Isoreticular Chemistry and Applications of Supramolecularly Assembled Copper-Adenine Porous Materials.

The useful concepts of reticular chemistry, rigid and predictable metal nodes together with strong and manageable covalent interactions between metal centers and organic linkers, have made the so-called metal-organic frameworks (MOFs) a flourishing area of enormous applicability. In this work, the extension of similar strategies to supramolecularly assembled metal-organic materials has allowed us to obtain a family of isoreticular compounds of the general formula [Cu7(µ-adeninato-κN3:κN9)6(µ3-OH)6(µ-OH2)6](OOC-R-COO)·nH2O (R: ethylene-, acetylene-, naphthalene-, or biphenyl-group) in which the rigid copper-adeninato entities and the organic dicarboxylate anions are held together not by covalent interactions but by a robust and flexible network of synergic hydrogen bonds and π-π stacking interactions based on well-known supramolecular synthons (SMOFs). All compounds are isoreticular, highly insoluble, and water-stable and show a porous crystalline structure with a pcu topology containing a two-dimensional (2D) network of channels, whose dimensions and degree of porosity of the supramolecular network are tailored by the length of the dicarboxylate anion. The partial loss of the crystallization water molecules upon removal from the mother liquor produces a shrinkage of the unit cell and porosity, which leads to a color change of the compounds (from blue to olive green) if complete dehydration is achieved by means of gentle heating or vacuuming. However, the supramolecular network of noncovalent interactions is robust and flexible enough to reverse to the expanded unit cell and color after exposure to a humid atmosphere. This humidity-driven breathing behavior has been used to design a sensor in which the electrical resistance varies reversibly with the degree of humidity, very similar to the water vapor adsorption isotherm of the SMOF. The in-solution adsorption properties were explored for the uptake and release of the widely employed 5-fluorouracil, 4-aminosalycilic acid, 5-aminosalycilic acid, and allopurinol drugs. In addition, cytotoxicity activity assays were completed for the pristine and 5-fluorouracil-loaded samples.

The T1D-associated lncRNA Lnc13 modulates human pancreatic ß cell inflammation by allele-specific stabilization of STAT1 mRNA.

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 ß-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 ß-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 ß-cells in an allele-specific manner. Our results show that the T1D-associated Lnc13 may contribute to the pathogenesis of T1D by increasing pancreatic ß-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.

Gluten-induced RNA methylation changes regulate intestinal inflammation via allele-specific XPO1 translation in epithelial cells.

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-nucleotide 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 (m6A)-related knock-out and HLA-DQ2 mice, and human samples from patients with CD. RESULTS: Individuals harbouring the risk allele had higher m6A 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 m6A methylation in humans as well as in in vitro and in vivo models. CONCLUSION: We identify a novel m6A-XPO1-NFkB pathway that is activated in CD patients. The findings will prompt the development of new therapeutic approaches directed at m6A proteins and XPO1, a target under evaluation for the treatment of intestinal disorders.

Functional Implications of Intergenic GWAS SNPs in Immune-Related LncRNAs.

Genome wide association studies (GWAS) have identified many loci contributing to genetic variation of complex traits. Immune mediated disorders are complex diseases for which hundreds of risk alleles have been identified by GWAS. However, the intergenic location of most of the signals has make it difficult to decipher their implication in disease pathogenesis. A significant number of immune disease-associated SNPs are located within long noncoding RNAs (lncRNAs). LncRNAs have gained importance due to their involvement in the regulation of a wide range of biological processes, including immune responses. GWAS SNPs located within lncRNAs can affect their regulatory capacity by modifying their secondary structure, altering their expression levels or regulating the transcription of different isoforms. In this review we discuss the functional implications of immune-related lncRNAs harboring disease associated SNPs on various disease conditions.

DEXI, a candidate gene for type 1 diabetes, modulates rat and human pancreatic beta cell inflammation via regulation of the type I IFN/STAT signalling pathway.

AIMS/HYPOTHESIS: 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. METHODS: Functional studies based on silencing or overexpression of DEXI were performed in rat and human pancreatic beta cells. Beta cell inflammation and apoptosis, driven by a synthetic viral double-stranded RNA, were evaluated by real-time PCR, western blotting and luciferase assays. RESULTS: DEXI-silenced beta cells exposed to a synthetic double-stranded RNA (polyinosinic:polycytidylic acid [PIC], a by-product of viral replication) showed reduced activation of signal transducer and activator of transcription (STAT) 1 and lower production of proinflammatory chemokines that was preceded by a reduction in IFNß levels. Exposure to PIC increased chromatin-bound DEXI and IFNß promoter activity. This effect on IFNß promoter was inhibited in DEXI-silenced beta cells, suggesting that DEXI is implicated in the regulation of IFNß transcription. In a mirror image of knockdown experiments, DEXI overexpression led to increased levels of STAT1 and proinflammatory chemokines. CONCLUSIONS/INTERPRETATION: These observations support DEXI as the aetiological gene in the type 1 diabetes-associated 16p13 genomic region, and provide the first indication of a link between this candidate gene and the regulation of local antiviral immune responses in beta cells. Moreover, our results provide initial information on the function of DEXI.

Cytoplasmic Form of Carlr lncRNA Facilitates Inflammatory Gene Expression upon NF-κB Activation.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of inflammation. To further understand the interaction between inflammatory signaling pathways and lncRNAs, we characterized the function of cardiac and apoptosis-related lncRNA (Carlr), an lncRNA expressed in both mouse and human cells of diverse tissues. Carlr expression is increased following NF-κB signaling in macrophages, with concomitant translocation to, and enrichment of, the transcript in the cytoplasm. Knockdown of Carlr results in impaired expression of NF-κB pathway genes and influences the interaction between macrophages and intestinal cells in an inflammatory environment. In human celiac disease patient samples, increased levels of the Carlr transcript were detected in the cytoplasm, alongside elevated expression of NF-κB pathway genes. These findings suggest that increased Carlr expression and/or cytoplasmic localization is required for efficient NF-κB signaling and is associated with the inflamed tissue state observed in human celiac disease.

Celiac Diasease-associated lncRNA Named HCG14 Regulates NOD1 Expression in Intestinal Cells.

OBJECTIVE: The aim of the study is to identify additional celiac disease associated loci in the major histocompatibility complex (MHC) independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level. METHODS: We performed a high-resolution single-nucleotide polymorphism (SNP) genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84. RESULTS: MHC genotyping revealed 2 associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci. Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells. CONCLUSIONS: We have successfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HCG14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.

Coregulation and modulation of NFκB-related genes in celiac disease: uncovered aspects of gut mucosal inflammation.

It is known that the NFκB route is constitutively upregulated in celiac disease (CD), an immune-mediated disorder of the gut caused by intolerance to ingested gluten. Our aim was to scrutinize the expression patterns of several of the most biologically relevant components of the NFκB route in intestinal biopsies from active and treated patients and after in vitro gliadin challenge, and to assess normalization of the expression using an inhibitor of the MALT1 paracaspase. The expression of 93 NFκB genes was measured by RT-PCR in a set of uncultured active and treated CD and control biopsies, and in cultured biopsy series challenged with gliadin, the NFκB modulator, both compounds and none. Methylation of eight genes involved in NFκB signaling was analyzed by conventional pyrosequencing. Groups were compared and Pearson's correlation matrixes were constructed to check for coexpression and co-methylation. Our results confirm the upregulation of the NFκB pathway and show that constitutively altered genes usually belong to the core of the pathway and have central roles, whereas genes overexpressed only in active CD are more peripheral. Additionally, this is the first work to detect methylation level changes in celiac intestinal mucosa. Coexpression is very common in controls, whereas gliadin challenge and especially chronic inflammation present in untreated CD result in the disruption of the regulatory equilibrium. In contrast, co-methylation occurs more often in active CD. Importantly, NFκB modulation partially restores coregulation, opening the door to future therapeutic possibilities and targets.

m6A Methylated Long Noncoding RNA LOC339803 Regulates Intestinal Inflammatory Response.

Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6-methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele-specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803-YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.

A fast, cheap, and easy protocol for celiac disease HLA haplotype typing using buccal swabs.

Celiac disease (CeD) is a complex autoimmune disorder characterized by intestinal immune-derived injury that develops in response to dietary gluten consumption. Human Leucocyte Antigen (HLA) complex haplotype typing is one of the main tests for CeD diagnosis, together with anti-endomysium and anti-transglutaminase autoantibody detection in blood and inflammation observation in the intestine, being the former mainly used for the initial discarding of the pathogenesis. Among the many types of HLA proteins, HLA-DQ2.5 and HLA-DQ8 are considered essential for CeD development. These receptors are only expressed when specific alleles are present, which can be accurately predicted by the presence of the tagging SNPs rs2187668 and rs7454108, respectively. Taking advantage of this premise, we present here an easy workflow to assess HLA genotyping in saliva by a quick and cheap isopropanol-ethanol precipitation-based DNA extraction method followed by the genotyping of two tagging SNPs for the most frequent CeD risk-associated HLA haplotypes. All the actual diagnostic methods for CeD are performed after acquisition of intestine biopsies or blood samples by invasive techniques. Therefore, the development of non-invasive methods would be of a great improvement and advantage for patients, especially children, as an alternative method for initial CeD screening.

In vivo sensitization to gliadin by oral administration.

Celiac disease is a highly prevalent immune-mediated enteropathy that develops in genetically susceptible individuals expressing HLA-DQ2 or HLA-DQ8 after ingestion of gluten and results in decreased quality of life and increased morbidity. This pathology is triggered by immunogenic peptides generated from gliadins present in gluten, which act on the intestinal mucosa in a context of high intestinal permeability, activating the innate and adaptive response of the immune system. Several in vivo rodent models attempt to reproduce some phases of the intestinal inflammatory process that occurs in celiac disease. Allergic sensitization to gluten simulates, or enhances in some animal models, the loss of tolerance to gliadin peptides and the initial events that lead to celiac disease in a specific genetic or environmental context. Here we describe a simple method for performing gliadin sensitization in an in vivo animal model.

Functional evolutionary convergence of long noncoding RNAs involved in embryonic development.

Long noncoding RNAs have been identified in most vertebrates, but the functional characterization of these molecules is 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 (nonlinear similarity) and secondary structure-based approaches combining in silico, in vitro and in vivo validation analysis. From the Madagascar gecko genes, we have found a non-orthologous lncRNA with a similar k-mer content and structurally concordant with the human lncRNA EVX1AS. Analysis of function-related characteristics together with locus-specific targeting of human EVX1AS and gecko EVX1AS-like (i.e., CRISPR Display) in human neuroepithelial cells and chicken mesencephalon have confirmed that gecko EVX1AS-like lncRNA mimics human EVX1AS function and induces EVX1 expression independently of the target species. Our data shows functional convergence of non-homologous lncRNAs and presents a useful approach for the definition and manipulation of lncRNA function within different model organisms.

Preparation of pepsin trypsin digested gliadin for stimulation experiments.

Celiac disease (CD) is a complex immune disorder of the intestine that developes in genetically susceptible individuals. CD develops as an intolerance to ingested gluten proteins (gliadins, secalins, hordeins and avenins), being gliadin one of the most immunogenic. Here we present a protocol for the preparation of digested gliadin for laboratory use, a fundamental axis for in vitro and in vivo stimulation studies related to celiac disease research. The importance of a scrupulous handling of materials, products and laboratory instruments to achieve a lipopolysaccharide free gliadin is explained and emphasized. Therefore, in the present chapter, a step-by-step set-up of the protocol for pepsin trypsin gliadin digestion is explained.

A long non-coding RNA that harbors a SNP associated with type 2 diabetes regulates the expression of TGM2 gene in pancreatic beta cells.

Introduction: Most of the disease-associated single nucleotide polymorphisms (SNPs) lie in non- coding regions of the human genome. Many of these variants have been predicted to impact the expression and function of long non-coding RNAs (lncRNA), but the contribution of these molecules to the development of complex diseases remains to be clarified. Methods: Here, we performed a genetic association study between a SNP located in a lncRNA known as LncTGM2 and the risk of developing type 2 diabetes (T2D), and analyzed its implication in disease pathogenesis at pancreatic beta cell level. Genetic association study was performed on human samples linking the rs2076380 polymorphism with T2D and glycemic traits. The pancreatic beta cell line EndoC-bH1 was employed for functional studies based on LncTGM2 silencing and overexpression experiments. Human pancreatic islets were used for eQTL analysis. Results: We have identified a genetic association between LncTGM2 and T2D risk. Functional characterization of the LncTGM2 revealed its implication in the transcriptional regulation of TGM2, coding for a transglutaminase. The T2Dassociated risk allele in LncTGM2 disrupts the secondary structure of this lncRNA, affecting its stability and the expression of TGM2 in pancreatic beta cells. Diminished LncTGM2 in human beta cells impairs glucose-stimulated insulin release. Conclusions: These findings provide novel information on the molecular mechanisms by which T2D-associated SNPs in lncRNAs may contribute to disease, paving the way for the development of new therapies based on the modulation of lncRNAs.

Revisiting genome wide association studies (GWAS) in coeliac disease: replication study in Spanish population and expression analysis of candidate genes.

INTRODUCTION: Recent genome wide association studies (GWAS) on coeliac disease (CD) have identified risk loci harbouring genes that fit the accepted pathogenic model and are considered aetiological candidates. METHODS: Using Taqman single nucleotide polymorphism (SNP) and expression assays, the study genotyped 11 SNPs tagging eight GWAS regions (1q31, 2q11-2q12, 3p21, 3q25-3q26, 3q28, 4q27, 6q25 and 12q24) in a Spanish cohort of 1094 CD patients and 540 controls, and performed expression analyses of candidate genes (RGS1, IL18R1/IL18RAP, CCR3, IL12A/SCHIP1, LPP, IL2/IL21-KIAA1109, TAGAP, and SH2B3) in intestinal mucosa from 29 CD children and eight controls. RESULTS: Polymorphisms in 1q31, 2q11-2q12, and 3q25 showed association in our cohort, and also 3q28 and 4q27 when combined with a previous study. Expression levels of IL12A, IL18RAP, IL21, KIAA1109, LPP, SCHIP1, and SH2B3 were affected by disease status, but the correlation between genotype and mRNA levels was observed only in IL12A, LPP, SCHIP1, and SH2B3. CONCLUSIONS: Expression differences between treated CD patients and controls along with SNP expression associations suggest a possible primary role for these four genes and their variants in pathogenesis. The lack of SNP effect in the remaining genes is probably a consequence of arbitrary candidate gene selection within association signals that are not based on functional studies.

Towards the design of contrast-enhanced agents: systematic Ga3+ doping on magnetite nanoparticles.

The main objective of the preparation of the Fe3-xGaxO4 (0.14 ≤ x ≤ 1.35) system was to further the knowledge of the magnetic response of Ga3+-doped magnetite for application as MRI contrast agents. With this purpose, monodisperse nanoparticles between 7 and 10 nm with different amounts of gallium were prepared from an optimized protocol based on thermal decomposition of metallo-organic precursors. Thorough characterization of the sample was conducted in order to understand the influence of gallium doping on the structural, morphological and magnetic properties of the Fe3-xGaxO4 system. X-ray diffraction and X-ray absorption near-edge structure measurements have proved the progressive incorporation of Ga in the spinel structure, with different occupations in both tetrahedral and octahedral sites. Magnetization measurements as a function of field temperature have shown a clear dependence of magnetic saturation on the gallium content, reaching an Ms value of 110 Am2 kg-1 at 5 K for x = 0.14 (significantly higher than bulk magnetite) and considerably decreasing for amounts above x = 0.57 of gallium. For this reason, nanoparticles with moderate Ga quantities were water-transferred by coating them with the amphiphilic polymer PMAO to further analyse their biomedical potential. Cytotoxicity assays have demonstrated that Fe3-xGaxO4@PMAO formulations with x ≤ 0.57, which are the ones with better magnetic response, are not toxic for cells. Finally, the effect of gallium doping on relaxivities has been analysed by measuring longitudinal (T1-1) and transverse (T1-1) proton relaxation rates at 1.4 T revealing that nanoparticles with x = 0.14 Ga3+ content present remarkable T2 contrast and the nanoparticles with x = 0.26 have great potential to act as dual T1-T2 contrast agents.

Efficient Magneto-Luminescent Nanosystems based on Rhodamine-Loaded Magnetite Nanoparticles with Optimized Heating Power and Ideal Thermosensitive Fluorescence.

Nanosystems that simultaneously contain fluorescent and magnetic modules can offer decisive advantages in the development of new biomedical approaches. A biomaterial that enables multimodal imaging and contains highly efficient nanoheaters together with an intrinsic temperature sensor would become an archetypical theranostic agent. In this work, we have designed a magneto-luminescent system based on Fe3O4 NPs with large heating power and thermosensitive rhodamine (Rh) fluorophores that exhibits the ability to self-monitor the hyperthermia degree. Three samples composed of highly homogeneous Fe3O4 NPs of ∼25 nm and different morphologies (cuboctahedrons, octahedrons, and irregular truncated-octahedrons) have been finely synthesized. These NPs have been thoroughly studied in order to choose the most efficient inorganic core for magnetic hyperthermia under clinically safe radiofrequency. Surface functionalization of selected Fe3O4 NPs has been carried out using fluorescent copolymers composed of PMAO, PEG and Rh. Copolymers with distinct PEG tail lengths (5-20 kDa) and different Rh percentages (5, 10, and 25%) have been synthesized, finding out that the copolymer with 20 kDa PEG and 10% Rh provides the best coating for an efficient fluorescence with minimal aggregation effects. The optimized Fe3O4@Rh system offers very suitable fluorescence thermosensitivity in the therapeutic hyperthermia range. Additionally, this sample presents good biocompatibility and displays an excellent heating capacity within the clinical safety limits of the AC field (≈ 1000 W/g at 142 kHz and 44 mT), which has been confirmed by both calorimetry and AC magnetometry. Thus, the current work opens up promising avenues toward next-generation medical technologies.

Relative Quantification of Residue-Specific m6A RNA Methylation Using m6A-RT-QPCR.

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.