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Many cell-penetrating peptides (CPPs) bind to glycosaminoglycans (GAG) located on the extracellular side of biological tissues. CPP binding to the cell surface is intimately associated with clustering of surface molecules and is usually followed by uptake into the cell interior. We have investigated the uptake mechanism by comparing CPPs which bind, but cannot induce, GAG clustering with those which do induce GAG clustering. We have synthesized the tryptophan-labeled CPP nona-l-arginine (WR9) and its monodispersely PEGylated derivate (PEG27-WR9) and have compared them with respect to glycan binding, glycan clustering, and their uptake into living cells. Both CPPs bind to the GAG heparin with high affinity (KD 100 nM), but the PEGylation prevents the GAG clustering. Thus, it is possible to uncouple and analyze the contributions of GAG binding and GAG clustering to the biological CPP uptake. The uptake of PEG-WR9 into CH-K1 cells is confined to intracellular vesicles, where colocalization with transferrin attests to an endocytic uptake. Transfection experiments with plasmid DNA for GFP revealed poor GFP expression, suggesting that endocytic uptake of PEG-WR9 is compromised by insufficient release from endocytic vesicles. In contrast, WR9 shows two uptake routes. At low concentration (<5 μM), WR9 uptake occurs mainly through endocytosis. At higher concentration, WR9 uptake is greatly enhanced, showing a diffuse spreading over the entire cytoplasm and nucleus-a phenomenon termed "transduction". Transduction of WR9 leads to a higher GFP expression as compared to PEG-WR9 endocytosis but also damages the plasma membrane as evidenced by SYTOX Green staining. The results suggest that GAG binding without and with GAG clustering induce two different pathways of CPP uptake. [ABSTRACT FROM AUTHOR]

Abstract: Cell-penetrating peptides (CPPs) traverse the membrane of biological cells at low micromolar concentrations and are able to take various cargo molecules along with. Despite large differences in their chemical structure, CPPs share the structural similarity of a high cationic charge density. This property confers to them the ability to bind electrostatically membrane constituents such as anionic lipids and glycosaminoglycans (GAGs). Controversies exist, however, about the biological response after the interaction of CPPs with such membrane constituents. Present review compares thermodynamic binding studies with conditions of the biological CPP uptake. It becomes evident that CPPs enter biological cells by different and probably competing mechanisms. For example, some amphipathic CPPs traverse pure lipid model membranes at low micromolar concentrations — at least in the absence of cargos. In contrast, no direct translocation at these conditions is observed for non-amphipathic CPPs. Finally, CPPs bind GAGs at low micromolar concentrations with potential consequences for endocytotic pathways. [Copyright &y& Elsevier]

During cellular uptake of fluorescently labeled cell-penetrating peptides (CPPs), intense fluorescent signals are commonly observed in the nucleus of the cell, suggesting intracellular CPP relocation and potential binding to the genome of the host. We therefore investigated the interaction of the CPP HIV-1 Tat(47–57) with double-stranded DNA, and we also tested whether the fluorescence intensity of the labeled CPP allows for linear predictions of its intracellular concentration. Using isothermal titration calorimetry, we observe that the CPP has a high affinity for salmon sperm DNA as characterized by a microscopic dissociation constant of 126 nM. The binding is exothermic, with a reaction enthalpy of -4.63 kcal/mol CPP (28 °C). The dissociation constant and reaction enthalpy decrease further at higher temperatures. The affinity of the CPP for DNA is thus 1–2 magnitudes higher than for extracellular heparan sulfate, the likely mediator of the CPP uptake. Accordingly, the high affinity for DNA confers stability to extracellular transport complexes of CPP and DNA but potentially affects the regulation and molecular organization of the host's genome after nuclear uptake. Moreover, the CPP leads to the condensation of DNA as evidenced by the pronounced increase in light-scattering intensity. The fluorescence quantum yield of the FITC-labeled CPP decreases considerably at concentrations > 5 μmol/L, at pH < 7, and upon binding to DNA and glycosaminoglycans. This change in fluorescence quantum yield impedes the microscopic identification of uptake routes and the comparison of uptake efficiency of different CPPs, especially if the accumulation in subcellular compartments (self-quenching and pH difference) and transitory binding partners (quenching and condensation) is unknown. [ABSTRACT FROM AUTHOR]

Abstract: Recent findings on specific and non-specific interactions of glycosaminoglycans (GAGs) accentuate their pivotal role in biology and the call for improved sequencing tools. The present study evaluates size-exclusion chromatography (SEC) of heparin oligosaccharides at high and low pressure, requiring amounts as low as 0.2microgram, using conventional UV detection after depolymerization with heparin lyases. Because of their high charge at physiological pH, SEC elution volumes of heparin oligosaccharides depend on both molecular size and charge repulsion from the matrix. As a consequence, SEC elution volumes of GAGs are smaller than those of globular proteins of similar molecular weight, and this might be exploited. Accordingly, larger heparin oligosaccharides are best separated according to their size at high ionic strength of the mobile phase (>30mM); in contrast, disaccharides are best separated according to their charge at low ionic strength, compatible with on-line coupling to mass spectrometry. Optimized SEC affords separation of characteristic heparin trisaccharides that contain uronic acid at the reducing end and suggest cellular storage of heparin as a free glycan. [Copyright &y& Elsevier]

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV- 1 TAT protein transduction domain [Fg-CPPTAT(PTD)] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPPTAT(PTD) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPPTAT(PTD) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPPTAT(PTD) uptake and are detected only on fibroblasts showing Fg-CPPTAT(PTD) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg- CPPTAT(PTD) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z[sub p] = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (π > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is ΔH[sup º] ≈ -1.5 kcal/mol peptide and is almost temperature-independent with ΔC[sub p, sup º] ∼0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for ∼80% of the binding energy. The overall binding constant, K[sub app], is ∼10³ - 10[sup 4] M[sup -1]. The intrinsic binding constant (K[sub p]), corrected for electrostatic effects and describing the partitioning of the peptide between the lipidwater interface and the membrane, is small and is K[sub... [ABSTRACT FROM AUTHOR]

Reports on Ro 09-0198 (cinnamycin) that is a tetracyclic peptide antibiotic used to monitor the transbilayer movement of phosphatidylethanolamine (PE) in biological membranes during cell division and apoptosis. Measurement of the thermodynamic parameters of complex formation with high sensitivity isothermal titration calorimetry; Free energy of binding.

Early detection of atrial fibrillation (AF) enables initiation of anticoagulation and early rhythm control therapy to reduce stroke, cardiovascular death, and heart failure. In a cross-sectional, observational study, we aimed to identify a combination of circulating biomolecules reflecting different biological processes to detect prevalent AF in patients with cardiovascular conditions presenting to hospital. Twelve biomarkers identified by reviewing literature and patents were quantified on a high-precision, high-throughput platform in 1485 consecutive patients with cardiovascular conditions (median age 69 years [Q1, Q3 60, 78]; 60% male). Patients had either known AF (45%) or AF ruled out by 7-day ECG-monitoring. Logistic regression with backward elimination and a neural network approach considering 7 key clinical characteristics and 12 biomarker concentrations were applied to a randomly sampled discovery cohort (n = 933) and validated in the remaining patients (n = 552). In addition to age, sex, and body mass index (BMI), BMP10, ANGPT2, and FGF23 identified patients with prevalent AF (AUC 0.743 [95% CI 0.712, 0.775]). These circulating biomolecules represent distinct pathways associated with atrial cardiomyopathy and AF. Neural networks identified the same variables as the regression-based approach. The validation using regression yielded an AUC of 0.719 (95% CI 0.677, 0.762), corroborated using deep neural networks (AUC 0.784 [95% CI 0.745, 0.822]). Age, sex, BMI and three circulating biomolecules (BMP10, ANGPT2, FGF23) are associated with prevalent AF in unselected patients presenting to hospital. Findings should be externally validated. Results suggest that age and different disease processes approximated by these three biomolecules contribute to AF in patients. Our findings have the potential to improve screening programs for AF after external validation. [ABSTRACT FROM AUTHOR]

Early detection of atrial fibrillation (AF) enables initiation of anticoagulation and early rhythm control therapy to reduce stroke, cardiovascular death, and heart failure. In a cross-sectional, observational study, we aimed to identify a combination of circulating biomolecules reflecting different biological processes to detect prevalent AF in patients with cardiovascular conditions presenting to hospital. Twelve biomarkers identified by reviewing literature and patents were quantified on a high-precision, high-throughput platform in 1485 consecutive patients with cardiovascular conditions (median age 69 years [Q1, Q3 60, 78]; 60% male). Patients had either known AF (45%) or AF ruled out by 7-day ECG-monitoring. Logistic regression with backward elimination and a neural network approach considering 7 key clinical characteristics and 12 biomarker concentrations were applied to a randomly sampled discovery cohort (n = 933) and validated in the remaining patients (n = 552). In addition to age, sex, and body mass index (BMI), BMP10, ANGPT2, and FGF23 identified patients with prevalent AF (AUC 0.743 [95% CI 0.712, 0.775]). These circulating biomolecules represent distinct pathways associated with atrial cardiomyopathy and AF. Neural networks identified the same variables as the regression-based approach. The validation using regression yielded an AUC of 0.719 (95% CI 0.677, 0.762), corroborated using deep neural networks (AUC 0.784 [95% CI 0.745, 0.822]). Age, sex, BMI and three circulating biomolecules (BMP10, ANGPT2, FGF23) are associated with prevalent AF in unselected patients presenting to hospital. Findings should be externally validated. Results suggest that age and different disease processes approximated by these three biomolecules contribute to AF in patients. Our findings have the potential to improve screening programs for AF after external validation. [ABSTRACT FROM AUTHOR]

Short episodes of ischemia (ischemic preconditioning) protect the heart against ventricular arrhythmias during zero-flow ischemia and reperfusion. However, in clinics, many episodes of ischemia present a residual flow (low-flow ischemia). Here we examined whether ischemic preconditioning protects against ventricular arrhythmias during and after a low-flow ischemia and, if so, by what mechanism(s). Isolated rat hearts were subjected to 60 min of low-flow ischemia (12% residual coronary flow) followed by 60 min of reperfusion. Ischemic preconditioning was induced by two cycles of 5 min of zero-flow ischemia followed by 5 and 15 min of reperfusion, respectively. Arrhythmias were evaluated as numbers of ventricular premature beats (VPBs) as well as incidences of ventricular tachycardia (VT) and ventricular .brillation (VF) during low-flow ischemia and reperfusion. Ischemic preconditioning significantly reduced the number of VPBs and the incidence of VT and of VF during low-flow ischemia. This antiarrhythmic effect of preconditioning was abolished by HOE 140 (100 nM), a bradykinin B2 receptor blocker. Similar to preconditioning, exogenous bradykinin (10 nM) reduced the number of VPBs and the incidence of VT and of VF during low-flow ischemia. Furthermore, the antiarrhythmic effects of both ischemic preconditioning and bradykinin were abolished by glibenclamide (1 µM), a non-specific blocker of ATP-sensitive K+ (KATP) channels. Finally, the antiarrhythmic effects of both ischemic preconditioning and bradykinin were abolished by HMR 1098 (10 µM), a sarcolemmal KATP channel blocker but not by 5-hydroxydecanoate (100 µM), a mitochondrial KATP channel blocker. In conclusion, ischemic preconditioning protects against ventricular arrhythmias induced by low-flow ischemia, and this protection involves activation of bradykinin B2 receptors and subsequent opening of sarcolemmal but not of mitochondrial KATP channels. [ABSTRACT FROM AUTHOR]

Many biomarkers that could be used to assess ejection fraction, heart failure, or myocardial infarction fail to translate into clinical practice because they lack essential performance characteristics or fail to meet regulatory standards for approval. Despite their potential, new technologies have added to the complexities of successful translation into clinical practice. Biomarker discovery and implementation require a standardized approach that includes: identification of a clinical need; identification of a valid surrogate biomarker; stepwise assay refinement, demonstration of superiority over current standard-of-care; development and understanding of a clinical pathway; and demonstration of real-world performance. Successful biomarkers should improve efficacy or safety of treatment, while being practical at a realistic cost. Everyone involved in cardiovascular healthcare, including researchers, clinicians, and industry partners, are important stakeholders in facilitating the development and implementation of biomarkers. This article provides suggestions for a development pathway for new biomarkers, discusses regulatory issues and challenges, and suggestions for accelerating the pathway to improve patient outcomes. Real-life examples of successful biomarkers—high-sensitivity cardiac troponin, T2* cardiovascular magnetic resonance imaging, and echocardiography—are used to illustrate the value of a standardized development pathway in the translation of concepts into routine clinical practice. [ABSTRACT FROM AUTHOR]

Background: Large-scale screening for atrial fibrillation (AF) requires reliable methods to identify at-risk populations. Using an experimental semi-quantitative biomarker assay, B-type natriuretic peptide (BNP) and fibroblast growth factor 23 (FGF23) were recently identified as the most suitable biomarkers for detecting AF in combination with simple morphometric parameters (age, sex, and body mass index [BMI]). In this study, we validated the AF model using standardised, high-throughput, high-sensitivity biomarker assays.Methods and Findings: For this study, 1,625 consecutive patients with either (1) diagnosed AF or (2) sinus rhythm with CHA2DS2-VASc score of 2 or more were recruited from a large teaching hospital in Birmingham, West Midlands, UK, between September 2014 and February 2018. Seven-day ambulatory ECG monitoring excluded silent AF. Patients with tachyarrhythmias apart from AF and incomplete cases were excluded. AF was diagnosed according to current clinical guidelines and confirmed by ECG. We developed a high-throughput, high-sensitivity assay for FGF23, quantified plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) and FGF23, and compared results to the previously used multibiomarker research assay. Data were fitted to the previously derived model, adjusting for differences in measurement platforms and known confounders (heart failure and chronic kidney disease). In 1,084 patients (46% with AF; median [Q1, Q3] age 70 [60, 78] years, median [Q1, Q3] BMI 28.8 [25.1, 32.8] kg/m2, 59% males), patients with AF had higher concentrations of NT-proBNP (median [Q1, Q3] per 100 pg/ml: with AF 12.00 [4.19, 30.15], without AF 4.25 [1.17, 15.70]; p < 0.001) and FGF23 (median [Q1, Q3] per 100 pg/ml: with AF 1.93 [1.30, 4.16], without AF 1.55 [1.04, 2.62]; p < 0.001). Univariate associations remained after adjusting for heart failure and estimated glomerular filtration rate, known confounders of NT-proBNP and FGF23. The fitted model yielded a C-statistic of 0.688 (95% CI 0.656, 0.719), almost identical to that of the derived model (C-statistic 0.691; 95% CI 0.638, 0.744). The key limitation is that this validation was performed in a cohort that is very similar demographically to the one used in model development, calling for further external validation.Conclusions: Age, sex, and BMI combined with elevated NT-proBNP and elevated FGF23, quantified on a high-throughput platform, reliably identify patients with AF.Trial Registration: Registry IRAS ID 97753 Health Research Authority (HRA), United Kingdom. [ABSTRACT FROM AUTHOR]

Objectives: Biotin >20.0 ng/mL (81.8 nmol/L) can reduce Elecsys® Troponin T Gen 5 (TnT Gen 5; Roche Diagnostics) assay recovery, potentially leading to false-negative results in patients with suspected acute myocardial infarction (AMI). We aimed to determine the prevalence of elevated biotin and AMI misclassification risk from biotin interference with the TnT Gen 5 assay. Methods: Biotin was measured using an Elecsys assay in two cohorts: (i) 797 0-h and 646 3-h samples from 850 US emergency department patients with suspected acute coronary syndrome (ACS); (ii) 2023 random samples from a US laboratory network, in which biotin distributions were extrapolated for higher values using pharmacokinetic modeling. Biotin >20.0 ng/mL (81.8 nmol/L) prevalence and biotin 99th percentile values were calculated. AMI misclassification risk due to biotin interference with the TnT Gen 5 assay was modeled using different assay cutoffs and test timepoints. Results: ACS cohort: 1/797 (0.13%) 0-h and 1/646 (0.15%) 3-h samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 2.62 ng/mL (10.7 nmol/L; 0-h) and 2.38 ng/mL (9.74 nmol/L; 3-h). Using conservative assumptions, the likelihood of false-negative AMI prediction due to biotin interference was 0.026% (0-h result; 19 ng/L TnT Gen 5 assay cutoff). US laboratory cohort: 15/2023 (0.74%) samples had biotin >20.0 ng/mL (81.8 nmol/L); 99th percentile biotin was 16.6 ng/mL (68.0 nmol/L). Misclassification risk due to biotin interference (19 ng/L TnT Gen 5 assay cutoff) was 0.025% (0-h), 0.0064% (1-h), 0.00048% (3-h), and <0.00001% (6-h). Conclusions: Biotin interference has minimal impact on the TnT Gen 5 assay's clinical utility, and the likelihood of false-negative AMI prediction is extremely low. [ABSTRACT FROM AUTHOR]

• Validation of 99th percentile URL for TnT Gen 5 assay in a healthy US population. • TnT Gen 5 results were higher in healthy males vs females and increased with age. • The findings facilitate interpretation of TnT Gen 5 results in US clinical practice. Determining diagnostic thresholds for cardiac troponin assays is key to interpreting their clinical performance. We describe the calculation of 99th percentile upper reference limits (URLs) for the Elecsys® Troponin T Gen 5 (TnT Gen 5) assay. Plasma and serum samples from healthy US participants were prospectively evaluated using TnT Gen 5 Short Turn Around Time and 18-min assays on cobas e 411 and cobas e 601 analyzers (Roche Diagnostics); with, up to 8 TnT Gen 5 results per participant. A total of 10,402 TnT Gen 5 results from 1301 participants were included (50.4% female). Across 9 calculation methods, overall 99th percentile URL was 19.2 ng/l (females, 13.5–13.6 ng/l; males, 21.4–22.2 ng/l). Across different sample/assay/analyzer combinations, overall 99th percentile URLs ranged from 18.4–20.2 ng/l. Median TnT Gen 5 results increased with age, were higher in males, and ranged from 3.0–3.7 ng/l across races/ethnicities and from 3.0–3.6 ng/l across body mass index (BMI) classes. Applying additional exclusion criteria (N-terminal pro-brain natriuretic peptide, BMI and estimated glomerular filtration rate) resulted in lower 99th percentile URLs (overall, 16.9 ng/l; females, 11.8 ng/l; males, 18.5 ng/l). Our findings facilitate the interpretation of TnT Gen 5 results in US clinical practice. [ABSTRACT FROM AUTHOR]

Atherosclerosis results in the narrowing of arterial blood vessels and this causes significant changes in the endogenous shear stress between healthy and constricted arteries. Nanocontainers that can release drugs locally with such rheological changes can be very useful. Here, we show that vesicles made from an artificial 1,3-diaminophospholipid are stable under static conditions but release their contents at elevated shear stress. These vesicles have a lenticular morphology, which potentially leads to instabilities along their equator. Using a model cardiovascular system based on polymer tubes and an external pump to represent shear stress in healthy and constricted vessels of the heart, we show that drugs preferentially release from the vesicles in constricted vessels that have high shear stress. [ABSTRACT FROM AUTHOR]