Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy

Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity

G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment. © 2013 Perrone et al

Synthesis, Binding and Antiviral Properties of Potent Core-Extended Naphthalene Diimides Targeting the HIV-1 Long Terminal Repeat Promoter G-Quadruplexes

We have previously reported that stabilization of the G-quadruplex structures in the HIV-1 long terminal repeat (LTR) promoter suppresses viral transcription. Here we sought to develop new G-quadruplex ligands to be exploited as antiviral compounds by enhancing binding toward the viral G-quadruplex structures. We synthesized naphthalene diimide derivatives with a lateral expansion of the aromatic core. The new compounds were able to bind/stabilize the G-quadruplex to a high extent, and some of them displayed clear-cut selectivity toward the viral G-quadruplexes with respect to the human telomeric G-quadruplexes. This feature translated into low nanomolar anti-HIV-1 activity toward two viral strains and encouraging selectivity indexes. The selectivity depended on specific recognition of LTR loop residues; the mechanism of action was ascribed to inhibition of LTR promoter activity in cells. This is the first example of G-quadruplex ligands that show increased selectivity toward the viral G-quadruplexes and display remarkable antiviral activity

¿Es necesaria la quimio-radioterapia preoperatoria en todos los pacientes con cáncer del recto localmente avanzado? Análisis de una serie consecutiva de pacientes estadificados preoperatoriamente como T2N+ o T3

Antecedentes: actualmente la quimio-radioterapia preoperatoria es el método estándar de tratamiento de los pacientes con cáncer del recto localmente avanzado. Sin embargo, a partir de la estandarización de la técnica de la escisión total del mesorrecto, las recidivas locales se han reducido significativamente y algunos pacientes podrían evitar el sobretratamiento con quimio-radioterapia preoperatoria y su potencial efecto tóxico. El presente estudio ha sido diseñado con el objetivo de evaluar los factores predictivos de recidiva en una serie institucional de pacientes con cáncer rectal estadificado preoperatoriamente con ecografía rectal y/o resonancia magnética como cT2N+ o cT3 y tratados con escisión total del mesorrecto sin quimio-radioterapia preoperatoria. Métodos: en el periodo comprendido entre noviembre 1997 y noviembre 2008, nuestro grupo multidisciplinar estadificó preoperatoriamente 398 pacientes con cáncer del recto mediante ecografía rectal y/o resonancia magnética. En este análisis incluimos 152 pacientes consecutivos estadificados preoperatoriamente como cT2N+, cT3N0, o cT3N+, que fueron sometidos a escisión total del mesorrecto sin quimio-radioterapia preoperatoria. Se analizaron los factores potencialmente relacionados con recidiva local, supervivencia libre de enfermedad y supervivencia especifica de cáncer. Además se evaluó el margen de resección circunferencial y la calidad del mesorrecto Resultados: con una mediana de seguimiento de 39 meses, la tasa actuarial a los 5 años de recidiva local, supervivencia libre de enfermedad y supervivencia especifica de cáncer fue respectivamente 9,5%, 65,4% and 77,8% para la totalidad de la muestra. La amenaza de margen de resección circunferencial en la estadificación preoperatoria con ecografía rectal y/o resonancia magnética fue la única variable preoperatoria pronostica independiente para predecir un mayor riesgo de recidiva local (p = 0,007), menor supervivencia libre de enfermedad (p = 0,007) y menor supervivencia especifica de cáncer (p = 0,05). Así, la tasa actuarial a 5 años de RL en los pacientes con o sin margen de resección circunferencial amenazado fue respectivamente del 19,4% y 5,4%. Conclusiones: - el presente estudio indica que los pacientes con cáncer del recto clasificados preoperatoriamente como cT2N+ o cT3 con un margen de resección circunferencial > 2 mm en la estadificación preoperatoria pueden ser tratados con escisión total del mesorrecto sin quimio-radioterapia preoperatoria en ausencia de otros factores de mal pronóstico en la estadificación local. - la única variable preoperatoria predictiva de recidiva local, supervivencia libre de enfermedad y supervivencia especifica de cáncer fue la presencia de un margen circunferencial ≤ 2 mm o no evaluado según la estadificación con ecografía rectal o resonancia magnética. - La presencia de un margen circunferencial patológico afecto es la única variable pronostica postoperatoria independiente respecto a la recidiva local. - El margen circunferencial afecto y la presencia de metástasis ganglionares en el análisis anatomo-patológico fueron las únicas variables postoperatorias pronosticas independientes respecto a la supervivencia libre de enfermedad y la supervivencia especifica de cáncer. En resumen, los resultados de nuestro estudio abogan por una estrategia selectiva de neoadyuvancia en el cáncer de recto localmente avanzado intervenido con intención curativa, basada en una estadificación preoperatoria adecuada acerca del MRC. La decisión terapéutica basada en este parámetro parece más fiable que la evaluación cTN de forma aislada y lleva a replantear el criterio ampliamente aceptado en la práctica de irradiar todos los cánceres del recto clasificados preoperatoriamente como cT2N+ o cT3.Background: Preoperative chemoradiation is becoming the standard treatment for patients with locally advanced rectal cancer. However, since the introduction of total mesorectal excision (TME), local recurrence rates have been significantly reduced and some patients can be spared from potentially toxic overtreatment. This study was designed to assess factors predicting recurrence in an institutional series of rectal cancer patients with clinical stage cT2N+, cT3N0/+, treated by radical surgery without preoperative chemoradiation. Methods: Between November 1997 and November 2008, our multidisciplinary group preoperatively staged 398 rectal cancer patients by ERUS and/or MRI. This analysis included 152 consecutive patients with cT2N+, cT3N0, or cT3N+ rectal cancer, who underwent TME without preoperative chemoradiation. Macroscopical assessment of mesorectal excision and circumferential resection margin (CRM) were determined. Factors potentially related with local recurrence (LR), disease free survival (DFS) and cancer-specific survival (CSS) were analyzed. Results: After a median follow-up of 39 months, 5-year actuarial rate for LR, DFS and CSS were respectively 9.5%, 65.4% and 77.8% for the whole group. Threatened mesorectal fascia at preoperative staging was the only independent preoperative factor to predict a higher risk for LR (p=0.007), shorter DFS (p=0.007) and CSS (p=0.05). In particular, 5-year LR rate in patients with or without preoperative threatened CRM was 19.4 % and 5.4% respectively. Conclusion: Our study suggests that rectal cancer patients clinically staged as T3N0/+ or T2N+ with a free margin >2 mm from mesorectal fascia could be treated with TME alone, avoiding overtreatment with preoperative chemoradiation

UWB Path Loss Models for Ingestible Devices

[EN] Currently, some medical devices such as the Wireless Capsule Endoscopy (WCE) are used for data transmission from inside to outside the body. Nevertheless, for certain applications such as WCE, the data rates offered by current medical frequency bands can result insufficient. Ultra Wideband (UWB) frequency band has become an interesting solution for this. However, to date, there is not a formal channel path loss model for the UWB frequency band in the gastrointestinal (GI) scenario due to the huge differences between the proposed studies. There are three main methodologies to characterize the propagation channel, software simulations and experimental measurements either in phantom or in in vivo animals. Previous works do not compare all the methodologies or present some disagreements with the literature. In this paper, a dedicated study of the path loss using the three methodologies aforementioned (simulations, phantoms and in vivo measurements) and a comparison with previous researches in the literature is performed. Moreover, numerical values for a path loss model which agrees with the three methodologies and the literature are proposed. This paper aims at being the starting point for a formal path loss model in the UWB frequency band for WBANs in the GI scenarioThis work was supported in part by the European Union's H2020-MSCA-ITN Program for the "Wireless In-body Environment Communication" Project under Grant 675353, in part by the Programa de Ayudas de Investigacion y Desarrollo (PAID-01-16) from Universitat Politecnica de Valencia, and in part by the Ministerio de Economia y Competitividad, Spain under Grant TEC2014-60258-C2-1-R through the European FEDER Funds.Pérez-Simbor, S.; Andreu-Estellés, C.; Garcia-Pardo, C.; Frasson, M.; Cardona Marcet, N. (2019). UWB Path Loss Models for Ingestible Devices. IEEE Transactions on Antennas and Propagation. 67(8):5025-5034. https://doi.org/10.1109/TAP.2019.2891717S5025503467

Impact of Receivers Location on the Accuracy of Capsule Endoscope Localization

[EN] In recent years, localization for capsule endoscopy applications using Ultra-Wideband (UWB) technology has become an attractive field of study due to its potential benefits for patients. Performance analysis of RF-based localization techniques are very limited in literature. Most of the available studies rely on software simulations using digital human models. Nonetheless, no realistic studies based on in-vivo measurements has been reported yet. This paper investigates the performance of RSS-based technique for three-dimensional (3D) localization in the UWB frequency band. Impact of receivers selection as well as of the evaluated path loss model on the localization accuracy is investigated. Results obtained through CST-based simulations and from recently conducted in-vivo measurements are presented and compared.This work was supported by the European Union's H2020:MSCA:ITN program for the "Wireless In-body Environment Communication- WiBEC" project under the grant agreement no. 675353. This work was also funded by the Ministerio de Economia y Competitividad, Spain (TEC2014-60258-C2-1-R), by the European FEDER funds.Barbi, M.; Garcia-Pardo, C.; Cardona Marcet, N.; Andrea Nevárez; Vicente Pons Beltrán; Frasson, M. (2018). Impact of Receivers Location on the Accuracy of Capsule Endoscope Localization. IEEE. 340-344. https://doi.org/10.1109/PIMRC.2018.8580862S34034

A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription

I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated. Here, we report the characterization of i-motifs within the long terminal repeat (LTR) promoter of the HIV-1 proviral genome. Biophysical and biochemical analysis revealed formation of a predominant i-motif with an unprecedented loop composition. One-dimensional nuclear magnetic resonance investigation demonstrated formation of three G-C H-bonds in the long loop, which likely improve the structure overall stability. Pull-down experiments combined with mass spectrometry and protein crosslinking analysis showed that the LTR i-motif is recognized by the cellular protein hnRNP K, which induced folding at physiological conditions. In addition, hnRNP K silencing resulted in an increased LTR promoter activity, confirming the ability of the protein to stabilize the i-motif-forming sequence, which in turn regulates the LTR-mediated HIV-1 transcription. These findings provide new insights into the complexity of the HIV-1 virus and lay the basis for innovative antiviral drug design, based on the possibility to selectively recognize and target the HIV-1 LTR i-motif

Dielectric Characterization of In Vivo Abdominal and Thoracic Tissues in the 0.5 26.5 GHz Frequency Band for Wireless Body Area Networks

(c) 2019 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other users, including reprinting/ republishing this material for advertising or promotional purposes, creating new collective works for resale or redistribution to servers or lists, or reuse of any copyrighted components of this work in other works.[EN] The dielectric properties of biological tissues are of utmost importance in the development of wireless body area networks (WBANs), especially for implanted devices. The early design stages of medical devices like capsule endoscopy, pacemakers, or physiological sensors rely on precise knowledge of the dielectric properties of the tissues present in their surrounding medium. Many of these applications make use of electromagnetic phantoms, which are software or physical models that imitate the shape and the electromagnetic properties of the tissues. They are used for designing devices in software simulations and for testing them in laboratory trials, aiding in both the development of WBAN antennas or in communication link evaluations. The existing reports about dielectric in vivo properties are limited and have drawbacks like: low variety of characterized tissues, lacking some relevant ones, and limitations and inhomogeneity in the measured frequency range. This paper aims at filling that gap by providing a new database of dielectric properties of biological tissues measured in vivo . In particular, it is focused on the tissues of the thoracic and the abdominal regions, measured at the same wide frequency band, on the same animal specimen, and under the same conditions. The properties have been obtained by measuring porcine tissues in the 0.5¿26.5 GHz band with the open-ended coaxial technique. In this paper, we focus on those tissues that have been scarcely characterized so far in the literature, like heart, esophagus, stomach, and pancreas. The Cole¿Cole fitting parameters of the measured tissues and their uncertainties are provided.This work was supported in part by UPV-IIS LaFe Program (STuDER, 2016, and EMOTE, 2018), in part by the Programa de Ayudas de Investigacion y Desarrollo (PAID-01-16) from the Universitat Politecnica de Valencia, in part by the European Union's H2020: MSCA: ITN Programs for the "Wireless In-Body Environment Communication-WiBEC'' Project, under Grant 675353, and in part by the "mmWave Communications in the Built Environments-WaveComBE'' Project, under Grant 766231.Fornés Leal, A.; Cardona Marcet, N.; Frasson, M.; Castelló-Palacios, S.; Nevárez, A.; Pons Beltrán, V.; Garcia-Pardo, C. (2019). Dielectric Characterization of In Vivo Abdominal and Thoracic Tissues in the 0.5 26.5 GHz Frequency Band for Wireless Body Area Networks. IEEE Access. 7:31854-31864. https://doi.org/10.1109/ACCESS.2019.2903481S3185431864

Liver Gene Therapy: Employing Surgery and Radiology for Translational Research

Gene therapy is a therapeutic strategy that aims to employ nucleic acids as drugs for the transient or permanent treatment of inherited or acquired pathologies. Based on the type of vector employed for the gene transfer, gene therapy can be classified as viral gene therapy and nonviral gene therapy. Nonviral gene therapy is less efficient but safer than viral gene therapy. Hydrodynamic naked DNA transfer has shown great translational potential, achieving therapeutic levels of a human protein in the murine model. The translational process of the procedure has already been performed. Different radiologic and surgical approaches permitted pressurizing the liver in vivo by excluding its vascularization partially or totally. These approaches mediated a tissue rate of human alpha-1-antitrypsin protein translation (100–1000 copies per cell) close to those obtained with the mouse gold standard model in a safe mode that could be translated to human settings