Targeting of G-quadruplex DNA with 99mTc(I)/Re(I) Tricarbonyl Complexes Carrying Pyridostatin Derivatives.

The main goal of this work was to elucidate the potential relevance of (radio)metal chelates of 99mTc and Re targeting G-quadruplex structures for the design of new tools for cancer theranostics. 99mTc provides the complexes with the ability to perform single-photon-emission computed tomography imaging studies, while the Re complexes should act as anticancer agents upon interaction with specific G4 DNA or RNA structures present in tumor tissues. Towards this goal, we have developed isostructural 99mTc(I) and Re(I) tricarbonyl complexes anchored by a pyrazolyl-diamine (Pz) chelator carrying a pendant pyridostatin (PDS) fragment as the G4-binding motif. The interaction of the PDF-Pz-Re (8) complex with different G4-forming oligonucleotides was studied by circular dichroism, fluorescence spectroscopy and FRET-melting assays. The results showed that the Re complex retained the ability to bind and stabilize G4-structures from different DNA or RNA sequences, namely those present on the SRC proto-oncogene and telomeric RNA (TERRA sequence). PDF-Pz-Re (8) showed low to moderate cytotoxicity in PC3 and MCF-7 cancer cell lines, as typically observed for G4-binders. Biodistribution studies of the congener PDF-Pz-99mTc (12) in normal mice showed that the complex undergoes a fast blood clearance with a predominant hepatobiliary excretion, pointing also for a high in vitro stability.

Intraoperative indocyanine green angiography in preventing hypoparathyroidism after thyroid cancer surgery.

BACKGROUND: The aim of this study was to determine whether the use of indocyanine green angiography to identify devascularized parathyroids during thyroidectomy for thyroid cancer would decrease the rates of postoperative hypoparathyroidism. METHODS: Retrospective study of patients who had undergone total thyroidectomy for treatment of thyroid cancer between March 2021 and March 2023. The indocyanine group included patients with all four parathyroids identified and evaluated by indocyanine green angiography at the end of the procedure. Those with parathyroid glands classified with no vascularization had the glands autotransplanted. A group without indocyanine angiography was used to compare results. RESULTS: The analysis included 100 patients in each group. Indocyanine angiography identified 14.75% of devascularized parathyroids at surgery. The number of parathyroids with a score of 2 (i.e., good vascularization) was not a safe predictor of normal parathyroid hormone levels after surgery. Indeed, 29.2% of the patients with three parathyroids with a score of 2 developed transient hypoparathyroidism. Permanent hypoparathyroidism occurred in 7% of the patients without indocyanine group and in none of the patients in the indocyanine group (p = 0.014). CONCLUSION: Intraoperative angiography with indocyanine green could contribute to reduce the occurrence of permanent hypoparathyroidism in patients undergoing surgical treatment for thyroid cancer.

Torcular pseudomass is frequently detected on fetal magnetic resonance imaging and reduces with gestational age.

BACKGROUND: The torcular pseudomass is an incidental extra-axial midline mass located between the venous sinuses and the occipital squama in the pediatric population. Although this structure is presumed to be a developmental feature, it has not been characterized on fetal MRI. OBJECTIVE: To determine the frequency, imaging features and longitudinal in utero evolution of torcular pseudomass using fetal MRI. MATERIALS AND METHODS: We present a single-center retrospective study of fetal MRI performed at a tertiary hospital. Two independent reviewers first ordinally scored torcular pseudomass as absent, focal, crescentic or bulky based on morphology. We reviewed available follow-up fetal and postnatal MRI and further classified torcular pseudomass as stable, involuted or progressive. We also collected clinical and demographic data from electronic charts and compared them among categories, corrected for multiple comparisons. RESULTS: This study included a total of 219 fetuses with median gestational age of 28 weeks (interquartile range [IQR]: 23-32 weeks). Torcular pseudomass was absent in 8% (n=17) and present as a focal mass in 15% (n=33), crescentic in 45% (n=98) and bulky in 32% (n=71) of the cases. Median gestational age was statistically different among torcular pseudomass categories and inversely associated with size. Follow-up fetal MRI was available in 9.6% (n=21) of cases (median interval 4 weeks; IQR: 2-9 weeks) and torcular pseudomass in these cases was classified as stable in 67% (n=14), involuted in 29% (n=6) and progressive in 5% (n=1). Postnatal MRI was available in 5% (n=12) of fetuses (median interval 11.5 months, IQR: 3-17 months), and among these cases torcular pseudomass was classified as stable in 33% (n=4) and involuted in 67% (n=8). CONCLUSION: Torcular pseudomass is highly prevalent in the fetal population and shows a natural tendency to involute, even in utero, although it sometimes persists during early infanthood.

G-Quadruplex Aptamer-Ligand Characterization.

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.

Ligands as Stabilizers of G-Quadruplexes in Non-Coding RNAs.

The non-coding RNAs (ncRNA) are RNA transcripts with different sizes, structures and biological functions that do not encode functional proteins. RNA G-quadruplexes (rG4s) have been found in small and long ncRNAs. The existence of an equilibrium between rG4 and stem-loop structures in ncRNAs and its effect on biological processes remains unexplored. For example, deviation from the stem-loop leads to deregulated mature miRNA levels, demonstrating that miRNA biogenesis can be modulated by ions or small molecules. In light of this, we report several examples of rG4s in certain types of ncRNAs, and the implications of G4 stabilization using small molecules, also known as G4 ligands, in the regulation of gene expression, miRNA biogenesis, and miRNA-mRNA interactions. Until now, different G4 ligands scaffolds were synthesized for these targets. The regulatory role of the above-mentioned rG4s in ncRNAs can be used as novel therapeutic approaches for adjusting miRNA levels.

Disruption of amyloid precursor protein ubiquitination selectively increases amyloid ß (Aß) 40 levels via presenilin 2-mediated cleavage.

Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid ß (Aß) peptide, derived from cleavage of amyloid precursor protein (APP) by ß- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aß40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aß40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aß40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane.

ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

Masticatory evaluation of anterior open bite malocclusion using the colorimetric capsule method.

This pediatric study aimed to assess the masticatory function of patients with anterior open bite through a method that utilizes colorimetric capsules. The sample consisted of 106 patients aged 7-11 years, of whom 51 presented with anterior open bite (experimental group) and 55 presented with normal overbite (control group). The colorimetric capsules used in this evaluation contained basic fuchsin granules, which were used to produce a solution after they were ground during chewing. Each patient was asked to chew 1 capsule for 20 seconds. The absorbance of the obtained solutions was analyzed with a spectrophotometer. Greater masticatory efficiency is directly related to the greater light absorbance of a solution, since light absorbance increases with solution concentration. A nonparametric Wilcoxon test indicated that the solutions obtained from the control group presented significantly greater light absorbance than did those from the experimental group (P < 0.05). Therefore, children with an anterior open bite exhibited less masticatory efficiency than those with a normal overbite.

Lipids under stress--a lipidomic approach for the study of mood disorders.

The emerging field of lipidomics has identified lipids as key players in disease physiology. Their physicochemical diversity allows precise control of cell structure and signaling events through modulation of membrane properties and trafficking of proteins. As such, lipids are important regulators of brain function and have been implicated in neurodegenerative and mood disorders. Importantly, environmental chronic stress has been associated with anxiety and depression and its exposure in rodents has been extensively used as a model to study these diseases. With the accessibility to modern mass-spectrometry lipidomic platforms, it is now possible to snapshot the extensively interconnected lipid network. Here, we review the fundamentals of lipid biology and outline a framework for the interpretation of lipidomic studies as a new approach to study brain pathophysiology. Thus, lipid profiling provides an exciting avenue for the identification of disease signatures with important implications for diagnosis and treatment of mood disorders.

G-quadruplex forming motifs in the promoter region of the B-MYB proto-oncogene.

To combat cancer, a comprehensive understanding of the molecular mechanisms and behaviors involved in carcinogenesis is crucial, as tumorigenesis is a complex process influenced by various genetic events and disease hallmarks. The B-MYB gene encodes a transcription factor involved in cell cycle regulation, survival, and differentiation in normal cells. B-MYB can be transformed into an oncogene through mutations, and abnormal expression of B-MYB has been identified in various cancers, including lung cancer, and is associated with poor prognosis. Targeting this oncogene is a promising approach for anti-cancer drug design. B-MYB has been deemed undruggable in previous reports, necessitating the search for novel therapeutic options. In this study, we found that the B-MYB gene promoter contains several G/C rich motifs compatible with G-quadruplex (G4) formation. We investigated and validated the existence of G4 structures in the promoter region of B-MYB, first in vitro using a combination of bioinformatics, biophysical, and biochemical methods, then in cell with the recently developed G4access method.

Targeting proto-oncogene B-MYB G-quadruplex with a nucleic acid-based fluorescent probe.

The B-MYB gene encodes a transcription factor (B-MYB) that regulates cell growth and survival. Abnormal expression of B-MYB is frequently observed in lung cancer and poses challenges for targeted drug therapy. Oncogenes often contain DNA structures called G-quadruplexes (G4s) in their promoter regions, and B-MYB is no exception. These G4s play roles in genetic regulation and are potential cancer treatment targets. In this study, a probe was designed to specifically identify a G4 within the promoter region of the B-MYB gene. This probe combines an acridine derivative ligand with a DNA segment complementary to the target sequence, enabling it to hybridize with the adjacent sequence of the G4 being investigated. Biophysical studies demonstrated that the acridine derivative ligands C5NH2 and C8NH2 not only effectively stabilized the G4 structure but also exhibited moderate affinity. They were capable of altering the G4 topology and exhibited enhanced fluorescence emission in the presence of this quadruplex. Additionally, these ligands increased the number of G4s observed in cellular studies. Through various biophysical studies, the target sequence was shown to form a G4 structure, even with an extra nucleotide tail added to its flanking region. Cellular studies confirmed the co-localization between the target sequence and the developed probe.

Urology Residency Applicant Selection: Program Directors' New Criteria.

OBJECTIVE: To evaluate the impact of the recent changes to the urology residency application process on the criteria utilized by residency program directors (PDs) for interview invitations and their perspectives concerning these changes. METHODS: One hundred thirty-seven urology residency PDs were invited to participate in an anonymous survey to explore interview selection criteria and the impact of the increase in preference signals (PS) per applicant. RESULTS: Fifty-eight PDs (42.8%) completed the survey. The highest-ranked criteria were letters of recommendation (LoR) and successful sub-internship (sub-I) at the PD's institution, without statistically significant differences between these 2. Gender, ethnicity, and medical school prestige were the lowest rated criteria, without significant differences between these 3. Compared to before the increase in the number of PS per applicant, 80.7% of PDs reported that not receiving a PS from an applicant this cycle would more negatively impact the chances of offering an interview to that applicant. Moreover, 12.2% stated they would not interview any applicants who did not send a PS. Finally, 62.1% of PDs believed recent changes worsened the process. CONCLUSION: Recent changes impacted PDs applicant evaluation, with the highest ranked criteria being LoRs and sub-I. Paradoxically, the increase in the number of PS per applicant has increased their importance as applicants are much less likely to receive interview offers from programs they have not signaled. Lastly, most PDs believe changes have worsened the evaluation process.

Maternal pre- and postpartum supplementation of a Bacillus-based DFM enhanced cow and calf performance.

This study evaluated the effects of maternal supplementation of a Bacillus-based direct-fed microbial (DFM) on the physiology and growth performance of Bos indicus-influenced cow-calf pairs. On day 0 (~139 d before expected calving date), 72 fall-calving, Brangus crossbred beef heifers (20 to 22 mo of age) pregnant with first offspring were stratified by their initial body weight (BW; 431 ±â€…31 kg) and body condition score (BCS; 6.0 ±â€…0.36; scale 1 to 9), and randomly allocated into 1 of 12 bahiagrass pastures (1 ha and six heifers per pasture). Treatments were randomly assigned to pastures (six pastures per treatment) and consisted of heifers supplemented with 1 kg/d of soybean hulls (dry matter, DM) that was added (BAC) or not (CON) with DFM containing Bacillus subtilis and B. licheniformis (Bovacillus; Chr. Hansen A/S, Hørsholm, Denmark). Treatments were provided from days 0 to 242 (139 ±â€…4 d prepartum to 104 ±â€…4 d postpartum). Calves were weaned on day 242 (96 ±â€…30 d of age) and then allocated into 1 of 16 drylot pens and fed the same concentrate at 3.25% of BW (DM) until day 319. Maternal treatment effects were not detected (P ≥ 0.29) for herbage allowance and forage chemical composition. Heifer BCS on days 39 and 63 tended (P ≤ 0.09) to be greater for BAC vs. CON heifers, whereas heifer BCS on day 91 was greater (P = 0.01) for BAC vs. CON heifers. Heifer BCS did not differ (P ≥ 0.20) between treatments on days 179 and 242. Plasma glucose concentration did not differ from days 0 to 63 (P ≥ 0.14) but were greater (P < 0.01) on day 179 and tended (P = 0.09) to be greater on day 242 for BAC vs. CON heifers. Calf BW at birth, ADG from birth to weaning, and BW at weaning did not differ (P ≥ 0.19) between treatments, but calf BW at drylot exit (day 319) was greater (P = 0.05) for BAC vs. CON calves. Maternal treatment effects were not detected (P ≥ 0.42) for calf serum concentration of IgG at birth and postvaccination plasma concentrations of glucose, cortisol, and haptoglobin. Serum titers against bovine respiratory syncytial virus (BRSV) were greater (P = 0.04) for BAC vs. CON calves on day 287, whereas seroconversion against parainfluenza-3 virus (PI-3) was greater (P < 0.01) for BAC vs. CON calves on day 271. Thus, maternal supplementation of a Bacillus-based DFM increased prepartum BCS gain and postpartum plasma glucose concentration of heifers and led to positive carryover effects on postweaning BW gain and humoral immune response in their offspring.

Direct-fed microbials (DFM), such as Bacillus spp., have been shown to produce a wide variety of enzymes related to nutrient digestion and to support gastrointestinal tract immune function and integrity, leading to increased nutrient digestibility and cattle performance. Nutritional management of beef cows during gestation and early lactation has been associated with enhanced future offspring growth performance and immune response following birth. The present study combined the use of Bacillus-based DFM for pregnant heifers during critical production stages (late gestation and early lactation) to promote the overall performance of heifers and their offspring. Heifers offered Bacillus-based DFM had greater body condition score at calving and postpartum plasma concentration of glucose, whereas their offspring had similar body weight at birth, but greater growth performance when fed relatively high amounts of protein and energy in drylot compared to cohorts born from heifers that did not receive Bacillus-based DFM supplementation.

Promoting Digital Proficiency and Health Literacy in Middle-aged and Older Adults Through Mobile Devices With the Workshops for Online Technological Inclusion (OITO) Project: Experimental Study.

BACKGROUND: Digital inclusion and literacy facilitate access to health information and can contribute to self-care behaviors and informed decision-making. However, digital literacy is not an innate skill, but rather requires knowledge acquisition. OBJECTIVE: The present study aimed to develop, conduct, and measure the impact, on digital and health literacy, of a digital inclusion program aimed at community dwellers. METHODS: The program targeted the recruitment of people aged 55 and older that owned mobile devices with an internet connection in 3 cities in northern Portugal (Paredes de Coura, Guimarães, and Barcelos). The program was titled the Workshops for Online Technological Inclusion (OITO) project and, in each city, was promoted by the coordinator of municipal projects and organized as an in-person 8-workshop program, using mobile devices, smartphones, or tablets. A quasi-experimental design was used with a nonrandomized allocation of participants in each set of 8 workshops. Sociodemographic, health status, and mobile use information were collected at baseline. Digital and health literacy were measured via the Mobile Device Proficiency Questionnaire and the Health Literacy Scale questionnaires, respectively, at baseline (T1), program completion (T2), and a 1-month follow-up (T3). A self-reported measure of autonomy was evaluated at T1 and T2 using a visual scale. RESULTS: Most participants were women with primary schooling (up to 4 years) aged between 65 and 74 years and retired. The intervention had an 81% (97/120) recruitment rate, 53% (43/81) adherence, and 94% (67/71) satisfaction rate, with 81 participants completing the entire 8-workshop program. Most participants had owned their mobile device for more than one year (64/81, 79%), were frequent daily users (70/81, 86%), and had received their mobile device from someone else (33/64, 52%). Over 80% (71/81) of the participants who completed the intervention used Android smartphones. At baseline, participants had low baseline scores in digital literacy, but medium-high baseline scores in health literacy. They showed significant improvement in digital literacy at T2 and T3 compared to T1, but without a significant difference between T2 and T3, regardless of sex, age, or schooling. A significant improvement in self-reported autonomy was observed at T3 compared with baseline. Regarding health literacy, no significant differences were found at T2 or T3 compared to the baseline. CONCLUSIONS: The feasibility indicators showed that the OITO project methodology had a substantial rate of recruitment and satisfaction. Program participants had significant improvement in digital literacy after 8 workshops and maintained their score 1 month after completing the intervention. There was no significant change in health literacy during the project period.

Iodine Availability through Iodized Salt in Portugal: 2010-2021 Sales Evolution and Distribution.

Salt iodization programs are considered the most cost-effective measures to ensure adequate iodine intake in iodine-deficient populations. Portuguese women of childbearing age and pregnant women were reported to be iodine-deficient, which led the health authorities, in 2013, to issue a recommendation for iodine supplementation during preconception, pregnancy and lactation. In the same year, iodized salt became mandatory in school canteens. Of note, no regulation or specific programs targeting the general population, or the impact of iodized salt availability in retailers, are known. The present study analyzed iodized salt supermarket sales from 2010 to 2021 from a major retailer, identifying the proportion of iodized salt in total salt sales and its distribution in mainland Portugal. Data on iodine content were collected through the nutritional label information. Of a total of 33 salt products identified, 3 were iodized (9%). From 2010 to 2021, the weighted sales of iodized salt presented a growing tendency, reaching the maximum of 10.9% of total sales (coarse plus fine salt) in 2021. Iodized salt reached a maximum of 11.6% of total coarse salt in 2021, a maximum of 2.4% of the total fine salt in 2018. The overall sales of iodized salt and their contribution to iodine intake are extremely low, prompting additional studies to understand the consumer's choice and awareness of the benefits of iodized salt.

Neuroimaging of pediatric tumors of the sellar region-A review in light of the 2021 WHO classification of tumors of the central nervous system.

Sellar/suprasellar tumors comprise about 10% of all pediatric Central Nervous System (CNS) tumors and include a wide variety of entities, with different cellular origins and distinctive histological and radiological findings, demanding customized neuroimaging protocols for appropriate diagnosis and management. The 5th edition of the World Health Organization (WHO) classification of CNS tumors unprecedently incorporated both histologic and molecular alterations into a common diagnostic framework, with a great impact in tumor classification and grading. Based on the current understanding of the clinical, molecular, and morphological features of CNS neoplasms, there have been additions of new tumor types and modifications of existing ones in the latest WHO tumor classification. In the specific case of sellar/suprasellar tumors, changes include for example separation of adamantinomatous and papillary craniopharyngiomas, now classified as distinct tumor types. Nevertheless, although the current molecular landscape is the fundamental driving force to the new WHO CNS tumor classification, the imaging profile of sellar/suprasellar tumors remains largely unexplored, particularly in the pediatric population. In this review, we aim to provide an essential pathological update to better understand the way sellar/suprasellar tumors are currently classified, with a focus on the pediatric population. Furthermore, we intend to present the neuroimaging features that may assist in the differential diagnosis, surgical planning, adjuvant/neoadjuvant therapy, and follow-up of this group of tumors in children.

Meniscus Sign in Patients with Anterior Circulation Large Vessel Occlusion Stroke does not Predict Outcome.

PURPOSE: The angiographic appearance of the occlusion site was suggested to influence outcomes of stroke patients with large vessel occlusion (LVO) who undergo endovascular treatment (EVT). We aimed to study the impact of the meniscus sign (MS) on outcomes of stroke patients with anterior circulation LVO. METHODS: Based on two prospective registries of acute ischemic stroke, we selected patients with carotid­T, M1 or M2 occlusion who underwent EVT. Clinical characteristics and outcomes were collected from the registries or from individual records. Two independent observers blinded to outcomes assessed the presence of MS in digital subtraction angiography before thrombectomy. Angiographic and clinical outcomes of patients with and without MS were compared. RESULTS: We included 903 patients, with median age of 78 years, 59.8% were male, median baseline NIHSS was 14 and 39.5% received intravenous thrombolysis. Patients with MS (n = 170, 18.8%) were more frequently female, presented with higher NIHSS scores and more frequently underwent intravenous thrombolysis. Presence of MS was significantly associated with cardioembolic etiology. Successful reperfusion, number of passes, first pass effect, procedural time, symptomatic intracerebral hemorrhage, in-hospital mortality and favorable 3­month functional outcome were similar in the groups of patients with and without MS. In the multivariable analyses, MS was not associated with successful reperfusion (odds ratio, OR = 1.08, 95% confidence interval, CI = 0.76-1.55), first pass effect (OR = 0.96, 95%CI = 0.48-1.92) or favorable 3­month outcome (OR = 1.40, 95%CI = 0.88-2.24). CONCLUSION: The presence of MS in acute ischemic stroke patients with anterior circulation large vessel occlusion who undergo EVT does not appear to influence angiographic or clinical outcomes.

Targeting a G-quadruplex from let-7e pre-miRNA with small molecules and nucleolin.

Let-7e precursor microRNA has the potential to adopt a G-quadruplex (rG4) structure and recently, its roles in oncology have been the focus of much attention, as it is now known that let-7e pre-miRNA is frequently dysregulated in cancers. Therefore, it is crucial to unveil and fully characterize its ability to adopt a rG4 structure, which could be stabilized or destabilized by small molecules and proteins such as nucleolin, a protein that is deeply associated with miRNA biogenesis. Herein, by combining a set of different methods such as circular dichroism (CD), nuclear magnetic resonance (NMR), UV spectroscopy (thermal difference spectra (TDS) and isothermal difference spectra (IDS)) and polyacrylamide gel electrophoresis (PAGE), we demonstrate the formation of the rG4 structure found in let-7e pre-miRNA sequence in the presence of K+ (5'-GGGCUGAGGUAGGAGG-3'). The ability of eight small molecules (or ligands) to bind to and stabilize this rG4 structure was also fully assessed. The dissociation constants for each RNA G-quadruplex/ligand complex, determined by surface plasmon resonance (SPR), ranged in the 10-6 to 10-9 M range. Lastly, the binding of the rG4 structure to nucleolin in the presence and absence of ligands was evaluated via CD, SPR, PAGE and confocal microscopy. The small molecules 360 A and PDS demonstrated attractive properties to targetthe rG4 structure of let-7e pre-miRNA and control its biology. Our findings also highlighted that the interaction of TMPyP4 with the G-quadruplex of let-7e precursor miRNA could block the formation of the complex between the rG4 and nucleolin. Overall, this study introduces an approach to target the rG4 found in let-7e pre-miRNA which opens up a new opportunity to control the microRNA biogenesis.

Stabilization of a DNA aptamer by ligand binding.

G-rich aptamers such as AS1411 are small oligonucleotides that present several benefits comparatively to monoclonal antibodies, since they are easier to manufacture and store, have small size and do not stimulate an immune response. We analyzed AT11-B1, a modified sequence of AT11 (itself a modified version of AS1411), in which one thymine was removed from the bulge region. We studied G-quadruplex (G4) formation/stabilization using PhenDC3, PDS, BRACO-19, TMPyP4 and 360A ligands by different biophysical techniques, namely circular dichroism (CD), Förster resonance energy transfer (FRET-melting) and nuclear magnetic resonance (NMR). The CD spectra showed that AT11-B1 adopts a predominant G4 of parallel topology when the buffer contains KCl or when ligands are added. PhenDC3 induced a ΔTm of 30 °C or more of the G4 structure as shown by CD- and FRET-melting experiments. The ligands demonstrate high affinity for AT11-B1 G4 and the NMR studies revealed that the AT11-B1 G4 involves four G-tetrad layers. The in silico studies suggest that all ligands bind AT11-B1 G4, namely, by stacking interactions, with the possible exception of PDS that may bind to the loop/groove interface. In addition, molecular dynamics simulations revealed that nucleolin (NCL) interacts with the AT11-B1 G4 structure through the RNA binding domain (RBD) 2 and the 12-residue linker between RBD1,2. Moreover, AT11-B1 G4 was internalized into a NCL-positive tongue squamous cell carcinoma cell line. In a nutshell, this study may help the identification of the ligands scaffolds to bind and stabilize AT11-B1, improving the targeting towards NCL that is overexpressed in cancer cells.