Dynamics of Hedgehog signaling filopodia during Drosophila melanogaster epithelia morphogenesis

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 31-07-2017Esta tesis tiene embargado el acceso al texto completo hasta el 31-01-201

Synthetic materials at the forefront of gene delivery

This is the Author's Accepted Manuscript of the following article: Lostalé-Seijo, I., & Montenegro, J. (2018). Synthetic materials at the forefront of gene delivery. Nature Reviews Chemistry. doi: 10.1038/s41570-018-0039-1The delivery of nucleic acids with transient activity for genetic engineering is a promising methodology with potential applications in the treatment of diseases ranging from cancer and infectious diseases to heritable disorders. Restoring the expression of a missing protein, correcting defective splicing of transcripts and silencing or modulating the expression of genes are powerful approaches that could have substantial benefits in biological research and medicine. Impressive progress in improving gene delivery has been made in the past decade, and several products have reached the market. However, translating the results of in vitro and preclinical studies into functional therapies is hindered by the suboptimal performance of gene delivery vehicles in capturing, protecting and delivering nucleic acid cargoes safely and efficaciously. Chemistry has a key role in the development of innovative synthetic materials to overcome the challenges of producing next-generation gene delivery therapies and protocols. In this Review, we discuss the latest chemical advances in the production of materials for the delivery of nucleic acids to cells and for gene therapyThe group of J.M. was partially supported by the Spanish Agencia Estatal de Investigación (AEI) (CTQ2014-59646-R and SAF2017-89890-R), the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the European Regional Development Fund (ERDF). J.M. received a Ramón y Cajal grant (RYC-2013-13784), a European Research Council (ERC) Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

Estudio del sistema del interferón en células aviares infectadas con el reovirus aviar S1133

Os reovirus aviarios son virus sen envoltura lipídica e xenoma de ARN bicatenario que infectan a aves, causando enfermidades coma a artrite infecciosa ou a síndrome de malabsorción. Neste traballo estudouse a resposta inmune innata que este virus desencadea en dous tipos celulares do seu hospedador natural, o polo, e comparouse coa xerada por outros virus coma o virus vaccinia e o virus da estomatite vesicular. O reovirus aviario é o único dos virus estudados capaz de inducir a produción de interferón (IFN) e aumentar os niveis da proteína quinasa R (PKR) en cultivos primarios de fibroblastos embrionarios de polo (CEF), mentres que é incapaz de facelo na liña celular DF1 derivada destes, o que podería depender de diferenzas nos niveis dos receptores de recoñecemento de patrón (PRRs) ou a unha menor eficiencia de infección destas células. A indución de IFN en CEF infectadas co reovirus aviario depende da decapsidación do virus pero non da expresión dos seus xenes. O reovirus aviario é resistente ao IFN e é capaz de impedir a activación de PKR e a fosforilación de eIF2, ao contrario que os outros dous virus estudados. Esta capacidade para impedir a activación de PKR depende da capacidade da proteína A para unir ARN bicatenario. Por outra banda, observouse que o pretratamento con IFN de células DF1 aumenta a cantidade de células infectadas co reovirus aviario mediante un mecanismo independente da activación das caspasas. Finalmente, estudáronse algúns posibles mecanismos da inhibición da síntese das proteínas celulares durante a infección con reovirus aviario

Where in the cell is our cargo? Current methods to study intracellular cytosolic localization

NOTICE: This is the Accepted Version of the following article: Méndez Ardoy, A., Lostalé-Seijo, I., & Montenegro, J. (2018). Where in the cell is our cargo? Current methods to study intracellular cytosolic localization. Chembiochem. doi: 10.1002/cbic.201800390 © 2018 WILEY‐VCH Verlag GmbH & Co. KGaA, WeinheimThe internalization and delivery of active substances into cells is a field of growing interest for chemical biology and therapeutics. As we move from small‐molecule based drugs towards bigger cargos, such as antibodies, enzymes, nucleases or nucleic acids, the development of efficient delivery systems becomes critical for their practical application. Different strategies and synthetic carriers have been developed including cationic lipids, gold nanoparticles, polymers, cell‐penetrating peptides, protein surface modification, etc. However, all these methodologies still present limitations related to the precise targeting of the different intracellular compartments and, in particular, the difficult access to the cellular cytosol. Additionally, the precise quantification of the cellular uptake of a molecule is not enough to demonstrate delivery and/or functional activity. Therefore, methods to determine the cellular distribution of cargos and carriers are of critical importance to identify the barriers that are blocking the activity. In this mini‐review, we survey the different techniques that can be currently used to track and monitor the subcellular localization of the synthetic molecules that we deliver inside cellsWe acknowledge funding from the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, SAF2017-89890-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. A. M.-A. received a MCIF from the EC (GLYCONANOPEP-750248). J. M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells

We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cellsThe Spanish Ministerio de Economía y Competitividad provided funding to Javier Benavente and José Martínez-Costas under grant numbers BFU2010-22228 and BFU2013-43513-R. Support was also provided by the Xunta de Galicia (CN 2012/018). Irene Lostalé-Seijo was a recipient of a predoctoral FPU fellowship (Ministerio de Educación y Ciencia) and a Research Fellowship (Bolsa de Investigación; Deputación Provincial da Coruña)S

Interferon induction by avian reovirus

We have previously shown that the replication of avian reovirus (ARV) in chicken embryo fibroblasts (CEF) is more resistant to the antiviral action of interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study we examined the capacity of these three viruses to induce the expression of IFN when infecting avian cells. Efficient expression of both type-α and type-β IFNs, as well as of the double-stranded RNA (dsRNA)-activated protein kinase (PKR), takes place in ARV-infected CEF, but not in cells infected with VSV or VV. PKR expression is not directly induced by ARV infection, but by the IFN secreted by ARV-infected cells. IFN induction in ARV-infected cells requires viral uncoating, but not viral gene expression, a situation similar to that reported for apoptosis induction by ARV-infected cells. However, our results demonstrate that IFN induction by ARV-infected CEF occurs by a caspase-independent mechanismThis work was funded by grants from the Spanish Ministerio de Economia y Competitividad (BFU2010-22228 and BFU2013-43513-R) and from the Xunta de Galicia (CN 2012/018). I.L.S. was a recipient of a predoctoral FPU fellowship (Ministerio de Educación y Ciencia) and a Research Fellowship (Bolsa de Investigación) (Deputación Provincial da Coruña)S

Oligoalanine helical callipers for cell penetration

This is the accepted manuscript of the following article: Pazo, M., Juanes, M., Lostalé-Seijo, I., & Montenegro, J. (2018). Oligoalanine helical callipers for cell penetration. Chem. Commun., 2018, 54, 6919-6922. doi: 10.1039/c8cc02304b. This article may be used for non-commercial purposes in accordance with RSC Terms and Conditions for self-archivingEven for short peptides that are enriched in basic amino acids, the large chemical space that can be spanned by combinations of natural amino acids hinders the rational design of cell penetrating peptides. We here report on short oligoalanine scaffolds for the fine-tuning of peptide helicity in different media and the study of cell penetrating properties. This strategy allowed the extraction of the structure/activity features required for maximal membrane interaction and cellular penetration at minimal toxicity. These results confirmed oligoalanine helical callipers as optimal scaffolds for the rational design and the identification of cell penetrating peptidesThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, SAF2017-89890-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. M. P. thanks the Xunta de Galicia for a pre-doctoral fellowship (ED481A-2017/142), and M. J. MINECO for a F. P. I. fellowship. J. M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition

The discovery of RNA guided endonucleases has emerged as one of the most important tools for gene edition and biotechnology. The selectivity and simplicity of the CRISPR/Cas9 strategy allows the straightforward targeting and editing of particular loci in the cell genome without the requirement of protein engineering. However, the transfection of plasmids encoding the Cas9 and the guide RNA could lead to undesired permanent recombination and immunogenic responses. Therefore, the direct delivery of transient Cas9 ribonucleoprotein constitutes an advantageous strategy for gene edition and other potential therapeutic applications of the CRISPR/Cas9 system. The covalent fusion of Cas9 with penetrating peptides requires multiple incubation steps with the target cells to achieve efficient levels of gene edition. These and other recent reports suggested that covalent conjugation of the anionic Cas9 ribonucleoprotein to cationic peptides would be associated with a hindered nuclease activity due to undesired electrostatic interactions. We here report a supramolecular strategy for the direct delivery of Cas9 by an amphiphilic penetrating peptide that was prepared by a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. The peptide/protein non-covalent nanoparticles performed with similar efficiency and less toxicity than one of the best methods described to date. To the best of our knowledge this report constitutes the first supramolecular strategy for the direct delivery of Cas9 using a penetrating peptide vehicle. The results reported here confirmed that peptide amphiphilic vectors can deliver Cas9 in a single incubation step, with good efficiency and low toxicity. This work will encourage the search and development of conceptually new synthetic systems for transitory endonucleases direct delivery.This work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R], the Xunta de Galicia (ED431G/09 and 2016-AD031) and the ERDF. M. J. received a F. P. I. fellowship from MINECO. J. M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

Supramolecular Recognition and Selective Protein Uptake by Peptide Hybrids

This manuscript has been accepted after peer review and appears as anAccepted Article online prior to editing, proofing, and formal publicationof the final Version of Record (VoR). This work is currently citable byusing the Digital Object Identifier (DOI) given below. The VoR will bepublished online in Early View as soon as possible and may be differentto this Accepted Article as a result of editing. Readers should obtainthe VoR from the journal website shown below when it is publishedto ensure accuracy of information. The authors are responsible for thecontent of this Accepted ArticleThe intracellular transport of exogenous proteins has emerged as one of the most promising methodologies for biotechnology and chemical biology. Current protein delivery is mainly approached by liposome encapsulation, translational fusion and ionic/hydrophobic non‐covalent aggregation with transporting molecular vehicles. We here introduce the concept of supramolecular recognition and selective transport of proteins by peptide hybrid materials. We have designed a helical amphiphilic cationic peptide that bears two orthogonal alkoxyamines for the precise anchoring of protein ligands. After the attachment of these protein ligands, the peptide showed a high binding affinity for its protein target (i.e. mannose/Concanavalin A, Biotin/Streptavidin). The resulting peptide/protein hybrids were taken up by human cells such as HeLa and HepG2. The concept described in this manuscript could potentially be adapted, through the appropriate choice of ligands, to the transport of other proteins with suitable supramolecular binding motifsThis work was partially supported by the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, CTQ2013-43264-R, SAF2017-89890-R], the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. We thank Pili Canoa (CACTI, UVIGO) for assistance with the SPR measurements. M.J. received a F.P.I. fellowship from MINECO (CTQ2014-59646-R). J.M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP- 677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S

Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cellsThis research was supported by grants from the Spanish Ministerio de Ciencia y Tecnología (BFU2007-61330/BMC) and from the Xunta de Galicia (08CSA009203PR). L. V-I. and I. L-S. were recipients of predoctoral fellowships from the FPI and FPU programs of the Spanish Ministerio de Ciencia y TecnologíaS