot-Ferriols, M., Roig-Pérez, L., Lanceros-Mendez, S., Gallego-Ferrer, G., & Gómez Ribelles, J. L. (2019). Freeze-extraction microporous electroactive supports for cell culture. European Polymer Journal, 119, 531-540. doi:10.1016/j.eurpolymj.2019.07.011Camacho, N. P., West, P., Torzilli, P. A., & Mendelsohn, R. (2000). FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage. Biopolymers, 62(1), 1-8. doi:10.1002/1097-0282(2001)62:13.0.co;2-oRibeiro, C., Panadero, J. A., Sencadas, V., Lanceros-Méndez, S., Tamaño, M. N., Moratal, D., … Gómez Ribelles, J. L. (2012). Fibronectin adsorption and cell response on electroactive poly(vinylidene fluoride) films. Biomedical Materials, 7(3), 035004. doi:10.1088/1748-6041/7/3/035004Ribeiro, C., Pärssinen, J., Sencadas, V., Correia, V., Miettinen, S., Hytönen, V. P., & Lanceros‐Méndez, S. (2014). Dynamic piezoelectric stimulation enhances osteogenic differentiation of human adipose stem cells. Journal of Biomedical Materials Research Part A, 103(6), 2172-2175. doi:10.1002/jbm.a.35368Sobreiro-Almeida, R., Tamaño-Machiavello, M., Carvalho, E., Cordón, L., Doria, S., Senent, L., … Sempere, A. (2017). Human Mesenchymal Stem Cells Growth and Osteogenic Differentiation on Piezoelectric Poly(vinylidene fluoride) Microsphere Substrates. International Journal of Molecular Sciences, 18(11), 2391. doi:10.3390/ijms18112391Moise, S., Céspedes, E., Soukup, D., Byrne, J. M., El Haj, A. J., & Telling, N. D. (2017). The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles. Scientific Reports, 7(1). doi:10.1038/srep3992

Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients

<p>Abstract</p> <p>Background</p> <p>DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed.</p> <p>Methods</p> <p>Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide.</p> <p>Results</p> <p>Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation.</p> <p>Conclusions</p> <p>An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.</p

Rol de los pastizales naturales en la retención de nutrientes provenientes de la agricultura = Role of natural grasslands in retaining nutrients from agriculture

La intensificación de los usos del suelo en las últimas décadas en Uruguay ha producido un aumento en la exportación de nutrientes a los cuerpos de agua, lo que explicaría el proceso de eutrofización observado en el período. La Laguna de Rocha es un sitio prioritario para la conservación por su biodiversidad, por lo que ingresó al Sistema Nacional de Áreas Protegidas. Sin embargo, no escapa a las presiones mencionadas y se han registrado floraciones de cianobacterias y de macrófitas sumergidas ocasionalmente. La cobertura vegetal en la interfase entre los cuerpos de agua y los sistemas productivos puede funcionar como zona de amortiguación, disminuyendo la carga de nutrientes provenientes de cultivos o sistemas de producción animal por escorrentía, contribuyendo a la provisión del servicio ecosistémico de mantenimiento de la calidad de agua. Mediante el uso de parcelas de escorrentía experimentales se estimó en este trabajo el servicio ecosistémico provisto por el pastizal natural de retención de nutrientes del agua de escorrentía proveniente de una pradera artificial. Se halló una retención de fosfato de 55 %, nitrógeno inorgánico disuelto 44 %, sólidos en suspensión 28 %, nitrógeno total 47 % y fósforo total 42 %

The Raman Laser Spectrometer for the ExoMars Rover Mission to Mars

The Raman Laser Spectrometer (RLS) on board the ESA/Roscosmos ExoMars 2020 mission will provide precise identification of the mineral phases and the possibility to detect organics on the Red Planet. The RLS will work on the powdered samples prepared inside the Pasteur analytical suite and collected on the surface and subsurface by a drill system. Raman spectroscopy is a well-known analytical technique based on the inelastic scattering by matter of incident monochromatic light (the Raman effect) that has many applications in laboratory and industry, yet to be used in space applications. Raman spectrometers will be included in two Mars rovers scheduled to be launched in 2020. The Raman instrument for ExoMars 2020 consists of three main units: (1) a transmission spectrograph coupled to a CCD detector; (2) an electronics box, including the excitation laser that controls the instrument functions; and (3) an optical head with an autofocus mechanism illuminating and collecting the scattered light from the spot under investigation. The optical head is connected to the excitation laser and the spectrometer by optical fibers. The instrument also has two targets positioned inside the rover analytical laboratory for onboard Raman spectral calibration. The aim of this article was to present a detailed description of the RLS instrument, including its operation on Mars. To verify RLS operation before launch and to prepare science scenarios for the mission, a simulator of the sample analysis chain has been developed by the team. The results obtained are also discussed. Finally, the potential of the Raman instrument for use in field conditions is addressed. By using a ruggedized prototype, also developed by our team, a wide range of terrestrial analog sites across the world have been studied. These investigations allowed preparing a large collection of real, in situ spectra of samples from different geological processes and periods of Earth evolution. On this basis, we are working to develop models for interpreting analog processes on Mars during the mission. Key Words: Raman spectroscopy—ExoMars mission—Instruments and techniques—Planetary sciences—Mars mineralogy and geochemistry—Search for life on Mars. Astrobiology 17, 627–65

Actualidad de las Plantas Medicinales en Terapéutica

RESUMENLas plantas medicinales han sido utilizadas por el hombre desde tiempo inmemorial para el alivio o curación de sus enfermedades. En la actualidad no solo se utilizan como punto de partida para la obtención de medicamentos industriales sino que mantienen plena vigencia para el tratamiento de numerosas dolencias. A pesar de su origen natural no están exentas de riesgos para la salud pues algunas provocan reacciones adversas e interacciones con otros fármacos que hay que tener en cuenta.En el presente trabajo se repasan las principales plantas medicinales, de gran utilidad terapéutica y amplio uso, clasificadas de acuerdo con los sistemas funcionales sobre los que actúan.Palabras claveFitoterapia, medicamentos, acción farmacológica, principios activos, interacciones medicamento-planta medicinal. ABSTRACTMedicinal plants have been used for the relief or cure of several diseases for centuries. Most of the medicines we used today are derived straight from plants and medicinal plants are used as a drug themselves, with great utility in therapeutics. Despite their natural origin, herbal drugs are not free of side effects or interactions with other drugs that must be taken into account.This study review the efficacy and safety of the most used medicinal plants classified according to the functional systems where they act.Key words:Phytotherapy, medicines, pharmacological action, active principles, interactions drug-medicinal plant

The Obestatin/GPR39 System Is Up-regulated by Muscle Injury and Functions as an Autocrine Regenerative System

Background: Satellite cell activation is orchestrated by several signals, which induce their differentiation into skeletal muscle fibers. Results: Obestatin and the GPR39 receptor exert an autocrine role on the control of myogenesis. Conclusion: Our data indicate that obestatin/GPR39 is an injury-regulated signal that functions as a myogenic regenerative system. Significance: Strategies to enhance obestatin-mediated signaling could be useful in treating trauma-induced muscle injuries and skeletal muscle myopathies

Generation of NKX2.5(GFP) Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5(GFP) reporter cardiac fibroblasts. We first established a new NKX2.5(GFP) reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5(GFP) hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5(GFP) cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.This work was supported by PID 2019-107150RB-I00/AEI/ 10.13039/501100011033 to XC-V; by the “Ramón y Cajal” State Program, Ministry of Economy and Competitivenes

Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

11 pages, 6 figures, 1 table.Background The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of 1phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported. Methodology/Principal Findings Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ. Conclusion/Significance This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.This work has been supported by grants to JCL from Comunidad de Madrid (GR-SAL-0821-2004), Ministerio de Ciencia e Innovación (SAF2008-03750, RD06/0020/0016), Fundación Mutua Madrileña, and by a grant to ARM from Fundación Mutua Madrileña.Peer reviewe

Selective predisposition to bacterial infections in IRAK-4–deficient children: IRAK-4–dependent TLRs are otherwise redundant in protective immunity

Human interleukin (IL) 1 receptor–associated kinase 4 (IRAK-4) deficiency is a recently discovered primary immunodeficiency that impairs Toll/IL-1R immunity, except for the Toll-like receptor (TLR) 3– and TLR4–interferon (IFN)-a/b pathways. The clinical and immunological phenotype remains largely unknown. We diagnosed up to 28 patients with IRAK-4 deficiency, tested blood TLR responses for individual leukocyte subsets, and TLR responses for multiple cytokines. The patients' peripheral blood mononuclear cells (PBMCs) did not induce the 11 non-IFN cytokines tested upon activation with TLR agonists other than the nonspecific TLR3 agonist poly(I:C). The patients' individual cell subsets from both myeloid (granulocytes, monocytes, monocyte-derived dendritic cells [MDDCs], myeloid DCs [MDCs], and plasmacytoid DCs) and lymphoid (B, T, and NK cells) lineages did not respond to the TLR agonists that stimulated control cells, with the exception of residual responses to poly(I:C) and lipopolysaccharide in MDCs and MDDCs. Most patients (22 out of 28; 79%) suffered from invasive pneumococcal disease, which was often recurrent (13 out of 22; 59%). Other infections were rare, with the exception of severe staphylococcal disease (9 out of 28; 32%). Almost half of the patients died (12 out of 28; 43%). No death and no invasive infection occurred in patients older than 8 and 14 yr, respectively. The IRAK-4–dependent TLRs and IL-1Rs are therefore vital for childhood immunity to pyogenic bacteria, particularly Streptococcus pneumoniae. Conversely, IRAK-4–dependent human TLRs appear to play a redundant role in protective immunity to most infections, at most limited to childhood immunity to some pyogenic bacteria