FPU Fellowship Research Articles

Characterization of a new arrhythmogenic cardiomyopathy mouse model due to filamin c haploinsufficiency

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Spanish Ministry of Universities FPU fellowship (FPU19/04044), Spanish Ministry of Science and Innovation Digital Transition call (TED2021-129774B-C22), EU HORIZON EIC Pathfinder Challenges 2022 Cardiogenomics call (Project 101115416). Background Filamin C (FLNC) is widely expressed in heart and skeletal muscle. It participates in cell junction and sarcomere assembly and function. We have previously shown that heterozygous FLNC truncating variants (FLNCtv) cause arrhythmogenic/dilated cardiomyopathy (ACM/DCM) in patients. Diverse studies have suggested haploinsufficiency as the potential mechanism of this disease. However, animal models of FLNCtv have only been reported to develop ACM/DCM in homozygosity. Purpose To develop a new model of ACM/DCM caused by a FLNCtv and study the progression of the disease in heterozygosity. Methods Two gRNAs flanking exon 15 of the Flnc gene and SpCas9 protein were microinjected into mouse zygotes to generate a mouse line with a constitutive deletion of Flnc exon 15 (FlncWt/Ex15del). mRNA and protein stability were determined by qRT-PCR and western blot, respectively. Cardiac function was analysed by ECG and echocardiography every 2 months, starting at 8 weeks of age (Fig. 1B). The mean ECG values were calculated using 3 time intervals and standard ECG waves. Results FlncWt/Ex15del mice showed a reduction in Flnc mRNA and protein similar to that observed in a human ACM/DCM patient bearing a similar mutation, suggesting that this FLNCtv causes haploinsufficiency. Both male and female FlncWt/Ex15del mice developed cardiac electrophysiological defects, including reduced amplitude of the QRS complex and the P-wave, notched R and/or S waves, and increased QT interval times, and these defects worsened with time. At 40 weeks of age, FlncWt/Ex15del mice showed some initial signs of left ventricular dilatation. Conclusions Mice carrying FLNCtv in heterozygosity develop electrophysiological changes resembling pathological features of ACM/DCM patients due to haploinsufficiency. The development of this pathological phenotype in heterozygosity, as opposed to previous models, paves the way for the development of new gene therapies using this model.

NEW MOLECULAR RECEPTORS FOR DIAGNOSIS AND THERAPY OF PANCREATIC DUCTAL ADENOCARCINOMA

Abstract INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) is one of the cancers with worst prognosis due to the intense desmoplastic reaction that occurs in this disease, which hinders the action of therapeutic agents and contributes to cancer progression. A possible way to develop new strategies for PDAC diagnosis and therapy is the study of the abnormal components of the tumor stroma. However, new receptors capable of detecting these components are needed. MATERIALS AND METHODS Two types of molecular receptors, monoclonal antibodies and aptamers, were developed in parallel targeting an extracellular matrix protein with potential as a PDAC marker. Antibodies were generated using mice of the DBA/1J strain, while aptamers were obtained by means of an in vitro selection method called SELEX. The analytical characteristics of the developed receptors were studied by ELISA-like assays, as well as cytochemistry on positive and negative cell lines for the protein of interest. RESULTS A total of 44 potential aptamers were identified. After a first bioinformatic screening, 9 candidates were chosen for evaluating their binding affinity in vitro. Two of them showed dissociation constants in the nanomolar range. One of the aptamers also showed specific staining of cells positive for the marker protein, as did one of the generated antibody clones. CONCLUSIONS The newly developed receptors have shown a great potential in the study of PDAC. However, their diagnostic and therapeutic usefulness requires a more in-depth evaluation, currently in progress. ACKNOWLEDGEMENTS This research was funded by the Spanish Government (RTI-2018-095756-B-I00) and Principado de Asturias Government (IDI2018-000217), co-financed by FEDER funds. R.L.G. thanks the Spanish Government for a predoctoral FPU fellowship (FPU16/05670).

Refined Three-Dimensional Strut-and-Tie Model for Analysis and Design of Four-Pile Caps

The authors wish to thank the Spanish Ministry of Education for the FPU fellowship FPU12-01459 received by the first author and the funding provided for his research stay at the University of Surrey. The authors also wish to thank the Spanish Ministry of Economy and Competitiveness for funding Project BIA 2012-32300.

Effect of different extenders for donkey sperm vitrification in straws.

Aseptic vitrification of semen samples packed in straws has been successfully developed in human but not in donkeys. The aim of this study was to compare the effect of two extenders for donkey sperm vitrification using straws. Ejaculates from four Andalusian donkeys were collected, and samples were extended in INRA-96 (I) or Gent (G) supplemented with sucrose 0.25M and 1% bovine serum albumin (BSA). Extended samples were cooled for one hour at 5°C. For vitrification, samples were filled in covered 0.25ml straws and then plunged directly into liquid nitrogen. For warming, straws were immersed in INRA-96 at 43°C. Results showed no significant differences between I and G treatments for TM (34.2%±8.7 vs. 30.7%±9.6) and PM (26.8%±7.3 vs. 24.6%±7.9), respectively. In conclusion, donkey sperm could be vitrified in straws either with INRA-96 or with Gent in combination with sucrose and BSA.

Host plant associations and geographical factors in the diversification of the Macaronesian Rhopalomesites beetles (Coleoptera: Curculionidae)

AbstractAimTo obtain nuclear and mitochondrial phylogenies of Macaronesian Rhopalomesites weevils and test the monophyly and time of origin of two species groups feeding on Euphorbiaceae and other plants. Additionally, to investigate the population structure within species, and its associations with geographical isolation versus trophic selection.LocationMacaronesian Islands and the British Isles.MethodsMaximum likelihood and Bayesian phylogenetic analyses were undertaken using mitochondrial (cox1 and cytb) and nuclear (ITS‐2 and 28S RNA) genomic sequences. Ancestral Rhopalomesites host plant associations and divergence times were inferred from Bayesian analyses and population data.ResultsEvidence was found for two Rhopalomesites clades. One was associated with Euphorbia host plants, having vicariant species in the Madeira and Canary archipelagos. In this lineage, an ancestral association with Euphorbia mellifera in the two island groups was deduced, which has subsequently undergone shifts to related host plant species in marginal areas. The second clade was found to be an ecological generalist, exploiting decaying wood from the Lauraceae or other forest trees – is also present on such islands along with the Azores and parts of Atlantic Western Europe.Main conclusionsThe results point to a quasi‐parallel colonization by the two ecologically distinct lineages in Macaronesia, dating to the early Pliocene, followed by allopatric isolation according to the presence of suitable habitats on particular islands in more recent times.

Customizing smart environments: A tabletop approach

We would like to thank all the volunteers that participated in the empirical study. Our thanks are also due to the ASIC/Polimedia team for their computer hardware support. This work was partially funded by the Spanish Ministry of Science and Innovation under the National R&D&I Program within the project CreateWorlds (TIN2010-20488). It also received support from a postdoctoral fellowship within the VALi+d Program of the Conselleria d'Educacio, Cultura I Esport (Generalitat Valenciana) awarded to Alejandro Catala (APOSTD/2013/013). The work of Patricia Pons has been supported by the Universitat Politecnica de Valencia under the Beca de Excelencia program, and currently by an FPU fellowship from the Spanish Ministry of Education, Culture and Sports (FPU13/03831).

ChemInform Abstract: Dihydrobiphenylenes Through Ruthenium‐Catalyzed [2 + 2 + 2] Cycloadditions of ortho‐Alkenylarylacetylenes with Alkynes.

AbstractThe internal alkyne 1‐(hex‐1‐yn‐1‐yl)‐2‐vinylbenzene fails to dimerize.

ChemInform Abstract: Enantioselective Organocatalytic Reactions with Isatin.

The authors gratefully acknowledge FSE, MICINN CTQ2009-11172BQU, Junta de Castilla and Leon (SA162A12-1) for financial support. MFF is grateful to the JCyL-ESF for her FPI fellowship. JP is grateful to MINECO for a FPU fellowship. P.G-G. is grateful for a JAE-DOC contract from CSIC co-funded by the European Social Fund (ESF)

Enantioselective Organocatalytic Reactions with Isatin

The authors gratefully acknowledge FSE, MICINN CTQ2009-11172BQU, Junta de Castilla and Leon (SA162A12-1) for financial support. MFF is grateful to the JCyL-ESF for her FPI fellowship. JP is grateful to MINECO for a FPU fellowship. P.G-G. is grateful for a JAE-DOC contract from CSIC co-funded by the European Social Fund (ESF)

Women as dedicatees of artes de canto in the early modern Iberian world: imposed knowledge or women's choice?

Ascension Mazuela-Anguita is a recipient of a FPU Fellowship (2009–12) of the Spanish Ministerio de Educacion, Cultura y Deporte at the Consejo Superior de Investigaciones Cientificas, CSIC (Spanish National Research Council), Mila i Fontanals Institution in Barcelona; her 2012 PhD dissertation at the Universitat de Barcelona studies printed music handbooks of the 16th-century Iberian world. She is a research fellow in the R&D Project The ‘Other’ in Spanish musical sources (16th–18th centuries): foreigners, women, and Amerindians (HAR2009-07706, sponsored by the Spanish Ministerio de Ciencia e Innovacion), and member of the Research Group Music, heritage, and society, both ascribed to the CSIC, Mila i Fontanals Institution, in Barcelona.

Controlling the Number of Proteins with Dip‐Pen Nanolithography

This work was supported by projects MAT2009-13977-C03, Nanomateria-DGA, and PI091/08. D.M. thanks the Ministerio de Ciencia y Tecnologia for a RyC contract. A. L. thanks ARAID for financial support. R. de M. is indebted to MICINN for receiving a predoctoral FPU fellowship. Funding from the European Network of Excellence MAGMANet is also acknowledged.

Lack of DREAM Protein Enhances Learning and Memory and Slows Brain Aging

Memory deficits in aging affect millions of people and are often disturbing to those concerned. Dissection of the molecular control of learning and memory is paramount to understand and possibly enhance cognitive functions. Old-age memory loss also has been recently linked to altered Ca(2+) homeostasis. We have previously identified DREAM (downstream regulatory element antagonistic modulator), a member of the neuronal Ca(2+) sensor superfamily of EF-hand proteins, with specific roles in different cell compartments. In the nucleus, DREAM is a Ca(2+)-dependent transcriptional repressor, binding to specific DNA signatures, or interacting with nucleoproteins regulating their transcriptional properties. Also, we and others have shown that dream mutant (dream(-/-)) mice exhibit marked analgesia. Here we report that dream(-/-) mice exhibit markedly enhanced learning and synaptic plasticity related to improved cognition. Mechanistically, DREAM functions as a negative regulator of the key memory factor CREB in a Ca(2+)-dependent manner, and loss of DREAM facilitates CREB-dependent transcription during learning. Intriguingly, 18-month-old dream(-/-) mice display learning and memory capacities similar to young mice. Moreover, loss of DREAM protects from brain degeneration in aging. These data identify the Ca(2+)-regulated "pain gene" DREAM as a novel key regulator of memory and brain aging.

Centrosome Dysfunction in Drosophila Neural Stem Cells Causes Tumors that Are Not Due to Genome Instability

Genome instability (GI) and centrosomal alterations are common traits in human cancer [1, 2]. It is suspected that centrosome dysfunction may cause tumors by bringing about GI, but direct experimental proof is still lacking [3]. To explore the possible functional link between centrosome function and overgrowth, we have assayed the tumorigenic potential of a series of mutants that affect different centrosomal proteins in Drosophila. We have found that a significant number of such mutant conditions are tumorigenic in larval brain tissue, where self-renewing asymmetric division of neural stem cells is frequent, but not in symmetrically dividing epithelial cells. We have also found that mutations that increase GI without causing centrosome dysfunction are not tumorigenic in our assay. From these observations, we conclude that the tumors caused by centrosome dysfunction cannot be explained solely by the resulting genome instability. We propose that such tumors might be caused by impaired asymmetric division of neural stem cells [4]. These results show that centrosome loss, far from being innocuous, is a potentially dangerous condition in flies.