Micropositioning and microscopic observation of individual picoliter-sized containers within SU-8 microchannels

We describe the fabrication and application of a bioanalytical chip, made of SU-8 photoresist, comprising integrated, high aspect-ratio microfluidic channels, suitable to manipulate and investigate vesicles, cell fragments and biological cells. A central micrometer-sized aperture allows electrical particle counting and planar membrane experiments, microfluids allow (sub)micrometer-sized objects to be transported and addressed with different chemicals. Here we show how lipid vesicles are positioned with micrometer precision within the micro-channels by means of pressure and electrophoretic movement. Our approach is suited for controlling and investigating (bio)chemical synthesis and cellular signalling processes in ultrasmall individual vesicles by electro-optical technique

Directed invasion of cancer cell spheroids inside 3D collagen matrices oriented by microfluidic flow in experiment and simulation

Invasion is strongly influenced by the mechanical properties of the extracellular matrix. Here, we use microfluidics to align fibers of a collagen matrix and study the influence of fiber orientation on invasion from a cancer cell spheroid. The microfluidic setup allows for highly oriented collagen fibers of tangential and radial orientation with respect to the spheroid, which can be described by finite element simulations. In invasion experiments, we observe a strong bias of invasion towards radial as compared to tangential fiber orientation. Simulations of the invasive behavior with a Brownian diffusion model suggest complete blockage of migration perpendicularly to fibers allowing for migration exclusively along fibers. This slows invasion toward areas with tangentially oriented fibers down, but does not prevent it

Engineering of carbohydrate oxidoreductases for sensors and bio-fuelcells

Pyranose dehydrogenase (PDH) and pyranose 2-oxidase (POx) are flavoproteins that catalyze the oxidation of free, non-phosphorylated sugars to the corresponding ketosugars. Pyranose dehydrogenase has a broad substrate specificity for monosaccharides (and few disaccharides), but is limited to a narrow range of electron acceptors and reacts extremely slowly with dioxygen, whereas pyranose 2-oxidase shows pronounced specificity for glucose and displays high oxidase as well as dehydrogenase activity. For bio-fuelcell and sensor applications, oxygen reactivity is undesirable as it leads to electron leakage and the formation of damaging hydrogen peroxide; for biocatalytic applications, oxygen reactivity is advantageous, as oxygen is freely available and obviates downstream removal of undesired electron acceptors. Site-saturation mutagenesis libraries of eleven (POx) and twelve (PDH) residues around the active sites were screened for oxidase and dehydrogenase activities. In POx, variants T166R, Q448H, L545C, L547R and N593C displayed reduced oxidase activities (between 40% and 0.2% of the wildtype) concomitant with unaffected or even increased dehydrogenase activity, dependent on the electron acceptor used (DCPIP, 1,4-benzoquinone or ferricenium ion). Kinetic characterization showed that both affinity and turnover numbers can be affected. The switch from oxidase to dehydrogenase activity was also observed electrochemically using screen-printed electrodes. In this miniaturized set-up with a reaction volume of only 50 µL the dehydrogenase activity of variant N593C was entirely preserved in the presence of a suitable mediator, shuttling electrons from the FAD cofactor to the electrode surface. The oxidase activity, utilizing molecular oxygen as acceptor, is abolished in this variant. Of all variants of PDH that were produced by saturation mutagenesis, only variants of one position displayed increased oxygen reactivity to a minor degree. Histidine 103, carrying the covalently attached FAD cofactor, was substituted by tyrosine, phenylalanine, tryptophan and methionine. Variant H103Y displayed a five-fold increase of oxygen reactivity. Stopped flow analysis revealed that the mutation slowed down the reductive half-reaction whereas the oxidative half-reaction was affected to a minor degree. No alterations in the secondary structure were observed, but disruption of the FAD bond had negative effects on thermal and conformational stability. We also engineered PDH by systematically removing several N-glycosylation sites, in order to improve performance by reducing the distance of the active site to the electrode surface, improving accessibility for redox polymers and facilitate denser enzyme packing on the electrode. One glycosylation site, N319, was found to be indispensable for functional expression and folding of the enzyme, as no active variants could be obtained. A variant with two sites, N75 and N175 near the active site entrance, exchanged against G and Q, respectively, showed significantly improved properties when used on electrodes with Osmium-based redox polymers (Mediated Electron Transfer) and a low level of Direct Electron Transfer. The lack of two glycosylation sites results in minor negative effects on expression yield and stability. Removal of a third site, N252, on the opposite side of the active site entrance, does not bring further improvements in catalysis and electron transfer, but is detrimental to functional expression and stability. The bulk of hyperglycosylation of the recombinantly expressed enzyme (observed in both Pichia pastoris and Saccharomyces cerevisiae) is located only on this one glycosylation site. Please click Additional Files below to see the full abstract

Die Staatsrechtslehre und die Veränderung ihres Gegenstandes. Gewährleistung von Freiheit und Sicherheit im Lichte unterschiedlicher Staats- und Verfassungsverständnisse. Risikosteuerung durch Verwaltungsrecht. Transparente Verwaltung - Konturen...Berichte und Diskussionen auf der Tagung der Vereinigung der Deutschen Staatsrechtslehrer in Hamburg vom 1. bis 4. Oktober 2003

This volume presents the lectures and discussions held at the conference of the "Vereinigung der deutschen Staatsrechtslehrer" (the association of german professors for public law), October 1st - Ocober 3rd, 2003, Hamburg

Young endurance training starting age in non-elite athletes is associated with higher proximal aortic distensibility.

OBJECTIVE Decreased proximal aortic distensibility (AD) is known to significantly predict all-cause mortality and cardiovascular events among individuals without overt cardiovascular disease. This cross-sectional study investigated the association of endurance training (ET) parameters, namely, ET starting age, ET years and yearly ET volume with AD in non-elite endurance athletes. METHODS Healthy, normotensive, male Caucasian participants of a 10-mile race were assessed with a 2D echocardiogram and comprehensive interview. Ascending aortic diameters were measured simultaneously with pulse pressure. Aortic strain, AD and aortic stiffness index were calculated. Predictors of AD were investigated among training parameters by linear regression models corrected for age, resting heart rate, stroke volume index and mean blood pressure. RESULTS Ninety-two of 121 athletes (aged 42±8 years) had sufficient echocardiogram quality and were used for analysis. ET starting age (range 6-52 years) and years of ET (range 2-46 years) were highly collinear and used in two separate models for AD. Significant factors for AD were ET starting age, 10-mile race time and resting heart rate in model I, and age, years of ET, 10-mile race time and heart rate in model II (all p<0.01). CONCLUSIONS In our cohort of healthy, non-elite, middle-aged runners, AD was significantly higher in athletes with younger ET starting age or more years of ET (in the model adjusted for confounders). In the model with years of ET, age had a negative contribution to AD, suggesting that with older age, the benefit of more years of ET on AD decreased. Future studies assessing the effect of exercise training on arterial properties should include training starting age

Imaging of Bioprosthetic Valve Dysfunction after Transcatheter Aortic Valve Implantation.

Transcatheter aortic valve implantation (TAVI) has become the standard of care in elderly high-risk patients with symptomatic severe aortic stenosis. Recently, TAVI has been increasingly performed in younger-, intermediate- and lower-risk populations, which underlines the need to investigate the long-term durability of bioprosthetic aortic valves. However, diagnosing bioprosthetic valve dysfunction after TAVI is challenging and only limited evidence-based criteria exist to guide therapy. Bioprosthetic valve dysfunction encompasses structural valve deterioration (SVD) resulting from degenerative changes in the valve structure and function, non-SVD resulting from intrinsic paravalvular regurgitation or patient-prosthesis mismatch, valve thrombosis, and infective endocarditis. Overlapping phenotypes, confluent pathologies, and their shared end-stage bioprosthetic valve failure complicate the differentiation of these entities. In this review, we focus on the contemporary and future roles, advantages, and limitations of imaging modalities such as echocardiography, cardiac computed tomography angiography, cardiac magnetic resonance imaging, and positron emission tomography to monitor the integrity of transcatheter heart valves

Infectious Endocarditis of a Heterotopic Caval Valved Stent.

Right-sided infective endocarditis (IE) accounts for 5% to 10% of all IE cases. Compared with left-sided IE, it is more often associated with intravenous drug abuse and intracardiac devices, whereas the latter has become more prevalent in recent decades. The authors report the first case of IE in a heterotopic caval valved stent used for treating torrential tricuspid regurgitation. (Level of Difficulty: Advanced.)

Framing climate change in frontline communities: anthropological insights on how mountain dwellers in the USA, Peru, and Italy adapt to glacier retreat

We report on anthropological research conducted in three mountain communities (in the USA, Italy and Peru), which have been directly affected by glacier retreat for over 40 years. Our mixed methods include ethnographic research, analysis of transcripts of interviews, focus groups and community meetings, and case studies of adaptation projects. Our findings indicate that local people are acute observers of change. They draw on two frames (climate change and community) in their discussions and projects but rely much more heavily on the latter frame. This pattern of drawing on the community frame, characteristic of all discussions, is most marked in the community meetings. The effectiveness of the community frame in supporting projects calls into question some widely shared notions about the role of belief in climate change as a crucial precondition for adaptation and challenges the “perceive–predict–act” model of climate change response

Reproducibility of 4D cardiac computed tomography feature tracking myocardial strain and comparison against speckle-tracking echocardiography in patients with severe aortic stenosis.

BACKGROUND Myocardial strain is an established parameter for the assessment of cardiac function and routinely derived from speckle tracking echocardiography (STE). Novel post-processing tools allow deformation imaging also by 4D cardiac computed tomography angiography (CCT). This retrospective study aims to analyze the reproducibility of CCT strain and compare it to that of STE. METHODS Left (LV) and right ventricular (RV), and left atrial (LA) ejection fraction (EF), dimensions, global longitudinal (GLS), circumferential (GCS) and radial strain (GRS) were determined by STE and CCT feature tracking in consecutive patients with severe aortic stenosis evaluated for transcatheter aortic valve implantation. RESULTS 106 patients (mean age 79.9 ​± ​7.8, 44.3% females) underwent CCT at a median of 3 days (IQR 0-28 days) after STE. In CCT, strain measures showed good to excellent reproducibility (intra- and inter-reader intraclass correlation coefficient ≥0.75) consistently in the LV, RV and LA. In STE, only LV GLS and LA GLS yielded good reproducibility, whereas LV GCS and LV GRS showed moderate, and RV GLS and free wall longitudinal strain (FWLS) poor reproducibility. Agreement between CCT and STE was strong for LV GLS only, while other strain features displayed moderate (LV GCS, LA GLS) or weak (LV GRS, RV GLS and FWLS) inter-modality correlation. CONCLUSION LV, RV and LA CCT strain assessments were highly reproducible. While a strong agreement to STE was found for LV GLS, inter-modality correlation was moderate or weak for LV GCS, LV GRS, and RV and LA longitudinal strain, possibly related to poor reproducibility of STE measurements