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SummaryType 1 diabetes-associated single nucleotide polymorphisms are mainly located in non-coding regions of the human genome. Single nucleotide polymorphisms located in long non-coding RNAs may result in the disruption of their secondary structure, affecting their function.Here, we functionally characterized the virus-induced type 1 diabetes-associated lncRNAARGI(Antiviral Response Gene Inducer).ARGIupregulation in pancreatic β cells leads to the transcriptional activation of antiviral and pro-inflammatory genes. Upon a viral insult,ARGIis upregulated in the nuclei of pancreatic β cells and binds to CTCF to interact with the regulatory regions ofIFNβand interferon-stimulated genes, promoting their transcriptional activation in an allele-specific manner.The presence of the risk allele for type 1 diabetes inARGIinduces an hyperactivation of type I IFN response in β cells, an expression signature that is present in the pancreas of diabetic patients. These data shed light on the molecular mechanisms by which type 1 diabetes-related single nucleotide polymorphisms in long non-coding RNAs influence pathogenesis at the pancreatic β cell level.

Long noncoding RNAs (lncRNAs) have been identified in almost all vertebrates, but the functional characterization of these RNA molecules is being challenging, mainly due to the lack of linear sequence homology between species. In this work, we aimed to find functional evolutionary convergent lncRNAs involved in development by screening of k-mer content (non linear similarity) and secondary structure-based approaches combined within silico, in vitroandin vivovalidation analysis. From the currently identified Madagascar gecko genes, we found a lncRNA with a similar k-mer content and structurally concordant with the human lncRNAEVX1AS. Analysis of function related characteristics together with locus-specific targeting of human and geckoEVX1AS(i.e. CRISPR Display) in human neuroepithelial cells and chicken mesencephalon confirmed that geckoEvx1as-likelncRNA mimics humanEVX1ASfunction and inducesEVX1expression independently of the target species. Our data show functional conservation of non-homologous lncRNAs and presents a useful approach for the definition and manipulation of lncRNA function within different model organisms.

Local heat generation from magnetic nanoparticles (MNPs) exposed to alternating magnetic fields can revolutionize cancer treatment. However, the application of MNPs as anticancer agents is limited by serious drawbacks. Foremost among these are the fast uptake and biodegradation of MNPs by cells and the unpredictable magnetic behavior of the MNPs when they accumulate within or around cells and tissues. In fact, several studies have reported that the heating power of MNPs is severely reduced in the cellular environment, probably due to a combination of increased viscosity and strong NP agglomeration. Herein, we present an optimized protocol to coat magnetite (Fe3O4) NPs larger than 20 nm (FM-NPs) with high molecular weight PEG molecules that avoid collective coatings, prevent the formation of large clusters of NPs and keep constant their high heating performance in environments with very different ionic strengths and viscosities (distilled water, physiological solutions, agar and cell culture media). The great reproducibility and reliability of the heating capacity of this FM-NP@PEG system in such different environments has been confirmed by AC magnetometry and by more conventional calorimetric measurements. The explanation of this behavior has been shown to lie in preserving as much as possible the magnetic single domain-type behavior of nearly isolated NPs. In vitro endocytosis experiments in a colon cancer-derived cell line indicate that FM-NP@PEG formulations with PEGs of higher molecular weight (20 kDa) are more resistant to endocytosis than formulations with smaller PEGs (5 kDa), showing quite large uptake mean-life (τ > 5 h) in comparison with other NP systems. The in vitro magnetic hyperthermia was performed at 21 mT and 650 kHz during 1 h in a pre-endocytosis stage and complete cell death was achieved 48 h posthyperthermia. These optimal FM-NP@PEG formulations with high resistance to endocytosis and predictable magnetic response will aid the progress and accuracy of the emerging era of theranostics.

[EN] Objectives Coeliac disease (CD) is a complex autoimmune disorder that develops in genetically susceptible individuals. Dietary gluten triggers an immune response for which the only available treatment so far is a strict, lifelong gluten free diet. Human leucocyte antigen (HLA) genes and several non-HLA regions have been associated with the genetic susceptibility to CD, but their role in the pathogenesis of the disease is still essentially unknown, making it complicated to develop much needed non-dietary treatments. Here, we describe the functional involvement of a CD-associated single-nudeotide polymorphism (SNP) located in the 5'UTR of XPO1 in the inflammatory environment characteristic of the coeliac intestinal epithelium. Design The function of the CD-associated SNP was investigated using an intestinal cell line heterozygous for the SNP, N6-methyladenosine (m(6)A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. Results Individuals harbouring the risk allele had higher m(6)A methylation in the 5'UTR of XPO1 RNA, rendering greater XPO1 protein amounts that led to downstream nuclear factor kappa B (NFkB) activity and subsequent inflammation. Furthermore, gluten exposure increased overall m(6)A methylation in humans as well as in in vitro and in vivo models. Conclusion We identify a novel m(6)A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m(6)A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders. This study was supported by a grant from the Spanish Ministry of Science, Universities and Innovation (PGC2018-097573-A-I00) to AC-R. JRB was funded by ISCIII Research project PI16/00258, cofinanced by the Spanish Ministry of Economy and Competitiveness and by the European Union ERDF/ESF 'A way to make Europe'. AO-G and MS-D were funded by predoctoral fellowships from the Basque Government and the University of the Basque Country respectively. DS and LH were funded by the Spanish Ministry (MINECO) (SAF2017-83813-C3-1-R) and cofunded by the ERDF, the Centro de Investigacion Biomedica en Red de Fisiopatologia de la Obesidad y la Nutricion (CIBEROBN) (Grant CB06/03/0001 to DS), the Government of Catalonia (2017SGR278 to DS), and the Fundacio La Marato de TV3 (201627-30 to DS). CH is a Howard Hughes Medical Institute Investigator and has been funded by the National Institute of Health HG008935. We would like to thank Xuechen Yu and Justin Vargas for processing the adult CD biopsy samples obtained from Columbia University. EFV is supported by a CIHR grant 168840 and holds a Canada Research Chair.

Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m

Technological advances in high-throughput sequencing in combination with antibody enrichment and/or induced nucleotide-specific chemical modifications have accelerated the mapping of epitranscriptomic modifications. However, site-specific detection and quantification of m6A are still technically challenging. Here, we describe a simple RT-QPCR-based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues.

Celiac disease (CD) is an immune-mediated disease that develops in genetically susceptible individuals upon gluten exposure. Human Leukocyte Antigen (HLA) genes in the Major Histocompatibility Complex (MHC) have been described to represent the 40% of the genetic risk to develop CD. Aiming to gain understanding of the genetic involvement in CD, high throughput studies have been performed, revealing that many CD-associated variants are located in non-coding regions, hindering the study of the functional implications of these single nucleotide polymorphisms (SNPs). In the last decade, long non-coding RNAs (lncRNAs) have been described to be influenced by disease-associated SNPs and to drive many important mechanisms involved in the development of inflammatory diseases. Here we describe the lncRNAs identified and characterized in the context of celiac disease and highlight the importance of the study of these molecules in inflammatory and autoimmune disorders.

mRNA stability influences gene expression and translation in almost all living organisms, and the levels of mRNA molecules in the cell are determined by a balance between production and decay. Maintaining an accurate balance is crucial for the correct function of a wide variety of biological processes and to maintain an appropriate cellular homeostasis. Long non-coding RNAs (lncRNAs) have been shown to participate in the regulation of gene expression through different molecular mechanisms, including mRNA stabilization. In this review we provide an overview on the molecular mechanisms by which lncRNAs modulate mRNA stability and decay. We focus on how lncRNAs interact with RNA binding proteins and microRNAs to avoid mRNA degradation, and also on how lncRNAs modulate epitranscriptomic marks that directly impact on mRNA stability. LncRNA work in author’s laboratory is supported by European Foundation for the Study of Diabetes (EFSD)-EFSD/JDRF/Lilly Programme on Type 1 Diabetes Research and the Spanish Ministry of Science, Innovation and Universities (PID2019-104475GA-I00) to I.S; and Spanish Ministry of Science, Innovation and Universities (SAF2017-91873-EXP and PGC2018-097573-A-I00) to ACR. M.S.D. and I.G.M. are supported by a Predoctoral Fellowship Grant from the UPV/EHU (Universidad del País Vasco/Euskal Herriko Unibertsitatea) and A.O.G. is supported by a Predoctoral Fellowship Grant from the Education Department of Basque Government.

The currently existing magnetic hyperthermia treatments usually need to employ very large doses of magnetic nanoparticles MNPs and or excessively high excitation conditions H f gt; 1010 A m s to reach the therapeutic temperature range that triggers cancer cell death. To make this anticancer therapy truly minimally invasive, it is crucial the development of improved chemical routes that give rise to monodisperse MNPs with high saturation magnetization and negligible dipolar interactions. Herein, we present an innovative chemical route to synthesize Zn doped magnetite NPs based on the thermolysis of two kinds of organometallic precursors i a mixture of two monometallic oleates FeOl ZnOl , and ii a bimetallic ironzinc oleate Fe3 amp; 8722;yZnyOl . These approaches have allowed tailoring the size 10 amp; 8722;50 nm , morphology spherical, cubic, and cuboctahedral , and zinc content ZnxFe3 amp; 8722;xO4, 0.05 lt; x lt; 0.25 of MNPs with high saturation magnetization amp; 8805;90 Am2 kg at RT . The oxidation state and the local symmetry of Zn2 and Fe2 3 cations have been investigated by means of X ray absorption near edge structure XANES spectroscopy, while the Fe center distribution and vacancies within the ferrite lattice have been examined in detail through Mo amp; 776;ssbauer spectroscopy, which has led to an accurate determination of the stoichiometry in each sample. To achieve good biocompatibility and colloidal stability in physiological conditions, the ZnxFe3 amp; 8722;xO4 NPs have been coated with high molecular weight poly ethylene glycol PEG . The magnetothermal efficiency of ZnxFe3 amp; 8722;xO4 PEG samples has been systematically analyzed in terms of composition, size, and morphology, making use of the latest generation AC magnetometer that is able to reach 90 mT. The heating capacity of Zn0.06Fe2.94O4 cuboctahedrons of 25 nm reaches a maximum value of 3652 W g at 40 kA m and 605 kHz , but most importantly, they reach a highly satisfactory value 600 W g under strict safety excitation conditions at 36 kA m and 125 kHz . Additionally, the excellent heating power of the system is kept identical both immobilized in agar and in the cellular environment, proving the great potential and reliability of this platform for magnetic hyperthermia therapies

The SARS-CoV-2 pandemic has evolved far more aggressively in countries lacking a robust testing strategy to identify infected individuals. Given the global demand for fast and reliable diagnosis to determine the carrier individuals, a stock-out scenario for a number of essential reagents/kits used along the diagnostic process has been foreseen by many organizations. Having identified the RNA extraction step as one of the key bottlenecks, we tested several alternatives that avoid the use of commercial kits for this step. The analysis showed that 2-propanol precipitation of the viral RNA, followed by one-step RT-qPCR results in a sensitivity and specificity comparable to that provided currently by automatized systems such as the COBAS 6800 system. Therefore, this simple protocol allows SARS-CoV-2 testing independently of commercial kit providers in a time and cost-effective manner. It can be readily implemented in research and/or diagnostic laboratories worldwide, provided that patient confidentiality and researcher safety are ensured. Scaling up the testing capabilities of hospitals and research facilities will identify larger numbers of infected individuals to paint a clear picture of the COVID-19 prevalence, a pre-requisite for informed policy decision making.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13. Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic β-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in β-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in β-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic β-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting.

The vast majority of type 1 diabetes (T1D) genetic association signals lie in noncoding regions of the human genome. Many have been predicted to affect the expression and secondary structure of long noncoding RNAs (lncRNAs), but the contribution of these lncRNAs to the pathogenesis of T1D remains to be clarified. Here, we performed a complete functional characterization of a lncRNA that harbors a single nucleotide polymorphism (SNP) associated with T1D, namely, Lnc13. Human pancreatic islets harboring the T1D-associated SNP risk genotype in Lnc13 (rs917997*CC) showed higher STAT1 expression than islets harboring the heterozygous genotype (rs917997*CT). Up-regulation of Lnc13 in pancreatic β-cells increased activation of the proinflammatory STAT1 pathway, which correlated with increased production of chemokines in an allele-specific manner. In a mirror image, Lnc13 gene disruption in β-cells partially counteracts polyinosinic-polycytidylic acid (PIC)-induced STAT1 and proinflammatory chemokine expression. Furthermore, we observed that PIC, a viral mimetic, induces Lnc13 translocation from the nucleus to the cytoplasm promoting the interaction of STAT1 mRNA with (poly[rC] binding protein 2) (PCBP2). Interestingly, Lnc13-PCBP2 interaction regulates the stability of the STAT1 mRNA, sustaining inflammation in β-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic β-cell inflammation. These findings provide information on the molecular mechanisms by which disease-associated SNPs in lncRNAs influence disease pathogenesis and open the door to the development of diagnostic and therapeutic approaches based on lncRNA targeting., This work was supported by a Research Project Grant from the Basque Department of Health (2015111068) and a Research Grant from Fundación de la Sociedad Española de Diabetes (FSED) to I.S. I.G.-M. and A.O.-G. were supported by Predoctoral Fellowship Grants from the UPV/EHU (Universidad del Pais Vasco/Euskal Herriko Unibertsitatea) and the Basque Department of Education, respectively. A.C.-R. was funded by a Grant from the Spanish Ministry of Science, Innovation and Universities (PGC2018-097573-A-I00). D.L.E. received support from the Fonds National de la Recherche 420 Scientifique (FNRS), Welbio Grant CR-2015A-06. B.R.G. was funded by the Spanish Ministry of Science, Innovation and Universities; the European Fund for Regional Development (FEDER) (Grant BFU2017-83588-P); and the Juvenile Diabetes Research Foundation (Grants 17-2013-372 and 2-SRA-2019-837-S-B).

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3′UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic β-cells exposed to inflammatory stimuli. J.R.B. is funded by Project ISCIII-PI16/00258 and co-funded by the European Union ERDF/ESF “A way to make Europe”. I.S. is funded by a research project grant 2015111068 of the Basque Department of Health. A.C.R. was funded by a Juan de la Cierva reincorporation and an Ikerbasque fellowships and a research project grant from Asociación Celiacos Madrid. I.R.G., A.O.G. and A.J.M. are supported by predoctoral fellowship grants from the UPV/EHU and the Basque Department of Education.

Medulloblastoma is the most common and malignant pediatric brain tumor in childhood. It originates from dysregulation of cerebellar development, due to an excessive proliferation of cerebellar granule neuron precursor cells (CGNPs). The underlying molecular mechanisms, except for the role of SHH and WNT pathways, remain largely unknown. ERBB4 is a tyrosine kinase receptor whose activity in cancer is tissue dependent. In this study, we characterized the role of ERBB4 during cerebellum development and medulloblastoma progression paying particular interests to its role in CGNPs and medulloblastoma stem cells (MBSCs). Our results show that ERBB4 is expressed in the CGNPs during cerebellum development where it plays a critical role in migration, apoptosis and differentiation. Similarly, it is enriched in the population of MBSCs, where also controls those critical processes, as well as self-renewal and tumor initiation for medulloblastoma progression. These results are translated to clinical samples where high levels of ERBB4 correlate with poor outcome in Group 4 and all medulloblastomas groups. Transcriptomic analysis identified critical processes and pathways altered in cells with knock-down of ERBB4. These results highlight the impact and underlying mechanisms of ERBB4 in critical processes during cerebellum development and medulloblastoma.

The initial stages of type 1 diabetes are characterised by an aberrant islet inflammation that is in part regulated by the interaction between type 1 diabetes susceptibility genes and environmental factors. Chromosome 16p13 is associated with type 1 diabetes and CLEC16A is thought to be the aetiological gene in the region. Recent gene expression analysis has, however, indicated that SNPs in CLEC16A modulate the expression of a neighbouring gene with unknown function named DEXI, encoding dexamethasone-induced protein (DEXI). We therefore evaluated the role of DEXI in beta cell responses to 'danger signals' and determined the mechanisms involved., SCOPUS: ar.j, info:eu-repo/semantics/published