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Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Observing individual RNA molecules provides valuable insights into their regulation, interactions with other cellular components, organization, and functions. Although fluorescent light-up aptamers (FLAPs) have recently shown promise for RNA imaging, their wider applications have been mostly hindered by poor brightness and photostability. We recently developed an avidity-based FLAP known as biRhoBAST that allows for single-molecule RNA imaging in live or fixed cells and tracking individual mRNA molecules in living cells due to its excellent photostability and high brightness. Here, we present step-by-step detailed protocols starting from cloning biRhoBAST repeats into the target RNA sequence, to imaging dynamics of single mRNA molecules. Additionally, we address the validation of single-molecule imaging experiments through single-molecule fluorescence in situ hybridization (smFISH) and colocalization studies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Myocardial infarction (MI) is increasing at a younger age. Growth differentiation factor-15 (GDF-15) has been implicated in several key mechanisms of atherogenesis. Arterial stiffness parameters, including pulse wave velocity (PWV) and the augmentation index (AIx), can indicate the presence or progression of atherosclerosis. The aim of this study is to evaluate the GDF-15 level and arterial stiffness parameters in patients with premature MI., Method: Thirty patients aged ≤45 years (mean age: 39 ± 5 years, 23 male) who recovered from a MI and 15 age and sex-matched subjects were consecutively included. The serum GDF-15 concentration levels and arterial stiffness parameters of the patients and controls were measured., Results: GDF-15 levels were significantly higher in patients with premature MI, while there were no significant differences in PWV and AIx between the groups. The GDF-15 level was correlated negatively with high-density lipoprotein (HDL) cholesterol and positively with uric acid levels. Both GDF-15 ( p = 0.046, odds ratio: 1.092, 95% confidence interval: 1.003-1.196) and HDL cholesterol ( p = 0.037, odds ratio: 0.925, 95% confidence interval: 0.859-0.995) were found as independent factors associated with premature MI., Conclusions: GDF-15 could be a risk factor for premature MI. Further studies are needed to elucidate the central role of GDF-15 in the pathophysiology of early atherosclerosis and MI in the young population.

Percutaneous coronary intervention of bifurcation lesions is associated with an increased risk of complications in patients with acute coronary syndrome (ACS). The study aimed to evaluate the in-hospital outcomes of patients with ACS with culprit bifurcation lesions who were treated with either a "main vessel-oriented" (MVO) treatment strategy or an "open side branch" (OSB) treatment strategy. This retrospective study included 575 consecutive patients with ACS. "MVO" and "OSB" treatment strategies were defined as primary/urgent percutaneous coronary intervention procedures performed by either totally ignoring the side branch (SB) or trying to maintain both main vessel and SB open with thrombolysis in myocardial infarction 3 flow. Procedural success and major cardiac/cerebrovascular events during hospitalization were noted. MVO and OSB treatment strategies were performed on 384 and 191 patients, respectively. The procedural success rate was significantly higher in the OSB treatment strategy whereas major cardiac/cerebrovascular events rates were similar except for the contrast-induced nephropathy rate being slightly higher in OSB treatment strategy. Subgroup analysis revealed a significantly higher procedural success rate in OSB treatment strategy if the SB was located within the bifurcation core, especially in those where the diameter of SB was ≥2 mm. In conclusion, our results suggest a better procedural result with SB protection attempts in patients with ACS with a culprit bifurcation lesion if the SB is originating within the bifurcation core and its diameter is ≥2 mm. MVO treatment strategy may be preferred in most cases with minor SBs located distant to the bifurcation core because of the similar procedural success., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Coronary slow flow may not only affect the coronary arteries, but it may also be a vascular problem affecting the rest of the arterial system., Objective: The aim of this study was to determine peripheral arterial stiffness and the thickness of the choroid layer in patients with slow coronary flow., Methods: Fifty consecutive patients (age, 54.3 ± 11.4 years, 38 male) with coronary slow flow and 25 consecutive patients (age, 50.5 ± 9.9 years, 16 male) with normal coronary arteries both documented by coronary angiography were included. Arterial stiffness parameters were measured noninvasively using a Mobil-O-Graph arteriography system. The choroidal thickness was assessed using the enhanced depth imaging optical coherence tomography method., Results: The patients with coronary slow flow had significantly higher peripheral systolic blood pressure, peripheral pulse pressure, central pulse pressure, and pulse wave velocity (PWV) and significantly thinner choroidal thickness compared to the controls. Thrombolysis in myocardial infarction frame count was positively correlated with PWV (r: 0.237, p = 0.041) and negatively correlated with choroidal thickness (r: -0.249, p = 0.031). There was also a negative correlation between PWV and mean choroidal thickness (r: -0.565, p < 0.001). Linear regression analysis showed that coronary slow flow was an independent predictor of both PWV and choroidal thickness when adjusted by age and sex., Conclusions: The acceleration of average peripheral arterial PWV with a thinning of choroidal thickness in patients with coronary slow flow may support the idea that this phenomenon may be a coronary presentation of a systemic microvascular disorder., Competing Interests: All the authors declare no conflict of interest.

The development of fluorescent probes for visualizing endogenous RNAs in living cells is crucial to understand their complex biochemical roles. Recently, we developed RhoBAST, one of the most photostable and brightest fluorescence light-up aptamers (FLAPs), as a genetically encoded tag for imaging messenger RNAs (mRNAs). Here, we describe programmable RhoBAST sequences flanked by target-binding hybridization arms that light up only when bound to the untagged target RNA in trans . As part of the hybridization arm, we introduced a modular transducer sequence that switches the secondary structure of RhoBAST and renders it incapable of binding to its fluorogenic ligand TMR-DN. Only the specific binding of the hybridization arms to the target RNA triggers the correct folding of RhoBAST and fluorescence light-up after binding to TMR-DN. We characterized the structural switching of programmable RhoBAST sequences extensively in vitro and applied them to visualize untagged mRNAs in live bacteria.

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins., (© 2023. The Author(s).)

Fluorescent light-up aptamers (FLAPs) have emerged as valuable tools to visualize RNAs, but are mostly limited by their poor brightness, low photostability, and high fluorescence background in live cells. Exploiting the avidity concept, here we present two of the brightest FLAPs with the strongest aptamer-dye interaction, high fluorogenicity, and remarkable photostability. They consist of dimeric fluorophore-binding aptamers (biRhoBAST and biSiRA) embedded in an RNA scaffold and their bivalent fluorophore ligands (bivalent tetramethylrhodamine TMR 2 and silicon rhodamine SiR 2 ). Red fluorescent biRhoBAST-TMR 2 and near-infrared fluorescent biSiRA-SiR 2 are orthogonal to each other, facilitating simultaneous visualization of two different RNA species in live cells. One copy of biRhoBAST allows for simple and robust mRNA imaging with strikingly higher signal-to-background ratios than other FLAPs. Moreover, eight biRhoBAST repeats enable single-molecule mRNA imaging and tracking with minimal perturbation of their localization, translation, and degradation, demonstrating the potential of avidity-enhanced FLAPs for imaging RNA dynamics., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Behcet's disease (BD) may present with life threating complications including neurological and cardiovascular involvement. Neuro-Behcet's disease (NBD) is one of the most important causes of morbidity and mortality in patients with BD. The aim of the present study is to investigate whether patients with NBD are different than BD patients with other manifestations in terms of subclinical myocardial dysfunction., Methods: Forty patients with NBD (23 female, mean age: 42.4 ± 9.4 years), 40 patients with BD (9 female, mean age: 39.7 ± 9.0 years) and 40 controls (20 male, mean age: 41.8 ± 6.5 years) were consecutively included in the study. All subjects underwent a transthoracic echocardiography for evaluation of left ventricular (LV) and atrial (LA) functions with two-dimensional (2D) speckle tracking echocardiography (STE)., Results: Baseline characteristics, clinical data, LV dimensions, systolic and diastolic functions were all in normal range among the groups. LV global longitudinal strain (LV-GLS) was significantly lower in patients with NBD and BD patients without neurologic involvement compared to controls. LA conduit strain was significantly lower in patients with NBD compared to controls. Patients with both parenchymal NBD and vascular NBD manifestations had significantly lower LV-GLS and LA conduit strain compared to controls. Linear regression analysis demonstrated that among cardiovascular risk factors only presence of NBD was the independent predictor of LV-GLS., Conclusions: BD is associated with impaired LV and LA functions. LV-GLS and LA conduit strains of the patients with NBD were lower. NBD was an independent predictor of LV-GLS, suggesting a link between neurological manifestations and cardiac dysfunction in BD patients.

Purpose: Insomnia is a common sleep disorder which has high comorbidity with a number of cardiovascular diseases (CVD). As a possible risk factor for the CVDs, arterial stiffness may be assessed non-invasively by pulse wave velocity (PWV) and augmentation index (AI). The aim of this study was to evaluate any relation between insomnia and arterial stiffness., Methods: Patients with insomnia were included in the study after the exclusion of other sleep disorders by polysomnography. Sleep quality and the degree of insomnia symptoms were evaluated by the Pittsburgh sleep quality index (PSQI) and insomnia severity index (ISI), respectively. PWV and AI were assessed by Mobil-O-Graph arteriograph system., Results: Consecutive patients with insomnia (n = 72, 56 women, mean age 55.8 ± 9.1 years) were included. Patients were grouped as those with severe ISI scores (22-28) and those with mild to moderate ISI scores (8-21). Despite no significant difference in characteristics and clinical data, patients with severe ISI scores had significantly higher total PSQI scores and NREM-2 with significantly lower REM duration. They also had significantly higher systolic blood pressure, mean blood pressure, pulse pressure, PWV, and AI compared to patients with mild and moderate ISI scores. Correlation analysis revealed that PWV and AI were significantly correlated with the ISI score and PSQI score., Conclusion: There is a close relation between arterial stiffness and insomnia suggesting a risk for CVD in patients with insomnia., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background and Purpose: Neurogenic myocardial injury occurs as a result of dysregulation of autonomic nervous system. The aim of this study was to explore the frequency of elevated troponin and dynamic ST segment/T wave changes and their relation with left ventricular (LV) systolic functions in acute ischemic stroke patients., Methods: One hundred and twenty-five patients (mean age: 65.1±15.2years, 76 male) presenting with acute ischemic stroke were consecutively included. 12-lead electrocardiogram was taken to assess dynamic ST segment/T wave changes, conventional transthoracic echocardiography to determine LV ejection fraction (LVEF). High-sensitive cardiac troponin I (hs-cTnI) level>0.04ng/mL was accepted as elevated., Results: Twenty-seven patients (21.6%) had elevated hs-cTnI and 60 patients (48%) had dynamic ST segment/T wave changes. The stroke patients with elevated hs-cTnI had significantly higher NT-proBNP values (2302±3450pg/mL vs 799±2075pg/mL p<0.001) and higher frequency of ST segment/T wave changes (85.2% vs 37.8% p<0.001), and lower LVEF (52.2±13.6% vs 61.0±8.5% p=0.002) compared to patients with normal troponin levels. The patients with ST segment/T wave changes had significantly higher frequencies of hyper-lipidemia (31.7% vs 15.4% p=0.031) and coronary artery disease (CAD) (43.3% vs 13.8% p<0.001), hs-cTnI (0.19±0.55ng/mL vs 0.02±0.01ng/mL p<0.001) and NT-proBNP levels (1430±2564pg/mL vs 842±2425pg/mL p=0.016), and lower LVEF (56.1±11.7% vs 61.9±8.3% p=0.009). Linear regression analysis revealed presence of CAD, but not ST segment/T wave changes as an independent predictor of hs-cTnI (p=0.034). LVEF was independently associated with hs-cTnI (p=0.003) and presence of CAD (p=0.009) when adjusted by age, sex and presence of ST segment/T wave changes., Conclusion: Troponin elevation and ST segment/T wave changes occurring in patients suffering acute ischemic stroke, especially in those with CAD, may be a sign of neurogenic stunned myocardium.