Alterations of the Hippocampal Neurogenic Niche in a Mouse Model of Dravet Syndrome

Hippocampal neurogenesis, the process by which neural stem cells (NSCs) continuously generate new neurons in the dentate gyrus (DG) of most mammals including humans, is chiefly regulated by neuronal activity. Thus, severe alterations have been found in samples from epilepsy patients and in the hippocampal neurogenic niche in mouse models of epilepsy. Reactive-like and gliogenic NSCs plus aberrant newborn neurons with altered migration, morphology, and functional properties are induced by seizures in experimental models of temporal lobe epilepsy. Hippocampal neurogenesis participates in memory and learning and in the control of anxiety and stress. It has been therefore hypothesized that part of the cognitive symptoms associated with epilepsy could be promoted by impaired hippocampal neurogenesis. We here analyze for the first time the alterations of the neurogenic niche in a novel mouse model of Dravet syndrome (DS), a genetic encephalopathy with severe epilepsy in infancy and multiple neurological comorbidities. Scn1a(WT/A1783V)mice, hereafter referred to as DS, carrying a heterozygous and clinically relevant SCN1A mutation (A1783V) recapitulate the disease at the genetic and phenotypic levels. We demonstrate that in the neurogenic niche of young adult DS mice there are fewer NSCs, they have impaired cell division and bear reactive-like morphology. In addition, there is significant aberrant neurogenesis. Newborn immature neurons migrate abnormally, and several morphological features are drastically changed. Thus, this study shows for the first time important modifications in hippocampal neurogenesis in DS and opens venues for further research on this topic.This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) Grant/Award Numbers SAF-2015-70866-R (with FEDER Funds) and RyC-212-11137 to JE and RTI2018-097730-B-I00/MCI/AEI/FEDER, UE, and AC17/00029 (ISCIII)/FEDER to RH-A. SM-S received a Fundacion Tatiana predoctoral fellowship. OA is the recipient of a Basque Government postdoctoral fellowship

Danio Rerio as Model Organism for Adenoviral Vector Evaluation

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectorsThis research was funded by Xunta de Galicia, Local Government, grant number ED431C 2018/28, and Gobierno de Navarra, Local Government, grant numbers 0011-1383-2018-000011 PT038 and 0011-1383-2019-000006 PT013 (XANTOGEN)S

Understanding the Molecular Mechanism of miR-877-3p Could Provide Potential Biomarkers and Therapeutic Targets in Squamous Cell Carcinoma of the Cervix

Cervical cancer (CC) is managed mainly using subjective and conventional methods. Research about the molecular mechanisms of micro-RNA-877-3p (miR-877-3p) in other cancer types revealed that it interacts with events that are important for CC. Our aim was to understand the role of miR-877-3p in CC. We observed that it was overexpressed in cervical tumors compared with benign lesions, and that it promoted CC cell migration and invasion by modulating cytoskeletal protein folding, which potentiated the effects caused by paclitaxel, one of the most common therapeutic drugs used in CC. We demonstrated a functional link between miR-877-3p and one of its predicted targets, ZNF177. The expression and subcellular location of ZNF177 objectively distinguished two CC entities and predicted poor outcome in the most aggressive form. Therefore, the understanding of the molecular mechanisms driven by miR-877-3p provides useful tools for CC clinical management, currently lacking of molecular biomarkers and targeted therapies. No therapeutic targets and molecular biomarkers are available in cervical cancer (CC) management. In other cancer types, micro-RNA-877-3p (miR-877-3p) has been associated with events relevant for CC development. Thus, we aimed to determine miR-877-3p role in CC. miR-877-3p levels were examined by quantitative-PCR in 117 cervical lesions and tumors. Effects on CC cell proliferation, migration, and invasion were evaluated upon anti-miR-877-3p transfection. miR-877-3p dependent molecular mechanism was comprehensively explored by proteomics, dual-luciferase reporter assay, western blot, and immunohistochemistry. Cervical tumors expressed higher miR-877-3p levels than benign lesions. miR-877-3p promoted CC cell migration and invasion, at least partly by modulating cytoskeletal protein folding through the chaperonin-containing T-complex protein 1 complex. Notably, miR-877-3p silencing synergized with paclitaxel. Interestingly, miR-877-3p downregulated the levels of an in silico-predicted target, ZNF177, whose expression and subcellular location significantly distinguished high-grade squamous intraepithelial lesions (HSILs) and squamous cell carcinomas of the cervix (SCCCs). Cytoplasmic ZNF177 was significantly associated with worse progression-free survival in SCCC. Our results suggest that: (i) miR-877-3p is a potential therapeutic target whose inhibition improves paclitaxel effects; (ii) the expression and location of its target ZNF177 could be diagnostic biomarkers between HSIL and SCCC; and (iii) cytoplasmic ZNF177 is a poor-prognosis biomarker in SCCC

Broad virus inactivation using inorganic micro/nano-particulate materials

Inorganic materials can provide a set of tools to decontaminate solid, liquid or air containing viral particles. The use of disinfectants can be limited or not practical in scenarios where continuous cleaning is not feasible. Physicochemical differences between viruses raise the need for effective formulations for all kind of viruses. In the present work we describe two types of antimicrobial inorganic materials: i) a novel soda-lime glass (G3), and ii) kaolin containing metals nanoparticles (Ag or CuO), as materials to disable virus infectivity. Strong antiviral properties can be observed in G3 glass, and kaolin-containing nanoparticle materials showing a reduction of viral infectivity close to 99%. in the first 10 ​min of contact of vesicular stomatitis virus (VSV). A potent virucidal activity is also present in G3 and kaolin containing Ag or CuO nanoparticles against all kinds of viruses tested, reducing more than 99% the amount of HSV-1, Adenovirus, VSV, Influenza virus and SARS-CoV-2 exposed to them. Virucidal properties could be explained by a direct interaction of materials with viruses as well as inactivation by the presence of virucidal elements in the material lixiviates. Kaolin-based materials guarantee a controlled release of active nanoparticles with antiviral activity. Current coronavirus crisis highlights the need for new strategies to remove viruses from contaminated areas. We propose these low-cost inorganic materials as useful disinfecting antivirals in the actual or future pandemic threats.This research was performed with support from The Spanish National Research Council (CSIC) (Project No 202060E109). M.F. is grateful to the Comunidad Autonoma de Madrid for research project No. 2017-T1/BIO-4992 (“Atracción de Talento” Action) cofunded by Universidad Complutense de Madrid. This publication was also supported by the European Virus Archive GLOBAL (EVA-GLOBAL) project that has received funding from the European Union's Horizon 2020 research and innovation program ​under grant agreement 871029. S. R.-R. was supported by the the FPI fellowship funded by Universidad San Pablo CEU. J.A-H. was supported by the PFIS fellowship co-funded by the FEDER/FSE and the ISCIII

Inhibición del enzima colina quinasa como estrategia para el desarrollo de los nuevos agentes antitumorales

Tesis doctoral original inédita, leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 6 de Mayo de 199

In vivo antitumor activity of choline kinase inhibitors: A novel target for anticancer drug discovery

Transformation by some oncogenes is associated with increased activity of choline kinase (ChoK), resulting in elevated constitutive levels of phosphorylcholine, a proposed second messenger required for DNA synthesis induced by growth factors. Here we describe the characterization of ChoK inhibitors with antiproliferative properties against human tumor-derived cell lines. The new molecules were tolerated in mice at doses that showed in vivo antitumor activity against human tumor xenografts derived from HT-29 and A431 cell lines implanted s.c. in nude mice. This first generation of inhibitors provides in vivo evidence that blockade of phosphorylcholine production is a valid strategy for the development of new anticancer agents, opening a new avenue for the development of antitumor drugs with a novel mechanism of action.This work was supported by Grant SAF98-0112-C02-01 from CICYT, Grant PB94- 0009 from DGICYT, and Grant 08.1/0024/1997 from Consejería de Educación of Comunidad de Madrid. R. H-A. is a Fellow from Fundación Ramón Areces.Peer Reviewe

Identification of Replication-competent HSV-1 Cgal+ Strain Signaling Targets in Human Hepatoma Cells by Functional Organelle Proteomics*S⃞

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal+) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal+ infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca2+-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal+ infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK½, ERK½) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal+ in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest

Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors

Recessive dystrophic epidermolysis bullosa, a devastating skin fragility disease characterized by recurrent skin blistering, scarring, and a high risk of developing squamous cell carcinoma is caused by mutations in COL7A1, the gene encoding type VII collagen, which is the major component of the anchoring fibrils that bind the dermis and epidermis. Ex vivo correction of COL7A1 by gene editing in patients' cells has been achieved before. However, in vivo editing approaches are necessary to address the direct treatment of the blistering lesions characteristic of this disease. We have now generated adenoviral vectors for CRISPR-Cas9 delivery to remove exon 80 of COL7A1, which contains a highly prevalent frameshift mutation in Spanish patients. For in vivo testing, a humanized skin mouse model was used. Efficient viral transduction of skin was observed after excisional wounds generated with a surgical punch on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. Type VII collagen deposition in the basement membrane zone of the wounded areas treated with the vectors correlated with restoration of dermal-epidermal adhesion, demonstrating that recessive dystrophic epidermolysis bullosa (RDEB) patient skin lesions can be directly treated by CRISPR-Cas9 delivery in vivo.This study was supported by grant ER18TRL714 from CIBERER and grants PI20/00615, PI21/00171 and RICORS/TERAV (RD21/0017/0027) from ISCIII, co-funded by the European Regional Development Fund and European Union – NextGenerationEU). G.T., A.J.T., and S.S. were supported by the Wellcome Trust (217112/Z/19/Z) and the NIHR Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. G.T. was also supported by the University College London Therapeutic Acceleration Support fund. Authors are indebted to Esteban Chacón-Solano for help with protein analysis, Mirentxu Santos for design of animal experiments, Blanca Duarte, Nuria Illera, Eva Muñoz, and Almudena Holguín for grafting experiments, and to Edilia De Almeida for animal maintenance and care