Combining site-directed spin labeling in vivo and in-cell EPR distance determination

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E. coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.publishe

Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.<br /

Impact of Preprocedural Left Ventricular Ejection Fraction on 1-Year Outcomes After MitraClip Implantation (from the ACCESS-EU Phase I, a Prospective, Multicenter, Nonrandomized Postapproval Study of the MitraClip Therapy in Europe)

This report describes the 12-month outcomes of the a prospective, multicenter, nonrandomized post-approval study of the MitraClip therapy in Europe (ACCESS-EU postapproval study of MitraClip therapy) with respect to preprocedural left ventricular ejection fraction (LVEF). Transcatheter deployment of the MitraClip device may be considered for patients who are not suitable for conventional surgery. A total of 567 patients with significant mitral regurgitation (MR) underwent MitraClip therapy. Of those, 393 had functional MR (FMR) and were subdivided by preprocedural LVEF (A: 10% to 20%, B: >20% to 30%, C: >30% to 40%, D: >40%). Procedural safety and efficacy and treatment outcomes including MR grade, New York Heart Association (NYHA) functional class, 6-minute walk test, and the Minnesota Living with Heart Failure Questionnaire were analyzed at baseline, 30 days, and 12 months. Baseline mean logistic EuroSCORE was 25 ± 19; 87% of patients were in NYHA classes III or IV (A: 96%, B: 83%, C: 90%, D: 86%). There was no incidence of death or stroke intraprocedurally. Eleven patients died within 30 days with no differences among subgroups. Kaplan-Meier survival at 12 months was 81.8% (A: 71%, B: 79%, C: 87%, D: 86%). There was a significant improvement in MR severity at 30 days and 12 months (p <0.0001). At 12 months, all subgroups experienced similar improvements in NYHA class, 6-minute walk test, and Minnesota Living with Heart Failure Questionnaire. This real-world registry reports promising results of MitraClip therapy in patients with FMR. In conclusion, the low rates of hospital mortality and adverse events in patients with FMR-even in patients with severely reduced LVEF-provide additional evidence of substantial benefits after MitraClip implantation

Correlations of intermediate monocytes with cortisol, aldosterone and noradrenaline.

<p>Correlations (Spearman) between intermediate monocyte measured by flow cytometry and cortisol (cor), aldosterone (aldo) as well as noradrenaline (nor). (a) Intermediate monocyte pre TAVR vs. cor, aldo, nor pre TAVR. (b) Intermediate monocyte post TAVR vs. cor, aldo, nor post TAVR. Correlations among cor, aldo and nor are also shown. On the top of each graph: pairwise correlation value and relative significance (negative value indicates inverse correlation); on bottom: bivariate scatterplot. ** p<0.01.</p

Blood count and selected other laboratory values.

<p>Blood count and selected other laboratory values.</p

Intermediate CD14⁺⁺CD16⁺ monocytes decline after transcatheter aortic valve replacement and correlate with functional capacity and left ventricular systolic function

<div><p>Background</p><p>Transcatheter aortic valve replacement (TAVR) is the method of choice for patients with severe aortic valve stenosis, who are ineligible or at high risk for surgery. Though TAVR leads to a significant reduction in mortality, a notable amount of patients are re-hospitalized early after TAVR. Parameters or biomarkers predicting outcome are therefore needed to identify patients who benefit most. Specific monocyte subsets have been associated with cardiovascular diseases and were shown to possess prognostic value.</p><p>Methods</p><p>Peripheral blood was drawn before and after transfemoral TAVR with the self-expanding CoreValve, Boston Lotus or the balloon-expanding Edwards Sapien prosthesis. Classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16⁺) and non-classical (CD14⁺CD16⁺⁺) monocyte subsets were determined by flow cytometry. Transthoracic echocardiography was performed before, early after as well as 3 months after the TAVR procedure.</p><p>Results</p><p>No significant differences in the absolute monocyte counts were found after TAVR. A significant decline in the intermediate monocyte population was though observed early after TAVR (pre 4.01±0.38%, post 2.803±0.34%, <i>p≤0</i>.<i>05</i>). Creatinine levels stayed stable after TAVR procedure and intermediate monocytes were associated with worse renal function. Monocyte decline was not related to changes in CRP-, noradrenaline, cortisol or aldosterone-levels. The amount of intermediate monocytes correlated with worse cardiac function and predicted the possibility to reach an improvement in NYHA functional class at 3 months after TAVR.</p><p>Conclusions</p><p>A significant decline of intermediate monocytes occurs shortly after TAVR. High levels of intermediate monocytes were associated with worse cardiac function and predicted poor functional capacity, hinting at a possible prognostic value.</p></div

Correlations of intermediate monocytes with CRP and creatinine.

<p>(a) Correlations between intermediate monocytes measured by flow cytometry with serum CRP levels [mg/l] before and at day 4–7 after TAVR-procedure as well as intermediate monocytes before with maximum CRP-level between TAVR procedure and day 4–7. (b) Correlations between intermediate monocytes and creatinine-levels (μmol/l) before and early after TAVR-procedure.</p

General traits, comorbidities and functional parameters before TAVR.

<p>General traits, comorbidities and functional parameters before TAVR.</p