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.

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.

LncRNA ARGI Contributes to Virus-Induced Pancreatic ß Cell Inflammation Through Transcriptional Activation of IFN-Stimulated Genes.

Type 1 diabetes (T1D) is a complex autoimmune disease that develops in genetically susceptible individuals. Most T1D-associated single nucleotide polymorphisms (SNPs) are located in non-coding regions of the human genome. Interestingly, SNPs in long non-coding RNAs (lncRNAs) may result in the disruption of their secondary structure, affecting their function, and in turn, the expression of potentially pathogenic pathways. In the present work, the function of a virus-induced T1D-associated lncRNA named ARGI (Antiviral Response Gene Inducer) is characterized. Upon a viral insult, ARGI is upregulated in the nuclei of pancreatic ß cells and binds to CTCF to interact with the promoter and enhancer regions of IFNß and interferon-stimulated genes, promoting their transcriptional activation in an allele-specific manner. The presence of the T1D risk allele in ARGI induces a change in its secondary structure. Interestingly, the T1D risk genotype induces hyperactivation of type I IFN response in pancreatic ß cells, an expression signature that is present in the pancreas of T1D patients. These data shed light on the molecular mechanisms by which T1D-related SNPs in lncRNAs influence pathogenesis at the pancreatic ß cell level and opens the door for the development of therapeutic strategies based on lncRNA modulation to delay or avoid pancreatic ß cell inflammation in T1D.

Nucleophosmin interaction with APE1: Insights into DNA repair regulation.

Nucleophosmin (NPM1), an abundant, nucleolar protein with multiple functions affecting cell homeostasis, has also been recently involved in DNA damage repair. The roles of NPM1 in different repair pathways remain however to be elucidated. NPM1 has been described to interact with APE1 (apurinic apyrimidinic endonuclease 1), a key enzyme of the base excision repair (BER) pathway, which could reflect a direct participation of NPM1 in this route. To gain insight into the possible role(s) of NPM1 in BER, we have explored the interplay between the subnuclear localization of both APE1 and NPM1, the in vitro interaction they establish, the effect of binding to abasic DNA on APE1 conformation, and the modulation by NPM1 of APE1 binding and catalysis on DNA. We have found that, upon oxidative damage, NPM1 is released from nucleoli and locates on patches throughout the chromatin, perhaps co-localizing with APE1, and that this traffic could be mediated by phosphorylation of NPM1 on T199. NPM1 and APE1 form a complex in vitro, involving, apart from the core domain, at least part of the linker region of NPM1, whereas the C-terminal domain is dispensable for binding, which explains that an AML leukemia-related NPM1 mutant with an unfolded C-terminal domain can bind APE1. APE1 interaction with abasic DNA stabilizes APE1 structure, as based on thermal unfolding. Moreover, our data suggest that NPM1, maybe by keeping APE1 in an "open" conformation, favours specific recognition of abasic sites on DNA, competing with off-target associations. Therefore, NPM1 might participate in BER favouring APE1 target selection as well as turnover from incised abasic DNA.