Characterization and Purification of Polydisperse Reconstituted Lipoproteins and Nanolipoprotein Particles

Heterogeneity is a fact that plagues the characterization and application of many self-assembled biological constructs. The importance of obtaining particle homogeneity in biological assemblies is a critical goal, as bulk analysis tools often require identical species for reliable interpretation of the results—indeed, important tools of analysis such as x-ray diffraction typically require over 90% purity for effectiveness. This issue bears particular importance in the case of lipoproteins. Lipid-binding proteins known as apolipoproteins can self assemble with liposomes to form reconstituted high density lipoproteins (rHDLs) or nanolipoprotein particles (NLPs) when used for biotechnology applications such as the solubilization of membrane proteins. Typically, the apolipoprotein and phospholipids reactants are self assembled and even with careful assembly protocols the product often contains heterogeneous particles. In fact, size polydispersity in rHDLs and NLPs published in the literature are frequently observed, which may confound the accurate use of analytical methods. In this article, we demonstrate a procedure for producing a pure, monodisperse NLP subpopulation from a polydisperse self-assembly using size exclusion chromatography (SEC) coupled with high resolution particle imaging by atomic force microscopy (AFM). In addition, NLPs have been shown to self assemble both in the presence and absence of detergents such as cholate, yet the effects of cholate on NLP polydispersity and separation has not been systematically examined. Therefore, we examined the separation properties of NLPs assembled in both the absence and presence of cholate using SEC and native gel electrophoresis. From this analysis, NLPs prepared with and without cholate showed particles with well defined diameters spanning a similar size range. However, cholate was shown to have a dramatic affect on NLP separation by SEC and native gel electrophoresis. Furthermore, under conditions where different sized NLPs were not sufficiently separated or purified by SEC, AFM was used to deconvolute the elution pattern of different sized NLPs. From this analysis we were able to purify an NLP subpopulation to 90% size homogeneity by taking extremely fine elutions from the SEC. With this purity, we generate high quality NLP crystals that were over 100 μm in size with little precipitate, which could not be obtained utilizing the traditional size exclusion techniques. This purification procedure and the methods for validation are broadly applicable to other lipoprotein particles

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

The Conformation of Interfacially Adsorbed Ranaspumin-2 Is an Arrested State on the Unfolding Pathway

Ranaspumin-2 (Rsn-2) is a surfactant protein found in the foam nests of the t\'{u}ngara frog. Previous experimental work has led to a proposed model of adsorption which involves an unusual clam shell-like `unhinging' of the protein at an interface. Interestingly, there is no concomitant denaturation of the secondary structural elements of Rsn-2 with the large scale transformation of its tertiary structure. In this work we use both experiment and simulation to better understand the driving forces underpinning this unusual process. We develop a modified G\={o}-model approach where we have included explicit representation of the side-chains in order to realistically model the interaction between the secondary structure elements of the protein and the interface. Doing so allows for the study of the underlying energy landscape which governs the mechanism of Rsn-2 interfacial adsorption. Experimentally, we study targeted mutants of Rsn-2, using the Langmuir trough, pendant drop tensiometry and circular dichroism, to demonstrate that the clam-shell model is correct. We find that Rsn-2 adsorption is in fact a two-step process: the hydrophobic N-terminal tail recruits the protein to the interface after which Rsn-2 undergoes an unfolding transition which maintains its secondary structure. Intriguingly, our simulations show that the conformation Rsn-2 adopts at an interface is an arrested state along the denaturation pathway. More generally, our computational model should prove a useful, and computationally efficient, tool in studying the dynamics and energetics of protein-interface interactions.Comment: 8 figure

Prevalence of vasculare risk factors in different stages of prodromal Alzheimer’s disease and its influence on cognitive decline

peer reviewe

Körperliche Aktivität und das Risiko für chronische Erkrankungen : Entwicklung und Evaluierung eines Indexes zur Messung körperlicher Aktivität und Baseline-datenkalibrierung in EPIC-Deutschland

Körperliche Aktivität (KA) ist ein Lifestyle Faktor, der vor chronischen Erkrankungen zu schützen scheint. In der European Prospective Investigation into Cancer and Nutrition (EPIC)-Studie wurden zwei Fragebögen verwendet, mit denen anhand des Cambridge Indexes und des Total Physical Activity Indexes die KA der Teilnehmer kategorisiert werden kann. Die präzise Messung der PA ist essentiell, um die wahre Assoziationsstärke zwischen KA und chronischen Erkrankungen zu schätzen. Objektive Messmethoden, die heutzutage zum Einsatz kommen, vereinfachen die schwierige Aufgabe der präzisen Messung der KA. Basierend darauf, wurden die folgenden Ziele in dieser Doktorarbeit verfolgt: die Entwicklung und Evaluierung eines validen KA Indexes– den Improved Physical Activity Index (IPAI), welcher in der Lage sein soll, die KA der Personen zu kategorisieren aber auch eine kontinuierliche Messung der KA zu liefern. Diese soll das Bewegungsausmaß widerspiegeln. Des Weiteren wurde eine Kalibrierung der Basis Fragebogen-Daten zur PA und die Schätzung des Zusammenhangs zwischen nicht-kalibrierter und kalibrierter KA und chronischen Erkrankungen, Typ 2 Diabetes, Myokardinfarkt, Schlaganfall und Krebs, angestrebt. Diese Ziele wurden in den deutschen EPIC-Zentren Potsdam und Heidelberg verfolgt. In einer Substudie wurden 1615 Teilnehmer rekrutiert. Diese Substudie beinhaltete ein breites Fragenspektrum zur üblichen KA, sowie eine objektive 7-Tage Messung der KA mittels eines Herzfrequenz und Bewegungsmessers - Actiheart. Die Baseline Fragebogenangaben zur KA wurden, mittels der aus der Substudie gewonnenen Daten, kalibriert. Das Risiko für chronische Erkrankungen wurde in der Gesamtkohorte mit Hilfe von proportionaler Hazards-Regression nach Cox berechnet. Der IPAI besteht aus den folgenden Aktivitätsvariablen: Art der Berufstätigkeit, Fahrrad fahren (Stunden/Woche), dem Sporthäufigkeitsscore, sowie Fernsehscore und Computernutzung (Stunden/Wochenende ab 18Uhr). Korrelationen zwischen dem IPAI und objektiv gemessener KA betrugen r=0.39-0.44 für Aktivitätscounts und r=0.32-0.40 für Aktivitätsenergieausgabe (PAEE). Der Cambridge Index und der Total Physical Activity Index waren schwächer mit objektiv gemessener KA korreliert als der IPAI. In nicht-berufstätigen Teilnehmern war der IPAI auch stärker mit objektiv gemessener KA korreliert, als die bisher genutzten Indizes. Stückweise Regression wurde zur Kalibrierung der Baseleine KA Daten angewandt. In den kalibrierten Daten waren die Hazardraten (HR (95% Konfidenzeinterval)) zwischen der höchsten KA Kategorie, im Vergleich zur niedrigsten KA Kategorie, niedriger als in den nicht kalibrierten Daten für die meisten chronischen Erkrankungen HR=0.40(0.35-0.46), Typ 2 Diabetes HR=0.08(0.06-0.10), Myokardinfarkt HR=0.40(0.24-0.67) und Schlaganfall HR=0.54(0.33-0.87). Die nicht-kalibrierten HRs betragen, in der gleichen Reihenfolge: HR=0.72(0.66-0.80), HR=0.57(0.48-0.67), HR=0.62(0.44-0.88), und HR=0.86(0.62-1.19), Es wurden keine Zusammenhänge zwischen KA und Krebs gefunden, auch nicht nach Ausschluss von Erkrankungen, die in den ersten 3 Jahren der Nachbeobachtung aufgetreten sind. Zusammenfassend zeigen die vorliegenden Ergebnisse, dass der IPAI ein valides Instrument zur populationsbezogenen subjektiven Messung der üblichen KA/Bewegung (sowohl kontinuierlich als auch in Kategorien) ist und in Studien zu diesem Zweck angewandt werden kann. Die Datenkalibrierung liefert eine präzisere Schätzung des Zusammenhangs zwischen KA und chronischen Erkrankungen. Die präventiven Effekte von KA auf chronische Erkrankungen, Typ 2 Diabetes, Myokardinfarkt und Schlaganfall werden in Studien mit Fragebogenmessung der KA unterschätzt. Interventionen im Bereich Public Health, vor allem in der Gruppe von Typ 2 Diabetes Gefährdeten, sollten einen stärkeren Fokus auf KA legen.Physical activity (PA) is a lifestyle factor that has been shown to prevent chronic diseases. The accurate PA measurement is essential to estimate the true magnitude of the relationship between PA and disease risk. Nowadays, objective measurement methods for PA have been developed and improve the challenging task of measuring the individuals’ PA level. Therefore, the aims of this thesis were firstly, the development and evaluation of a valid physical activity index – the Improved Physical Activity Index (IPAI), which will be able to categorize people into activity categories but may also be used as a continuous measure that reflects one’s activity amount (movement). Secondly, the calibration of the available baseline PA questionnaire measurement and finally, the estimation of the associations between calibrated and non-calibrated baseline PA data and risk of overall chronic diseases, type 2 diabetes, myocardial infarction, stroke and overall cancer. These objectives were accomplished by applying an extensive physical activity questionnaire and a 7-day heart rate and acceleration sensor PA measurement to a sub-sample (n=1,615) of older adults from the European Prospective Investigation into Cancer and Nutrition (EPIC-Germany) study. Baseline self-reported PA was calibrated using statistical models based on the objective PA measurement in the sub-sample. The risk of chronic diseases was estimated using Cox proportional hazards regression in the whole EPIC-Germany cohort. The IPAI consists of items covering five areas including PA at work, sport, cycling, television viewing, and computer use. The correlations between the IPAI and accelerometer counts in the training and validation sample ranged from r=0.39 to 0.44 and with physical activity energy expenditure from r=0.32 to 0.40 and were higher than between the Cambridge Index or the Total Physical Activity Index with the objective measures. In non-working participants the IPAI also showed higher correlations than the established indices. For the baseline PA data calibration segmented regression analysis has been chosen. The hazard rates (HR (95% confidence intervals)) for the risk reduction in the highest calibrated PA category compared to the lowest for overall chronic diseases HR=0.40(0.35-0.46), type 2 diabetes HR=0.08(0.06-0.10), myocardial infarction HR=0.40(0.24-0.67) and stroke HR=0.54(0.33-0.87) were lower than the non-calibrated results HR=0.72(0.66-0.80), HR=0.57(0.48-0.67), HR=0.62(0.44-0.88), and HR=0.86(0.62-1.19), respectively. There were no associations between the calibrated PA and risk of cancer, even after excluding the first 3 years of follow up. In conclusion, a valid PA index which is able to express PA on a continuous scale as well as to categorize participants was developed. In populations with increasing rates of non-working people the performance of the IPAI is better than the established indices used in EPIC. The PA data calibration provides more precise risk estimates. The preventive effects of PA on overall chronic disease risk, type 2 diabetes, myocardial infarction and stroke, has been shown to be underestimated. Public health interventions should persistently focus on PA, especially in type 2 diabetes jeopardized individuals

Protein kinase regulation of sarcoplasmic reticulum function in isolated adult rat ventricular myocytes

The sarcoplasmic reticulum (SR) is one of the major regulators of the cytosolic Ca2+ concentration in cardiac ventricular muscle cells. In the myocardium, relaxation results from a decrease in cytoplasmic free Ca2+ levels mediated through an efflux of Ca2+ from the cell via the sarcolemmal Na+/Ca2+- exchanger and by the sequestration of 0a2+ by the network SR membranes through the actions of a calcium pump (a Mg2+dependent, Ca2+/K+activated adenosine triphosphatase; Ca2+/K+-ATPase). During an action potential, the Ca2+ stored in the SR is released to the cytoplasm via a Ca2+release channel present in the junctional SR and Ca2+ also enters the cell through voltage-controlled Ca2+ channels in the sarcolemmal membrane. These two processes result in an increase in cytoplasmic Ca2+ concentration which leads to contraction of the myocardium. SR membrane function is regulated in part by the phosphorylation of proteins present in this membrane. Subsequent to 3-adrenergic stimulation of the heart by catecholamines, levels of cAMP are increased leading to the activation of cAMP-dependent protein kinase (PK A). In isolated cardiac SR membrane vesicles and perfused hearts, phosphorylation of an indigenous SR protein, phospholamban, is mediated by PK A. Phosphorylated phospholamban acts as a modulator of the Ca2+/K+-ATPase to stimulate active Ca2+ uptake by increasing the affinity of this enzyme for Ca2+. This stimulation of Ca2+-uptake is the main mechanism by which catecholamines accelerate relaxation in the heart. When phospholamban is in the dephosphorylated state, a cytoplasmic portion of the molecule interacts near the phosphorylation site of the Ca2+-pump to inhibit Ca2+-transport. [More abstract follows]Pharmaceutical Sciences, Faculty ofGraduat

Entwicklung einer Administratoroberfläche für Lehrveranstaltungen mit Photovoltaik-Anlagen in der virtuellen Realität (Kurztitel: Leitstand für die Lehre in VR)

Diese Arbeit beschäftigt sich mit der Erstellung einer Administratoroberfläche für die Lehre bei Photovoltaik (PV)-Praktika in der virtuellen Realität (VR). Die erstellte Umgebung bietet, mittels Bildschirmspiegelungen, Möglichkeiten zur didaktischen Anleitung und Unterstützung der Studierenden. Das Thema wurde aufgrund einer bestehenden Lehranwendung in der VR bedeutungsvoll und zeigt deutliches Potenzial. Diese Lehranwendung wird bereits umfassend und verpflichtend in den Praktika eingesetzt. Sie bietet einen praxisnahen Aufbau von Solaranlagen und erhöht gefahrlos die Experimentierfreudigkeit. Mit ihr lassen sich die aufgebauten Anlagen technisch prüfen, simulieren und bewerten. Zudem werden die beiden Möglichkeiten zur Unterstützung der Studierenden beurteilt. Als Ergebnis wird die Umsetzung der nahezu automatisierten Administratorober-fläche verdeutlicht und ein Usability-Test aus den Praktika evaluiert. Schlagwörter: Administratoroberfläche, Bildschirmspiegelung, C, Didaktik, im-mersiv, Oculus Quest 2, Photovoltaik, Python, Tkinter, virtuelle RealitätThis work focuses on the creation of an administrator interface for teaching pho-tovoltaic (PV) practicals in the Virtual Reality (VR). The created environment pro-vides the possibilities for didactic guidance and support of the students by means of screen mirroring. The topic became significant due to an existing teaching ap-plication in VR and shows clear potential. The teaching application is already ex-tensively and mandatory used in the internships. It provides a hands-on construc-tion of solar systems and safely increases the willingness to experiment. It can be used to test, simulate and evaluate the constructed systems. In addition, the two existing possibilities for supporting the students are evaluated up on this ba-sis. As a result, the implementation of the almost automated administrator inter-face is clarified and a usability test from the practical courses is evaluated. Keywords: administrator interface, C, didactic, immersive, Oculus Quest 2, photovoltaics, Python, screen mirroring, Tkinter, virtual realit