Site-Specific Isotopic Labeling (SSIL): Access to High-Resolution Structural and Dynamic Information in Low-Complexity Proteins

Remarkable technical progress in the area of structural biology has paved the way to study previously inaccessible targets. For example, large protein complexes can now be easily investigated by cryo-electron microscopy, and modern high-field NMR magnets have challenged the limits of high-resolution characterization of proteins in solution. However, the structural and dynamic characteristics of certain proteins with important functions still cannot be probed by conventional methods. These proteins in question contain low-complexity regions (LCRs), compositionally biased sequences where only a limited number of amino acids is repeated multiple times, which hamper their characterization. This Concept article describes a site-specific isotopic labeling (SSIL) strategy, which combines nonsense suppression and cell-free protein synthesis to overcome these limitations. An overview on how poly-glutamine tracts were made amenable to high-resolution structural studies is used to illustrate the usefulness of SSIL. Furthermore, we discuss the potential of this methodology to give further insights into the roles of LCRs in human pathologies and liquid–liquid phase separation, as well as the challenges that must be addressed in the future for the popularization of SSIL.     

A simple mass-action model for the eukaryotic heat shock response and its mathematical validation

The heat shock response is a primordial defense mechanism against cell stress and protein misfolding. It proceeds with the minimum number of mechanisms that any regulatory network must include, a stress-induced activation and a feedback regulation, and can thus be regarded as the archetype for a cellular regulatory process. We propose here a simple mechanistic model for the eukaryotic heat shock response, including its mathematical validation. Based on numerical predictions of the model and on its sensitivity analysis, we minimize the model by identifying the reactions with marginal contribution to the heat shock response. As the heat shock response is a very basic and conserved regulatory network, our analysis of the network provides a useful foundation for modeling strategies of more complex cellular processes.     

Propagation and Dissemination Strategies of Transmissible Spongiform Encephalopathy Agents in Mammalian Cells

Transmissible spongiform encephalopathies or prion disorders are fatal infectious diseases that cause characteristic spongiform degeneration in the central nervous system. The causative agent, the so-called prion, is an unconventional infectious agent that propagates by converting the host-encoded cellular prion protein PrP into ordered protein aggregates with infectious properties. Prions are devoid of coding nucleic acid and thus rely on the host cell machinery for propagation. While it is now established that, in addition to PrP, other cellular factors or processes determine the susceptibility of cell lines to prion infection, exact factors and cellular processes remain broadly obscure. Still, cellular models have uncovered important aspects of prion propagation and revealed intercellular dissemination strategies shared with other intracellular pathogens. Here, we summarize what we learned about the processes of prion invasion, intracellular replication and subsequent dissemination from ex vivo cell models.     

Manganese chloride tetrahydrate (CMC-001) enhanced liver MRI: evaluation of efficacy and safety in healthy volunteers

Object

To evaluate the efficacy of three dose levels of the oral hepatobiliary manganese-based magnetic resonance imaging (MRI) contrast agent CMC-001, and assess its safety profile and patient acceptability.

Materials and methods

After ethics committee approval, 32 healthy volunteers (males/females: 18/14) were included. Liver MRI was performed before and 3?h after ingestion of 0.8, 0.4, and 0.2?g of CMC-001 on separate occasions. Liver-to-muscle signal intensity (SI) ratio from baseline to post-contrast and image quality was assessed. Adverse drug reactions/adverse events (ADRs/AEs) and clinico-laboratory tests were monitored.

Results

The increase in liver-to-muscle SI ratio was significantly higher after 0.8?g (0.696) compared to 0.4?g (0.458) and 0.2?g (0.223) (in all pair-wise comparisons, P?Conclusion Liver MRI using 0.8?g CMC-001 has the highest efficacy and still acceptable ADRs and should therefore be preferred.     

PET/MRI in cancer patients: first experiences and vision from Copenhagen

Combined PET/MRI systems are now commercially available and are expected to change the medical imaging field by providing combined anato-metabolic image information. We believe this will be of particular relevance in imaging of cancer patients. At the Department of Clinical Physiology, Nuclear Medicine & PET at Rigshospitalet in Copenhagen we installed an integrated PET/MRI in December 2011. Here, we describe our first clinical PET/MR cases and discuss some of the areas within oncology where we envision promising future application of integrated PET/MR imaging in clinical routine. Cases described include brain tumors, pediatric oncology as well as lung, abdominal and pelvic cancer. In general the cases show that PET/MRI performs well in all these types of cancer when compared to PET/CT. However, future large-scale clinical studies are needed to establish when to use PET/MRI. We envision that PET/MRI in oncology will prove to become a valuable addition to PET/CT in diagnosing, tailoring and monitoring cancer therapy in selected patient populations.     

Pre-dried refiner flakes allow very short or even continuous conching of milk chocolate

Chocolate conching is usually still performed in a time consuming batch process. For milk chocolate, its main purpose is to remove water and to adjust flow properties. As the conch is not an efficient dryer and flow properties are mainly influenced by shear treatment, the objective of this study was to separate and optimise both processes. Prior to liquefaction, refiner flakes were dried to <0.6% of water in an oven, which allows to omit the dry phase of conching. Conventional conching of 5 h was then reduced to a liquefaction process taking 20 min. Alternatively, continuous processing was tested applying a high shear mixer REFLECTOR^®. Sample properties were very close to a standard. Only continuous liquefaction at very high throughput resulted in slightly increased viscosity. Sensory analyses just found negligible differences. Thus neither pre-drying nor short or continuous liquefaction had a negative impact on product quality.     

Bestimmung von Gamma-Hydroxybuttersäure und Gamma- Hydroxybuttersäure- Metaboliten im Haar und deren forensische Bedeutung

Gamma-Hydroxybutyrate (GHB) has been subject of scientific research due to its endogenous occurrence in humans and at the same time sedating effect after exogenous uptake, whether intentional or not. Of particular interest in this context is the distinction between possible endogenous GHB-concentrations and those that were in hair after a (single) exogenous uptake. In the present study, methods of extraction and quantification by LC-MS/MS for GHB and its metabolites glycolic acid, 2,4-dihydroxybutanoic acid (2,4-DHB), 3,4-dihydroxybutanoic acid (3,4-DHB) and 4-(sulfooxy)butanoic acid (GHB-sulf) were established. After validation of those methods, hair samples of persons without known exogenous GHB uptake were analysed to define an endogenous reference range for each substance. Subsequently hair samples were analysed after single or regular uptake of GHB. GHB-concentrations of 928 hair samples from persons without known uptake of GHB were between < 0.1 and 6.3 ng/mg. Only 29 samples showed concentrations higher than 3.0 ng/mg. Interestingly, hair samples that were stored for two to eight years showed significantly higher GHB-concentrations between 1.5 and 130 ng/mg, with 48 of 52 analysed segments showing concentrations above 3.0 ng/mg. Thus, storing seems to have a greater effect on the observed GHB-concentration than the quantity of GHB taken. In accordance with that, of 136 analysed hair samples of persons with a daily uptake of 3 to 9 g GHB, only 25 of the measured concentrations were above 3.0 ng/mg and only three outside of the prior defined endogenous reference range. The association between the quantity of exogenous GHB-uptake and the observed GHB-concentration in hair was found to be very weak. In agreement with those results, no raise in GHB-concentration was observed in hair samples after a single uptake of GHB. All concentrations were in the lower range of the prior defined endogenous reference range. Regarding the four analysed GHB-metabolites, the following endogenous reference ranges (including outliers) were defined: glycolic acid 0.39 - 11 (24) ng/mg, GHB-sulf < 0.40 - 1.7 (12) ng/mg, 3,4-DHB < 0.10 - 0.63 (4.7) ng/mg and 2,4-DHB < 0.20 - 0.45 (0.65) ng/mg. In hair samples from persons with regular uptake of GHB, at least one metabolite was found to be elevated (i.e., above the prior defined endogenous reference range) in the average of each hair strand, in contrast to the observed GHB-concentrations. In four of eleven hair strands even all four metabolites were above their endogenous reference range. Nevertheless, no statistically significant association between the quantity of GHB-uptake and the observed concentration in hair was found. Hair strands examined after a single GHB-uptake partially showed elevated concentrations of GHB-metabolites. The elevated segments, however, were not in each case those one would expect assuming the average hair growth rate of 1 cm/month. In the present study, it was shown that the exogenous uptake of GHB does not necessarily result in an elevated GHB-concentration in hair. Thus, the sole quantification of GHB will most likely not allow a clear decision whether or not GHB has been taken up exogenously. Thus, the here developed analytical methods do not yield legal certainty. However, in combination with the detection of one or more elevated GHB-metabolite concentrations, a better evaluation of this issue seems possible.     

Efficient and inefficient aspects of residential energy behaviour: What are the policy instruments for change?

The empirical part of this study is based on a survey of 600 Swedish households and a number of interviews where questions about residential energy behaviour and possible policy instruments for change were raised. The study provides insight into current behavioural patterns and gives a bottom-up perspective on the realistic perspective potentials for change and ways to achieve them. Residential energy use accounts for a fifth of the total in Northern nations and patterns of behaviour may influence levels of energy use to the same extent as choice of appliances. The study revealed those behavioural patterns that are efficient and those that need to be improved for energy conservation. Several policy instruments for change were identified in the study and they include combinations of information, economic measures, administrative measures and more user friendly technology as well as equipment with sufficient esthetic quality. Policy instruments that have fostered energy efficient behaviour in Sweden include the massive information campaigns during the oil crises in the 1970s as well as energy labelling of appliances. Still, many households are “energy-unaware” and several energy efficient behaviours are motivated not by energy conservation concern but of a perceived lack of time. This shows that it is important to have a broad perspective in energy conservation, to evaluate trends and to use policy instruments timely to support or discourage them.     

Metallographic evidence of carbon diffusion in the growth of bainite

There are two paradigms regarding the formation of bainite. One is based on the first stage being rapid, diffusionless growth of acicular ferrite and the subsequent formation of carbide occurring by precipitation from the supersaturated ferrite. An assumption that the first stage occurs as a series of subsequent rapid steps resulting in sub-units plays an important role as an explanation of the not so rapid growth observed macroscopically. The other paradigm is based on the first stage being the formation of acicular ferrite under carbon diffusion and on the subsequent growth of carbide and ferrite side by side. Metallographic observations are presented that support the second paradigm. It is difficult to see how they can be accounted for by the first paradigm, in particular the observation of the shapes of sub-units.     

Texture as a reflection of microstructure

The perception of texture has been correlated to the microstructure of particulate whey protein gels. A full, two-level, factorial experimental design was used in which the processing conditions, pH, heating rate and addition of salt were used as design factors. The texture of the gels was analysed by a sensory panel, and the microstructure was analysed by light and electron microscopy. The microstructure was quantified by using different types of image analysis.

In this study of particulate whey protein gels, the test principles of analysing texture were divided into two groups: destructive tests and non-destructive tests. The microstructural parameters can also be divided into two groups: overall network dimensions (pore size and particle size) and strand characteristics. The texture as measured with destructive methods was sensitive to overall network dimensions, whereas texture as measured with non-destructive methods was sensitive to the strand characteristics of particulate protein gels.