Three-Dimensional Software- and MR-Imaging-Based Muscle Volumetry Reveals Overestimation of Supraspinatus Muscle Atrophy Using Occupation Ratios in Full-Thickness Tendon Tears.

Supraspinatus muscle atrophy is widely determined from oblique-sagittal MRI by calculating the occupation ratio. This ex vivo and clinical study aimed to validate the accuracy of 3D software- and MR-imaging-based muscle volumetry, as well as to assess the influence of the tear pattern on the occupation ratio. Ten porcine muscle specimens were volumetrized using the physical water displacement volumetry as a standard of reference. A total of 149 individuals with intact supraspinatus tendons, partial tears, and full-thickness tears had 3T MRI. Two radiologists independently determined occupation ratio values. An excellent correlation with a Pearson's r of 0.95 for the variables physical volumetry using the water displacement method and MR-imaging-based muscle volumetry using the software was found and formed the standard of reference for the patient study. The inter-reader reliability was 0.92 for occupation ratios. The correlation between occupation ratios and software-based muscle volumes was good in patients with intact tendons (0.84) and partial tears (0.93) but considerably lower in patients with full-thickness tears (0.68). Three-dimensional-software- and MR-imaging-based muscle volumetry is reliable and accurate. Compared to 3D muscle volumetry, the occupation ratio method overestimates supraspinatus muscle atrophy in full-thickness tears, which is most likely due to the medial retraction of the myotendinous unit.

Technology development to evaluate the effectiveness of viscosity reducing excipients.

Addition of pharmaceutical excipients is a commonly used approach to decrease the viscosity of highly concentrated protein formulations, which otherwise could not be subcutaneously injected or processed. The variety of protein-protein interactions, which are responsible for increased viscosities, makes a portfolio approach necessary. Screening of several excipients to develop such a portfolio is time and money consuming in industrial settings. Responsible protein-protein interactions were investigated using the interaction parameter kD obtained from dynamic light scattering measurements in the studies presented herein. Together with in-silico calculated excipient parameter, kD could be used as a screening tool accelerating screening and formulation development as kD is suitable to high-throughput formats using small quantities of protein and low concentrations. A qualitative correlation between kD and high-concentration viscosity behavior could be shown in our case.

Small quantum dots conjugated to nanobodies as immunofluorescence probes for nanometric microscopy.

Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new ∼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed ∼2 nm precision.

Conformational state distributions and catalytically relevant dynamics of a hinge-bending enzyme studied by single-molecule FRET and a coarse-grained simulation.

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.

Evaluation of model-based versus non-parametric monaural noise-reduction approaches for hearing aids.

OBJECTIVE: Single channel noise reduction has been well investigated and seems to have reached its limits in terms of speech intelligibility improvement, however, the quality of such schemes can still be advanced. This study tests to what extent novel model-based processing schemes might improve performance in particular for non-stationary noise conditions. DESIGN: Two prototype model-based algorithms, a speech-model-based, and a auditory-model-based algorithm were compared to a state-of-the-art non-parametric minimum statistics algorithm. A speech intelligibility test, preference rating, and listening effort scaling were performed. Additionally, three objective quality measures for the signal, background, and overall distortions were applied. For a better comparison of all algorithms, particular attention was given to the usage of the similar Wiener-based gain rule. STUDY SAMPLE: The perceptual investigation was performed with fourteen hearing-impaired subjects. RESULTS: The results revealed that the non-parametric algorithm and the auditory model-based algorithm did not affect speech intelligibility, whereas the speech-model-based algorithm slightly decreased intelligibility. In terms of subjective quality, both model-based algorithms perform better than the unprocessed condition and the reference in particular for highly non-stationary noise environments. CONCLUSION: Data support the hypothesis that model-based algorithms are promising for improving performance in non-stationary noise conditions.

Native and unfolded states of phosphoglycerate kinase studied by single-molecule FRET.

Single-molecule Förster resonance energy transfer (FRET) measurements with phosphoglycerate kinase from yeast were performed at different concentrations of guanidine hydrochloride. From these steady-state measurements we obtained FRET efficiency histograms characterizing structural properties of individual proteins at different stages between the native and the fully unfolded state. Native proteins exhibit a slightly more expanded structure under buffer conditions without denaturant as compared to conditions with denaturant. At 0.5 M GndHCl an unfolded state population that exhibits a significantly expanded structure as compared to the native state, emerges. The unfolded state is characterized by a pronounced broadening of the efficiency distribution, which indicates a large structural and/or dynamical heterogeneity within the population. At high denaturant concentrations, well above the unfolding transition at C(1/2)~0.7 M, we observe a progressive expansion of the protein structure, namely globule-coil transition.

Structural stability of soybean (Glycine max) α-amylase: properties of the unfolding transition studied with fluorescence and CD spectroscopy.

Stability and unfolding of mammalian and microbial α-amylases have been intensively investigated. However, there is only limited information available on the structural stability of plant α-amylases, namely of the two isoenzymes from barley AMY1 and AMY2, of the α-amylase from mung bean (Vigna radiata), and of the α-amylase from malted sorghum (Sorghum bicolor). We report here the stability of soyabean α-amylase (GMA), against elevated temperatures and chemical denaturants (GndHCl) by employing circular dichroism and fluorescence spectroscopy. Since it is well-known that calcium ions play a crucial role for enzymatic activity and stability of a-amylases, we performed our studies with calcium bound and calcium free GMA. The thermal unfolding transition temperature decreased from 72°C for calcium saturated samples to 57°C for the case of calcium depleted GMA. Similarly, the GndHCl transition concentration was lowered from 0.70 M for calcium bound GMA to 0.41 M in the absence of calcium. Thermal unfolding of GMA irreversible due to aggregation of the unfolded state. GMA unfolded in 6 M GndHCl shows high degree of reversibility after diluting the unfolded enzyme in native buffer containing 7 M glycerol. Furthermore, the refolded enzyme showed 93% of activity.

The effect of calcium binding on the unfolding barrier: A kinetic study on homologous alpha-amylases.

Extreme thermostabilities of proteins can be achieved by binding co-factors to the protein structures. For various alpha-amylases protein stabilization upon calcium binding is a well-known phenomenon. In the present study the mechanism of stabilization of three homologous alpha-amylases was investigated by measuring the unfolding kinetics with CD spectroscopy. For this purpose thermal unfolding kinetics of calcium saturated and calcium depleted enzymes were analyzed by means of Eyring-plots. The free energy change between the native and the transition state which characterized the unfolding barrier height was found to be proportional to the number of calcium ions bound to the protein structures. For the most thermostable alpha-amylases calcium binding caused a significant increase in the enthalpy change, which was partly compensated by increased entropy changes.

Uneven twins: comparison of two enantiocomplementary hydroxynitrile lyases with alpha/beta-hydrolase fold.

Hydroxynitrile lyases (HNLs) are applied in technical processes for the synthesis of chiral cyanohydrins. Here we describe the thorough characterization of the recently discovered R-hydroxynitrile lyase from Arabidopsis thaliana and its S-selective counterpart from Manihot esculenta (MeHNL) concerning their properties relevant for technical applications. The results are compared to available data of the structurally related S-HNL from Hevea brasiliensis (HbHNL), which is frequently applied in technical processes. Whereas substrate ranges are highly similar for all three enzymes, the stability of MeHNL with respect to higher temperature and low pH-values is superior to the other HNLs with alpha/beta-hydrolase fold. This enhanced stability is supposed to be due to the ability of MeHNL to form tetramers in solution, while HbHNL and AtHNL are dimers. The different inactivation pathways, deduced by means of circular dichroism, tryptophan fluorescence and static light scattering further support these results. Our data suggest different possibilities to stabilize MeHNL and AtHNL for technical applications: whereas the application of crude cell extracts is appropriate for MeHNL, AtHNL is stabilized by addition of polyols. In addition, the molecular reason for the inhibition of MeHNL and HbHNL by acetate could be elucidated, whereas no such inhibition was observed with AtHNL.

Observing proteins as single molecules encapsulated in surface-tethered polymeric nanocontainers.

Immobilizing biomolecules provides the advantage of observing them individually for extended time periods, which is impossible to accomplish for freely diffusing molecules in solution. In order to immobilize individual protein molecules, we encapsulated them in polymeric vesicles made of amphiphilic triblock copolymers and tethered the vesicles to a cover slide surface. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein-folding studies. The fact that polymeric vesicles possess an extreme stability under various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl and, thereby, ideally suited for unfolding and refolding studies with encapsulated proteins. We demonstrate this with encapsulated phosphoglycerate kinase, which was fluorescently labeled with Atto655, a dye that exhibits pronounced photoinduced electron transfer (PET) to a nearby tryptophan residue in the native state. Under unfolding conditions, PET was reduced, and we monitored alternating unfolding and refolding conditions for individual encapsulated proteins.

Price increase causes fewer sales of factory-made cigarettes and higher sales of cheaper loose tobacco in Germany.

Aim of this study is the analysis of the price responsiveness of demand for cigarettes and loose tobacco in Germany over the period 1991--2006. In this period the average consumption of all kinds of cigarettes per capita (German population > or = 15 years) declined from 634 pieces/quarter to 457pieces/quarter (-28%). Consumption of factory-made cigarettes decreased from about 545 pieces/quarter to 330 pieces/quarter in 2006 (-39%). In the same time consumption of self-made cigarettes increased from 89 pieces/quarter to 127 pieces/quarter (+42%). A one Euro Cent increase in price is associated with 28 cigarettes of all kinds consumed less per quarter. Data indicate that the different types of cigarettes are substitutes, e.g. there is evidence for a positive relationship between the price of factory-made cigarettes and the consumption of hand-made cigarettes. Thus, the increase in such consumption is rather driven by a positive cross-price effect of 17.01. Data indicate additionally an overall decrease in the cigarette consumption and a partial switch to cheaper loose tobacco. The availability of low-taxed loose tobacco may undermine the public health benefits of higher cigarette prices. Price differentials between tobacco products should be reduced in order to maximize the public health benefits of high cigarette prices.