Estudio de la estabilidad de aptámeros generados frente a la proteína 4E-BP1

El control de la síntesis de proteínas juega un papel importante en el crecimiento y proliferación celular. En la mayoría de los organismos eucariotas se lleva a cabo una traducción dependiente de cap en la cual el factor eucariótico de iniciación (eIF) 4E juega un papel muy importante. La actividad de este factor está regulada por la proteína 4E-BP1 (proteína de unión a eIF4E), entre otras, cuya fosforilación permite la liberación del factor y su participación en la traducción. La sobreexpresión del factor eIF4E produce irregularidades en el ciclo celular relacionadas con el cáncer, enfermedad que produce 8.2 millones de muertes anuales en el mundo. La actividad reguladora de la proteína 4E-BP1 la convierte en una posible diana terapéutica, por lo que en el laboratorio se seleccionaron tres aptámeros frente a dicha proteína a través del método SELEX. Los aptámeros se conocen como 1R, 1F y 20F y poseen estructuras secundarias complejas y con posibilidad de formación de G-cuádruplex, lo que les confiere una alta estabilidad. Por ello en el presente trabajo se ha ampliado la caracterización estructural y funcional de estos aptámeros mediante parámetros como susceptibilidad a nucleasas, IC50, niveles intracelulares, efecto sobre la síntesis de proteínas y constante de disociación. Los resultados posicionan a los aptámeros 1R y 20F como los más estables y los principales candidatos como potenciales herramientas terapéuticas. Sin embargo, es necesario realizar nuevos experimentos funcionales así como estudiar la especificidad de los aptámeros frente a otras proteínas y otros aspectos importantes para su aplicación clínic

Toward a clinical practice guide in pharmacogenomics testing for functional polymorphisms of drug-metabolizing enzymes. Gene/drug pairs and barriers perceived in Spain

The development of clinica lpractice recommendations or guidelines for the clinical use of biomarkers is an issue of great importance withr regard to adverse drug reactions.The poten-tial of pharmacogenomicbiomarkers has been extensively investigated in recent years.However,several barriers to implementing the use of pharmacogenomics testing exist.We conducted a survey among members of the Spanish Societies of Pharmacology and Clinical Pharmacology to obtain information about the perception of such barriers and to compare the perceptions of participants about the relative importance of majorgene/drug pairs.Of 11 potential barriers,the highest importance was attributed to lack of institutional support for pharmacogenomic stesting,and to the issues related to the lack of guidelines.Of the proposed gene/drug pairs the highest importance was assigned to HLA-B/abacavir, UGT1A1/irinotecan, and CYP2D6/tamoxifen.In this perspective article,we compare the relative importance of 29 gene/drugpairs in the Spanish study with that of the same pairs in the American Society for Clinical Pharmacology and Therapeutic sstudy,and we provide suggestions and areas of focus to develop a guide for clinical practice in pharmacogenomics testingThe work in the author’s laboratory is financed by Grants PS09/00943, PS09/00469, RETICS RIRAAF RD07/0064/0016, and CIBERehd from Instituto de Salud CarlosIII,Madrid, Spain, and by Grants GR10068 from Junta de Extremadura, Spain. Financed in part with FEDER funds from the European Unio

MAFG is a potential therapeutic target to restore chemosensitivity in cisplatin-resistant cancer cells by increasing reactive oxygen species

Adjuvant chemotherapy for solid tumors based on platinum-derived compounds such as cisplatin is the treatment of choice in most cases. Cisplatin triggers signaling pathways that lead to cell death, but it also induces changes in tumor cells that modify the therapeutic response, thereby leading to cisplatin resistance. We have recently reported that microRNA-7 is silenced by DNA methylation and is involved in the resistance to platinum in cancer cells through the action of the musculoaponeurotic fibrosarcoma oncogene family, protein G (MAFG). In the present study, we first confirm the miR-7 epigenetic regulation of MAFG in 44 normal- and/or tumor-paired samples in non–small-cell lung cancer (NSCLC). We also provide translational evidence of the role of MAFG and the clinical outcome in NSCLC by the interrogation of two extensive in silico databases of 2019 patients. Moreover, we propose that MAFG-mediated resistance could be conferred due to lower reactive oxygen species production after cisplatin exposure. We developed specifically selected aptamers against MAFG, with high sensitivity to detect the protein at a nuclear level probed by aptacytochemistry and histochemistry analyses. The inhibition of MAFG activity through the action of the specific aptamer apMAFG6F increased the levels of reactive oxygen species production and the sensitivity to cisplatin. We report first the specific nuclear identification of MAFG as a novel detection method for diagnosis in NSCLC, and then we report that MAFG modulates the redox response and confers cell protection against free radicals generated after platinum administration, thus also being a promising therapeutic target.This study was supported by the “Fondo de Investigación Sanitaria-Instituto de Salud Carlos III” [PI15/00186 and CP 08/000689 to I.I.C. ] ; and the European Regional Development Fund/European Social Fund FIS [FEDER/FSE, Una Manera de Hacer Europa] . MINECO funds support O.V., C.R.A. and O.P.contracts through RTC-2015-4362-1 and RTC-2016-5314-1 projects

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Use of Aptamers as Diagnostics Tools and Antiviral Agents for Human Viruses

Appropriate diagnosis is the key factor for treatment of viral diseases. Time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, Ebola and SARS. Chronic viral diseases such as HIV-1 or HCV are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. Over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretely, several strategies are currently being explored using aptamers against virus proteins. From a diagnostics point of view, aptamers are being designed as a bio-recognition element in diagnostic systems to detect viral proteins either in the blood (serum or plasma) or into infected cells. Another potential use of aptamers is for therapeutics of viral infections, interfering in the interaction between the virus and the host using aptamers targeting host-cell matrix receptors, or attacking the virus intracellularly, targeting proteins implicated in the viral replication cycle. In this paper, we review how aptamers working against viral proteins are discovered, with a focus on recent advances that improve the aptamers’ properties as a real tool for viral infection detection and treatment

A magnesium-induced RNA conformational switch at the internal ribosome entry site of hepatitis C virus genome visualized by atomic force microscopy

Supplementary data.The 5′ untranslated region of hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) element, composed of domains II–IV, which is required for cap-independent translation initiation. Little information on the 3D structure of the whole functional HCV IRES is still available. Here, we use atomic force microscopy to visualize the HCV IRES conformation in its natural sequence context, which includes the upstream domain I and the essential, downstream domains V and VI. The 574 nt-long molecule analyzed underwent an unexpected, Mg2+-induced switch between two alternative conformations: from ‘open’, elongated morphologies at 0–2 mM Mg2+ concentration to a ‘closed’, comma-shaped conformation at 4–6 mM Mg2+. This sharp transition, confirmed by gel-shift analysis and partial RNase T1 cleavage, was hindered by the microRNA miR-122. The comma-shaped IRES-574 molecules visualized at 4–6 mM Mg2+ in the absence of miR-122 showed two arms. Our data support that the first arm would contain domain III, while the second one would be composed of domains (I–II)+(V–VI) thanks to a long-range RNA interaction between the I-II spacer and the basal region of domain VI. This reinforces the previously described structural continuity between the HCV IRES and its flanking domains I, V and VI.Spanish Ministerio de Ciencia e Innovación and Ministerio de Economía y Competitividad [BIO2010–20696 and BIO2013–47228-R to C.B.; MAT2011–26534 to J.A.M.- G.; BFU2010–11612-E to J.G.; FIS2012–38866-C05–05 to L.V.]; Spanish National Research Council [CSIC- 200920I040 to C.B.]; Comunidad de Madrid [NANOAVANSENS S2013/MIT-3029 to L.V.]; FEDER funds from the European Union. CIBERehd is funded by the Instituto de Salud Carlos III. JAE-Doc postdoctoral fellowship from the CSIC Program Junta de Ampliación de Estudios, co-funded by the European Science Foundation [to M.M.]. Funding for open access charge: Spanish NationalResearch Council (CSIC).Peer reviewe

A magnesium-dependent RNA structural switch at the Internal Ribosome Entry Site of Hepatitis C Virus genome monitored by Atomic Force Microscopy

Comunicación presentada en el XII Congreso Nacional de Virología que tuvo lugar del 9 al 12 de junio de 2013 en Burgos (España).Hepatitis C virus (HCV) is the main etiological agent of chronic liver disease in humans. Both 5 ́and 3 ́ untranslatable regions (UTR) of the single-stranded RNA HCV genome are highly structured and include regulatory elements necessary for viral replication and translation (1). In particular, the 5 ́UTR is highly conserved among all HCV genotypes and contains an internal ribosome entry site (IRES) element responsible to drive cap-independent translation initiation (2). The ion-dependent tertiary fold of the minimal HCV IRES element (containing domains II to IV) has been investigated (3), and significant progress has been made in determining the three-dimensional structure of individual IRES domains and subdomains at high resolution (4). Nevertheless, little information is still available on the tertiary structure of the whole functional HCV IRES element. Atomic Force Microscopy (AFM) is a useful nanotechnology-based tool for the analysis of a wide range of biological entities, including nucleic acids and their complexes (5). We have optimized AFM technology for analysing HCV IRES structure in native conditions as well as for monitoring its conformational changes in diverse physicochemical environments, in particular at magnesium ion concentrations ranging from 0 to 10 mM. Here we report the magnesium-dependent folding of the HCV IRES in a sequence context that includes its structured, functionally relevant flanking regions (domains I, V and VI). In the 568 nt-long HCV genomic RNA molecule analyzed, a structural switch has been monitored when magnesium concentration increases from 2 to 4 mM. This effect has been confirmed by classical techniques for RNA structural characterization such as gel-shift analysis and partial RNase T1 cleavage. Our results suggest a magnesium-driven transition from an ‘open’ to a ‘closed’ conformation of the HCV IRES, at least partially similar to that caused by miR-122 (6). The functional relevance of such an RNA structural switch will be discussed

Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain

The aim of this study was to determine the presence of virulence genes and antibiotic resistance profiles in 164 Escherichia coli strains isolated from birds (feral pigeons, hybrid ducks, house sparrows and spotless starlings) inhabiting urban and rural environments. A total of eight atypical enteropathogenic E. coli strains were identified one in a house sparrow, four in feral pigeons and three in spotless starlings. Antibiotic resistance was present in 32.9% (54) of E. coli strains. The dominant type of resistance was to tetracycline (21.3%), ampicillin (19.5%) and sulfamethoxazole (18.9%). Five isolates had class 1 integrons containing gene cassettes encoding for dihydrofolate reductase A (dfrA) and aminoglycoside adenyltransferase A (aadA), one in a feral pigeon and four in spotless starlings. To our knowledge, the present study constitutes the first detection of virulence genes from E. coli in spotless starlings and house sparrows, and is also the first identification worldwide of integrons containing antibiotic resistance gene cassettes in E. coli strains from spotless starlings and pigeons. © 2014 © 2014 Houghton Trust Ltd

Selection and characterization of DNA aptamers for highly selective recognition of the major allergen of olive pollen Ole e 1

In this study, single-stranded DNA aptamers with binding affinity to Ole e 1, the major allergen of olive pollen, were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Binding of the aptamers was firstly established by enzyme-linked oligonucleotide assay (ELONA) and aptaprecipitation assays. Additionally, aptamer-modified monolithic capillary chromatography was used in order to evaluate the recognition of this allergenic protein against other non-target proteins. The results indicated that AptOle1#6 was the aptamer that provided the highest affinity for Ole e 1. The selected aptamer showed good selective recognition of this protein, being not able to retain other non-target proteins (HSA, cyt c, and other pollen protein such as Ole e 9). The feasibility of the affinity monolithic column was demonstrated by selective recognition of Ole e 1 in an allergy skin test. The stability and reproducibility of this monolithic column was suitable, with relative standard deviations (RSDs) in retention times and peak area values of 7.8 and 9.3%, respectively (column-to-column reproducibility). This is the first study that describes the design of an efficient DNA aptamer for this relevant allergen