Enzyme-Linked Oligonucleotide Assay (ELONA).

Aptamers are single-stranded oligonucleotides able to recognize a target with high affinity and specificity. Aptamers are used in different diagnostics applications, highlighting, among all, variations of the traditional enzyme-linked immunosorbent assay (ELISA). In this chapter, we show the procedures for the development of two types of indirect ELONA: a sandwich ELONA and a direct ELONA coupled to either real-time quantitative PCR as a direct and sensitive readout.

Business intelligence for the visualization and data analysis of Telepharmacy activity indicators in a hospital pharmacy service scorecard.

OBJECTIVE: The consolidation of Telepharmacy during the COVID-19 pandemic  has raised the need for managing large volumes of real-time activity data  through data analysis. The aim of this project was to design a dynamic, user- friendly, customizable scorecard in a hospital pharmacy service for the  visualization and analysis of Telepharmacy activity indicators through the use  of advanced business intelligence technology. METHOD: The software tool was developed by a multidisciplinary team between  April and May 2021, driven from the hospital pharmacy service. Once the Telepharmacy indicators of interest were established,  datasets were extracted from raw databases (administrative databases,  Telepharmacy database, outpatient dispensing software, drug catalogues)  through data analysis. The different data sources were integrated in a  scorecard using PowerBI®. The criteria for processing missing and duplicated  data were defined, and data pre-processing, normalization and transformation were performed. Once the pilot scorecard was validated by  different profiles of users, the structure was designed for the panels to  automatically update as databases were updated. RESULTS: Design and implementation of a scorecard of Telepharmacy activity:  general descriptive panel (demographic profile of patients, count and delivery  conditions, program and medical service); geolocation of destination;  pharmacological profile; relative analysis of patients involved in the  Telepharmacy program with respect to the total of outpatients. In the last  updating as of January 2022, data from 16,000 dispensations to more than  4,000 patients had been collected. This means that 21.93% of outpatients had  benefited at some time point from the Telepharmacy service. Filters enable the  visualization of timeline progress and patient characterization, and  measure Telepharmacy activity by program. CONCLUSIONS: The processing of large Telemedicine datasets from various  sources through Business Intelligence in a hospital pharmacy service makes it  possible to synthesize information, generate customized reports, and visualize  information in a dynamic and attractive format. The application of this new  technology will help us improve strategic clinical and management decision  making.

OBJETIVO: La consolidación de la Telefarmacia en el contexto de la pandemia por la COVID-19 exige manejar a tiempo real un gran volumen de datos de actividad mediante análisis de datos. El objetivo de este trabajo fue diseñar un cuadro de mando ágil, personalizable y dinámico para la visualización y análisis de indicadores de actividad en Telefarmacia en un servicio de farmacia de hospital, mediante el empleo de herramientas  avanzadas de inteligencia empresarial (business intelligence).Método: Un equipo de trabajo multidisciplinar desarrolló una herramienta de  software entre abril y mayo de 2021 impulsado desde el servicio de farmacia  de hospital. Una vez consensuados los indicadores de interés en Telefarmacia,  se extrajeron los datos a partir de bases de datos brutas (base de datos de  Telefarmacia, programa de dispensación de pacientes externos, bases de datos  administrativas, catálogos de fármacos) mediante análisis de datos. La  integración de las diferentes fuentes de datos en el cuadro de mando se realizó mediante PowerBI®. Se definió el manejo de los datos perdidos y duplicados y  se aplicó preprocesamiento, normalización y transformación de los datos. Una  vez  validado el piloto por diferentes tipos de usuarios, se diseñó la estructura para actualización automática de los paneles con las sucesivas actualizaciones de las fuentes de datos. RESULTADOS: Diseño e implementación de un cuadro de mando de la actividad  en Telefarmacia: panel descriptivo general (perfil demográfico de pacientes,  recuento y condiciones de envíos, programa y servicio médico); geolocalización  de destino; perfil farmacológico; análisis relativo de los  pacientes beneficiarios de Telefarmacia respecto del total de pacientes  externos. En el último corte, a enero de 2022, se habían incluido datos de  16.000 dispensaciones con entrega informada a más de 4.000 pacientes, lo  que supone que el 21,93% de los pacientes externos han estado en algún momento en el programa de Telefarmacia. La aplicación de filtros permite  visualizar la evolución temporal, caracterizar grupos de pacientes y  dimensionar la actividad por programas. CONCLUSIONES: El procesamiento de paquetes de datos de Telemedicina, de  gran volumen, difícil manejo y procedentes de diversas fuentes relativas a Telefarmacia mediante inteligencia empresarial, en un servicio de farmacia  de hospital, permite sintetizar la información y proporcionar informes  personalizados y visualizaciones dinámicas y atractivas. La aplicación de estas  nuevas tecnologías puede ayudarnos a mejorar la toma de decisiones  estratégicas, tanto clínicas como de gestión.

Inteligencia empresarial para la visualización y análisis de datos de indicadores de actividad de Telefarmacia en un cuadro de mando de un servicio de farmacia de hospital / Business intelligence for the visualization and data analysis of Telepharmacy activity indicators in a hospital pharmacy service scorecard

Objetivo: La consolidación de la Telefarmacia en el contexto de la pandemia por la COVID-19 exige manejar a tiempo real un gran volumen dedatos de actividad mediante análisis de datos. El objetivo de este trabajofue diseñar un cuadro de mando ágil, personalizable y dinámico para lavisualización y análisis de indicadores de actividad en Telefarmacia en unservicio de farmacia de hospital, mediante el empleo de herramientas avanzadas de inteligencia empresarial (business intelligence).Método: Un equipo de trabajo multidisciplinar desarrolló una herramienta de software entre abril y mayo de 2021 impulsado desde el servicio de farmacia de hospital. Una vez consensuados los indicadoresde interés en Telefarmacia, se extrajeron los datos a partir de bases dedatos brutas (base de datos de Telefarmacia, programa de dispensación de pacientes externos, bases de datos administrativas, catálogosde fármacos) mediante análisis de datos. La integración de las diferentesfuentes de datos en el cuadro de mando se realizó mediante PowerBI®.Se definió el manejo de los datos perdidos y duplicados y se aplicópreprocesamiento, normalización y transformación de los datos. Una vez validado el piloto por diferentes tipos de usuarios, se diseñó la estructurapara actualización automática de los paneles con las sucesivas actualizaciones de las fuentes de datos.Resultados: Diseño e implementación de un cuadro de mando de laactividad en Telefarmacia: panel descriptivo general (perfil demográficode pacientes, recuento y condiciones de envíos, programa y serviciomédico); geolocalización de destino; perfil farmacológico; análisis relativo de los pacientes beneficiarios de Telefarmacia respecto del totalde pacientes externos. En el último corte, a enero de 2022, se habíanincluido datos de 16.000 dispensaciones con entrega informada a másde 4.000 pacientes, lo que supone que el 21,93% de los pacientes externos han estado en algún momento en el programa de Telefarmacia. (AU)

Objective: The consolidation of Telepharmacy during the COVID-19pandemic has raised the need for managing large volumes of real-timeactivity data through data analysis. The aim of this project was to designa dynamic, user-friendly, customizable scorecard in a hospital pharmacyservice for the visualization and analysis of Telepharmacy activity indicators through the use of advanced business intelligence technology.Method: The software tool was developed by a multidisciplinary teambetween April and May 2021, driven from the hospital pharmacy service.Once the Telepharmacy indicators of interest were established, datasetswere extracted from raw databases (administrative databases, Telepharmacy database, outpatient dispensing software, drug catalogues) throughdata analysis. The different data sources were integrated in a scorecardusing PowerBI®. The criteria for processing missing and duplicated datawere defined, and data pre-processing, normalization and transformationwere performed. Once the pilot scorecard was validated by differentprofiles of users, the structure was designed for the panels to automaticallyupdate as databases were updated. Results: Design and implementation of a scorecard of Telepharmacyactivity: general descriptive panel (demographic profile of patients, countand delivery conditions, program and medical service); geolocation ofdestination; pharmacological profile; relative analysis of patients involvedin the Telepharmacy program with respect to the total of outpatients. Inthe last updating as of January 2022, data from 16,000 dispensations tomore than 4,000 patients had been collected. This means that 21.93%of outpatients had benefited at some time point from the Telepharmacyservice. (AU)

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.

Development of an antigen Enzyme-Linked AptaSorbent Assay (ELASA) for the detection of swine influenza virus in field samples.

Influenza viruses are highly variable pathogens that infect a wide range of mammalian and avian species. According to the internal conserved proteins (nucleoprotein: NP, and matrix proteins: M), these viruses are classified into type A, B, C, and D. Influenza A virus in swine is of significant importance to the industry since it is responsible for endemic infections that lead to high economic loses derived from poor weight gain, reproductive disorders, and the role it plays in Porcine Respiratory Disease Complex (PRDC). To date, swine influenza virus (SIV) diagnosis continues to be based in complex and expensive technologies such as RT-qPCR. In this study, we aimed to improve actual tools by the implementation of aptamers as capture molecules. First, three different aptamers have been selected using as target the recombinant NP of Influenza A virus expressed in insect cells. Then, these molecules have been used for the development of an Enzyme-Linked AptaSorbent Assay (ELASA) in combination with specific monoclonal antibodies for Influenza A detection. A total of 171 field samples (nasal swabs) have been evaluated with the newly developed assay obtaining a 79.7% and 98.1% sensitivity and specificity respectively, using real time RT-PCR as standard assay. These results suggest that the assay is a promising method that could be used for Influenza A detection in analysis laboratories facilitating surveillance labours.

DNA aptamers targeting Leishmania infantum H3 protein as potential diagnostic tools.

Leishmaniasis is a disease caused by a parasite of the genus Leishmania that affects millions of people worldwide. These parasites are characterized by the presence of a DNA-containing granule, the kinetoplastid, located in the single mitochondrion at the base of the cell's flagellum. Interestingly, these flagellates do not condense chromatin during mitosis, possibly due to the specific molecular features of their histones. Although histones are extremely conserved proteins, kinetoplastid core histone sequences diverge significantly from those of higher eukaryotes. This divergence makes kinetoplastid core histones potential diagnostic and/or therapeutic targets. Aptamers are short single-stranded nucleic acids that are able to recognize target molecules with high affinity and specificity. Their binding capacity is a consequence of the particular three-dimensional structure acquired depending on their sequence. These molecules are currently used for detection, diagnosis and therapeutic purpose. Starting from a previously obtained ssDNA aptamer population against rLiH3 protein we have isolated two individual aptamers, AptLiH3#4 and AptLiH3#10. Next, we have performed ELONA, Western blot and slot blot assays to establish aptamer specificity and affinity for LiH3 histone. In addition, ELONA assays using peptides corresponding to overlapped sequences of LiH3 were made to map the aptamers:LiH3 interaction. Finally, different assays using aptamers were performed in order to evaluate the possibility of using these aptamers as sensing molecule to recognize the endogenous protein LiH3. Our results indicate that both aptamers have high affinity and specificity for the target and are able to detect the endogenous LiH3 histone protein in promastigotes lysates. In silico analysis reveals that these two aptamers have different potential secondary structure among them, however, both of them are able to recognize the same peptide sequences present in the protein. In conclusion, our findings indicate that these aptamers could be used for LiH3 histone detection and, in consequence, as potential biosensing molecules in a diagnostic tool for leishmaniasis.

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.

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, Mg(2+)-induced switch between two alternative conformations: from 'open', elongated morphologies at 0-2 mM Mg(2+) concentration to a 'closed', comma-shaped conformation at 4-6 mM Mg(2+). 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 Mg(2+) 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.

End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3'X-tail region.

The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA-RNA contacts important in the establishment of viral infection. Microarray antisense oligonucleotide assays, improved dimethyl sulfate probing methods and 2' acylation chemistry (selective 2'-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3' end to be regulated by the internal ribosome entry site (IRES) element via direct RNA-RNA interactions. The essential cis-acting replicating element (CRE) and the 3'X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5' terminus. Further, the structural transition in the 3'X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3'X-tail region would therefore appear to occur. The preservation of this RNA-RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle.

The folding of the hepatitis C virus internal ribosome entry site depends on the 3'-end of the viral genome.

Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3'-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3'-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3'-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA-RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.

Structural basis for the biological relevance of the invariant apical stem in IRES-mediated translation.

RNA structure plays a fundamental role in internal initiation of translation. Picornavirus internal ribosome entry site (IRES) are long, efficient cis-acting elements that recruit the ribosome to internal mRNA sites. However, little is known about long-range constraints determining the IRES RNA structure. Here, we sought to investigate the functional and structural relevance of the invariant apical stem of a picornavirus IRES. Mutation of this apical stem revealed better performance of G:C compared with C:G base pairs, demonstrating that the secondary structure solely is not sufficient for IRES function. In turn, mutations designed to disrupt the stem abolished IRES activity. Lack of tolerance to accept genetic variability in the apical stem was supported by the presence of coupled covariations within the adjacent stem-loops. SHAPE structural analysis, gel mobility-shift and microarrays-based RNA accessibility revealed that the apical stem contributes to maintain IRES RNA structure through the generation of distant interactions between two adjacent stem-loops. Our results demonstrate that a highly interactive structure constrained by distant interactions involving invariant G:C base pairs plays a key role in maintaining the RNA conformation necessary for IRES-mediated translation.

Structural analysis provides insights into the modular organization of picornavirus IRES.

Picornavirus RNA translation is driven by the internal ribosome entry site (IRES) element. The impact of RNA structure on the foot-and-mouth disease virus (FMDV) IRES activity has been analyzed using Selective 2'Hydroxyl Acylation analyzed by Primer Extension (SHAPE) and high throughput analysis of RNA conformation by antisense oligonucleotides printed on microarrays. SHAPE reactivity revealed the self-folding capacity of domain 3 and evidenced a change of RNA structure in a defective GNRA mutant. A modified RNA conformation of this mutant was also evidenced by RNA accessibility to oligonucleotides. Interestingly, comparison of nucleotide reactivity with RNA accessibility revealed that SHAPE reactive nucleotides corresponding to the GNRA motif were not accessible to their respective target oligonucleotides. The differential response was observed both in domain 3 and the entire IRES. Our results demonstrate distant effects of the GNRA motif in the domain 3 RNA conformation, and highlight the modular organization of a picornavirus IRES.

A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination.

In eukaryotes, the production of mature messenger RNA that exits the nucleus to be translated into protein in the cytoplasm requires precise and extensive modification of the nascent transcript. Any failure that compromises the integrity of an mRNA may cause its retention in the nucleus and trigger its degradation. Multiple studies indicate that mRNAs with processing defects accumulate in nuclear foci or 'dots' located near the site of transcription, but how exactly are defective RNAs recognized and tethered is still unknown. Here, we present evidence suggesting that unprocessed ß-globin transcripts render RNA polymerase II (Pol II) incompetent for termination and that this quality control process requires the integrity of the nuclear exosome. Our results show that unprocessed pre-mRNAs remain tethered to the DNA template in association with Pol II, in an Rrp6-dependent manner. This reveals an unprecedented link between nuclear RNA surveillance, the exosome and Pol II transcriptional termination.

Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages.

Clk/STY is a LAMMER protein kinase capable to phosphorylate serine/arginine-rich (SR) proteins that modulate pre-mRNA splicing. Clk/STY alternative splicing generates transcripts encoding a full-length kinase and a truncated catalytically inactive protein. Here we showed that clk/STY, as well as other members of the family (e.g. clk2, clk3 and clk4), are up-regulated during HMBA-induced erythroleukemia cell differentiation. mRNAs coding for the full-length and the truncated forms were responsible for the overall increased expression. In clk/STY, however, a switch was observed for the ratio of the two alternative spliced products. In undifferentiated cells the full-length transcript was more abundant whereas the transcript encoding for the truncated form predominated at latter stages of differentiation. Surprisingly, overexpression of clk/STY did not alter the splicing switch upon differentiation in MEL cells. These results suggest that clk/STY might contribute to control erythroid differentiation by a mechanism that implicates a balance between these two isoforms.