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

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

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

¿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

Outcomes of emergency surgical interventions in right-sided colonic cancer:nationwide population-based study based on Danish Colorectal Cancer Group register

Aim: The aim of this study was to investigate the trends in morbidity and mortality of patients with right-sided colonic cancer who had an emergency surgical procedure in Denmark after the introduction of quality index parameters.Methods: This was a retrospective nationwide study based on a prospectively maintained Danish Colorectal Cancer Group database focused on right-sided colonic cancer in the interval from 1 May 2001 to 30 April 2018, who underwent emergency surgical intervention (within 48 h of hospital admission). The primary objective was to investigate the trends in morbidity and mortality throughout the study years. Multivariable estimates were adjusted for age, sex, smoking status, alcohol consumption, ASA score classification, tumour localization, type of access to abdominal cavity, surgeon's grade of specialization, and metastatic disease.Results: Out of 2839 patients, a total of 2740 patients fulfilled the inclusion criteria, of whom 2464 underwent right or transverse colon resection (89.9 per cent). The 30-day and 90-day postoperative mortality rates were significantly reduced over the time of the study (OR 0.943, 95 per cent c.i. 0.922 to 0.965, P &lt; 0.001 and OR 0.953, 95 per cent c.i. 0.934 to 0.972, P &lt; 0.001 respectively); however, the complication rates did not follow this trend. Older patients (OR 1.032, 95 per cent c.i. 1.009 to 1.055, P = 0.005) and patients with high ASA scores (OR 1.61, 95 per cent c.i. 1.422 to 1.830, P &lt; 0.001) had higher rates of severe grade 3b postoperative complications. A stoma was constructed in 276 patients (10 per cent), whereas a stent was used in only eight patients. Defunctioning procedures, including stoma construction or colonic stenting (without oncological resection), did not reduce the risk of complications compared with that of definitive surgery.Conclusion: The 30-day and 90-day postoperative mortality rates were significantly reduced over the time of the study. Age and ASA score were risk factors for severe postoperative complications.Aim: The aim of this study was to investigate the trends in morbidity and mortality of patients with right-sided colonic cancer who had an emergency surgical procedure in Denmark after the introduction of quality index parameters. Methods: This was a retrospective nationwide study based on a prospectively maintained Danish Colorectal Cancer Group database focused on right-sided colonic cancer in the interval from 1 May 2001 to 30 April 2018, who underwent emergency surgical intervention (within 48 h of hospital admission). The primary objective was to investigate the trends in morbidity and mortality throughout the study years. Multivariable estimates were adjusted for age, sex, smoking status, alcohol consumption, ASA score classification, tumour localization, type of access to abdominal cavity, surgeon's grade of specialization, and metastatic disease. Results: Out of 2839 patients, a total of 2740 patients fulfilled the inclusion criteria, of whom 2464 underwent right or transverse colon resection (89.9 per cent). The 30-day and 90-day postoperative mortality rates were significantly reduced over the time of the study (OR 0.943, 95 per cent c.i. 0.922 to 0.965, P &lt; 0.001 and OR 0.953, 95 per cent c.i. 0.934 to 0.972, P &lt; 0.001 respectively); however, the complication rates did not follow this trend. Older patients (OR 1.032, 95 per cent c.i. 1.009 to 1.055, P = 0.005) and patients with high ASA scores (OR 1.61, 95 per cent c.i. 1.422 to 1.830, P &lt; 0.001) had higher rates of severe grade 3b postoperative complications. A stoma was constructed in 276 patients (10 per cent), whereas a stent was used in only eight patients. Defunctioning procedures, including stoma construction or colonic stenting (without oncological resection), did not reduce the risk of complications compared with that of definitive surgery. Conclusion: The 30-day and 90-day postoperative mortality rates were significantly reduced over the time of the study. Age and ASA score were risk factors for severe postoperative complications.</p

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

Naphthalene Diimide-Tetraazacycloalkane Conjugates Are G-Quadruplex-Based HIV-1 Inhibitors with a Dual Mode of Action

Human immunodeficiency virus 1 (HIV-1) therapeutic regimens consist of three or more drugs targeting different steps of the viral life cycle to limit the emergence of viral resistance. In line with the multitargeting strategy, here we conjugated a naphthalene diimide (NDI) moiety with a tetraazacycloalkane to obtain novel naphthalene diimide (NDI)-tetraazacycloalkane conjugates. The NDI inhibits the HIV-1 promoter activity by binding to LTR G-quadruplexes, and the tetraazacycloalkane mimics AMD3100, which blocks HIV entry into cells by interfering with the CXCR4 coreceptor. We synthesized, purified, and tested the metal-free NDI-tetraazacycloalkane conjugate and the two derived metal-organic complexes (MOCs) that incorporate Cu2+ and Zn2+. The NDI-MOCs showed enhanced binding to LTR G4s as assessed by FRET and CD assays in vitro. They also showed enhanced activity in cells where they dose-dependently reduced LTR promoter activity and inhibited viral entry only of the HIV-1 strain that exploited the CXCR4 coreceptor. The time of addition assay confirmed the dual targeting at the different HIV-1 steps. Our results indicate that the NDI-MOC conjugates can simultaneously inhibit viral entry, by targeting the CXCR4 coreceptor, and LTR promoter activity, by stabilizing the LTR G-quadruplexes. The approach of combining multiple targets in a single compound may streamline treatment regimens and improve the overall patient outcomes

Assessing the Value of Explainable Artificial Intelligence for Magnetic Resonance Imaging

Recent advancements in Artificial Intelligence (AI) have often improved the accuracy of medical diagnostics in several fields, such as cancer detection and diagnosing cardiovascular or neuromuscular diseases. However, the opaque nature of AI decision-making can limit its adoption in the clinical setting, as physicians require clear and interpretable explanations to trust these tools. To address this issue, the field of eXplainable Artificial Intelligence (XAI) aims to clarify the rationale behind AI predictions while ensuring compliance with ethical standards and advanced regulations such as the GDPR and the AI Act. This study applies multiple explainability methods to a diagnostic support model for Distal Myopathies (DMs), a rare neuromuscular disorder marked by subtle, early-stage tissue alterations. Beyond classification, our approach generates detailed explanations for the model’s predictions. We propose novel techniques, including a hierarchical occlusion method and an ensemble framework that combines individual explanations to produce refined, interpretable visualizations. Feedback from expert radiologists is used to assess the effectiveness of these methods, highlighting their potential to enhance trust and usability in clinical practice. Our results show that pretrained convolutional networks achieve high classification accuracy, exceeding 88%, with perfect recall in identifying affected cases, while underscoring the need for adaptive and user-centric approaches to explainability in AI-driven diagnostic tools