Signature of a non-harmonic potential as revealed from a consistent shape and fluctuation analysis of an adherent membrane

The interaction of fluid membranes with a scaffold, which can be a planar surface or a more complex structure, is intrinsic to a number of systems - from artificial supported bilayers and vesicles to cellular membranes. In principle, these interactions can be either discrete and protein mediated, or continuous. In the latter case, they emerge from ubiquitous intrinsic surface interaction potentials as well as nature-designed steric contributions of the fluctuating membrane or from the polymers of the glycocalyx. Despite the fact that these nonspecific potentials are omnipresent, their description has been a major challenge from experimental and theoretical points of view. Here we show that a full understanding of the implications of the continuous interactions can be achieved only by expanding the standard superposition models commonly used to treat these types of systems, beyond the usual harmonic level of description. Supported by this expanded theoretical framework, we present three independent, yet mutually consistent, experimental approaches to measure the interaction potential strength and the membrane tension. Upon explicitly taking into account the nature of shot noise as well as of finite experimental resolution, excellent agreement with the augmented theory is obtained, which finally provides a coherent view of the behavior of the membrane in a vicinity of a scaffold.Comment: 15 pages, 12 figures, accepted by Physical Review

Verständlichkeit des bundeseinheitlichen Medikationsplans bei chronisch Kranken

Fragestellung: Im Oktober 2016 wurde ein bundeseinheitlicher Medikationsplan (MP) gesetzlich vorgeschrieben. In dieser Studie wurde die Verständlichkeit des Medikationsplans bei chronisch Kranken mit Polymedikation untersucht. Methoden: In die Querschnittsstudie wurden 100 Patienten eingeschlossen, die mit einer Dauermedikation von mindestens fünf Arzneimitteln behandelt wurden. Davon hatten 50 Patienten die Diagnose einer chronischen Herzinsuffizienz (CHF) und 50 Patienten die Diagnose eines Diabetes mellitus Typ 2 (DMT2). Es wurde ein strukturiertes Interview zur Handhabung des MP durchgeführt, die Patienten wurden nach ihrer Meinung zu dem MP befragt und die Beurteilung des Verständnisses des MP wurde durch das Befüllen von Dosierhilfen bewertet. Zur quantitativen Einschätzung der Handhabung des MP wurde ein Score-System entwickelt, der sogenannte ET-MP-Score („Evaluation Tool to test the handling oft he Medication Plan“). Zusätzlich wurden die Patienten mittels der PHQ-9-, Mini-Cog- und G9-EHFScBs-9- Fragebögen auf Zeichen einer Depression, einer kognitiven Beeinträchtigung und bei den chronisch Herzkranken auf das vorhandene Gesundheitsbewusstsein getestet. Ergebnisse: Nur jeder dritte Patient (32%) war in der Lage, anhand des MP die richtige Dosierung der sechs Arzneimittel (Wochentag, Tageszeit, Dosis) korrekt umzusetzen (ET-MP-Score >90%). Der mittlere ET-MP-Score lag bei 73 ± 27%. Zwischen den beiden Patientengruppen mit CHF und DMT2 ergab sich im Hinblick auf das Verständnis des MP kein relevanter Unterschied. Faktoren, die Einfluss auf den ET-MP-Score hatten, waren ein höheres Alter (≥ 70, p < 0,01), eine geringere Bildung (< 10 Jahre Bildung, p < 0,01), eine kognitive Beeinträchtigung (p < 0,001) und die Tatsache, ob Arzneimittel des MP vorher bekannt waren oder nicht (p ≤ 0,03). Keinen signifikanten Einfluss auf den ET-MP-Score hatten die Anzahl der täglichen Tabletten, die Lebenssituation, das Geschlecht, der Charlson Komorbiditätsindex, das Vorhandensein einer Depression, der Gebrauch einer Dosierhilfe oder das vorherige Benutzen eines MP. Schlussfolgerung: Die Studie zeigt, dass Patienten mit CHF oder DMT2 und Polymedikation Probleme im Umgang mit dem MP haben, unabhängig von der jeweils chronischen Erkrankung. Ein höheres Alter, ein geringerer Bildungsgrad und eine kognitive Beeinträchtigung stellten Risikofaktoren für Patienten im Hinblick auf eine korrekte Umsetzung des MP dar. Patienten mit diesen Risikofaktoren benötigen zusätzliche Unterstützung bei der Handhabung des MP.Purpose: A standardized medication plan (MP) was recently enacted into German Law. Its comprehensibility in the hands of patients with chronic diseases requiring polymedication is incompletely understood. Patients and methods: 100 patients who took at least five medicines regularly were prospectively included in a cross-sectional study: 50 patients with chronic heart failure (CHF) and 50 patients with diabetes mellitus type 2 (DMT2). We performed a structured test-scenario regarding handling of a provided MP, requested patients’ opinion and evaluated the execution of the information on the MP by filling pill boxes. An established weighted scoring system, the “Evaluation Tool to test the handling of the Medication Plan” (ET-MP) was applied to quantitate the ability of the patients to handle the MP. In addition, signs of depression, cognitive function and self-care behavior in chronic heart failure were characterized using the PHQ-9, Mini-Cog, G9-EHFScB-9 questionnaires, respectively. Results: The understanding of the MP was poor irrespective of the underlying condition. Only 32% of all patients were able to handle the MP without difficulties (ET-MP score >90%), the mean ET-MP score was 73±27%. Understanding of the MP was similar between patients with CHF and DMT2. Understanding the MP was better in patients aged <70 years compared to ≥70 years (p<0.01). Patients ≥10 years of education achieved higher ET-MP results (84±22%) than patients with <10 years of education (68±27%, p<0.01). Patients with signs of cognitive impairment exhibited significantly lower ET-MP scores (61±27%) than patients without cognitive impairment (80±24%, p<0.001). There were no significant correlations of ET-MP score with number of daily medications, living situation, sex, the Charlson Comorbidity Index, the PHQ-9 score, and usage of a dosing aid or possession of a medication list. Conclusion: The study shows that many patients with CHF or DMT2 on polymedication exhibit problems using the MP irrespective of the compared chronic diseases. Higher age, lower education and cognitive impairment identify patients with high risk of problems with the MP that require additional assistance

PDE7A1 hydrolyzes cCMP

AbstractThe degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP KM value of 135±19μM. The Vmax for cCMP hydrolysis reached 745±27nmol/(minmg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP

Single-molecule analysis of dynamics and interactions of the SecYEG translocon

Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm2·s−1. Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions

Nanoscale FasL Organization on DNA Origami to Decipher Apoptosis Signal Activation in Cells

Cell signaling is initiated by characteristic protein patterns in the plasma membrane, but tools to decipher their molecular organization and activation are hitherto lacking. Among the well-known signaling pattern is the death inducing signaling complex with a predicted hexagonal receptor architecture. To probe this architecture, DNA origami-based nanoagents with nanometer precise arrangements of the death receptor ligand FasL are introduced and presented to cells. Mimicking different receptor geometries, these nanoagents act as signaling platforms inducing fastest time-to-death kinetics for hexagonal FasL arrangements with 10 nm inter-molecular spacing. Compared to naturally occurring soluble FasL, this trigger is faster and 100x more efficient. Nanoagents with different spacing, lower FasL number or higher coupling flexibility impede signaling. The results present DNA origami as versatile signaling scaffolds exhibiting unprecedented control over molecular number and geometry. They define molecular benchmarks in apoptosis signal initiation and constitute a new strategy to drive particular cell responses