Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.     

Exploiting the Potential of Public Procurement: Opportunities for Circular Economy

The objective of a circular economy (CE) is to maintain the value of products, materials, and resources in the economy by closing material loops and minimizing waste generation. In recent years, the role of public procurement has been recognized as an important, but as yet not fully exploited, opportunity by cities and municipalities in their transition toward circular societies. This study analyzed public procurement opportunities to promote CE. Different approaches and examples of circular public procurement were identified using case studies. In addition, opportunities to promote CE through sustainable and green public procurement policy were identified analyzing predefined sustainable or green public procurement criteria. The study concludes that public procurement can promote CE and related business models by setting criteria and requirements for the extension of product life spans, efficiency and/or intensity of use, and efficient cycling of biological or technical materials, as well as for the securing of clean and nonrisky cycles. Circular procurement can occur through the procurement of better‐quality products in circular terms, the procurement of new circular products, the use of business concepts that support the CE, and investments in circular ecosystems. Several sectors and product groups were identified as having potential for circular procurement, such as construction, waste, and wastewater management, transportation, food, and catering, furniture, and textiles. The study also suggests that the use of certain tools, such as performance‐based procurement, life cycle approach, and life cycle costing, as well as criteria concerning reuse and recycling of materials, could promote circular procurement. Market dialogue and cooperation between procurers and actors in the supply chains are important for the future development of circular procurement.     

Light partitioning among species and species replacement in early successional grasslands

Abstract. We studied canopy structure, shoot architecture and light harvesting efficiencies of the species (photon flux captured per unit above‐ground plant mass) in a series of exclosures of different age (up to 4.5 yr) in originally heavily grazed grassland in N Japan.Vegetation height and Leaf Area Index (LAI) increased in the series and Zoysia japonica, the dominant in the beginning, was replaced by the much taller Miscanthus sinensis. We showed how this displacement in dominance can be explained by inherent constraints on the above‐ground architecture of these two species. In all stands light capture of plants increased with their above‐ground biomass but taller species were not necessarily more efficient in light harvesting. Some subordinate species grew disproportionally large leaf areas and persisted in the shady undergrowth. Some other species first grew taller and managed to stay in the better‐lit parts of the canopy, but ultimately failed to match the height growth of their neighbours in this early successional series. Their light harvesting efficiencies declined and this probably led to their exclusion. By contrast, species that maintained their position high in the canopy managed to persist in the vegetation despite their relatively low light harvesting efficiencies. In the tallest stands ‘later successional’ species had higher light harvesting efficiencies for the same plant height than ‘early successional’ species which was mostly the result of the greater area to mass ratio (specific leaf area, SLA) of their leaves. This shows how plant stature, plasticity in above‐ground biomass partitioning, and architectural constraints determine the ability of plants to efficiently capture light, which helps to explain species replacement in this early successional series.     

Hereditary spastic paraplegia SPG13 is associated with a mutation in the gene encoding the mitochondrial chaperonin Hsp60

SPG13, an autosomal dominant form of pure hereditary spastic paraplegia, was recently mapped to chromosome 2q24-34 in a French family. Here we present genetic data indicating that SPG13 is associated with a mutation, in the gene encoding the human mitochondrial chaperonin Hsp60, that results in the V72I substitution. A complementation assay showed that wild-type HSP60 (also known as "HSPD1"), but not HSP60 (V72I), together with the co-chaperonin HSP10 (also known as "HSPE1"), can support growth of Escherichia coli cells in which the homologous chromosomal groESgroEL chaperonin genes have been deleted. Taken together, our data strongly indicate that the V72I variation is the first disease-causing mutation that has been identified in HSP60.     

Local tissue temperature increase of a generic implant compared to the basic restrictions defined in safety guidelines

The objective of this study was to investigate if persons with implantable medical devices are intrinsically protected by the current electromagnetic safety guidelines. For inter-laboratory comparisons, the U.S. Food and Drug Administration has defined a generic implant as consisting of an insulated wire with noninsulated tips, simulating active implants composed of a metallic case, and insulated wires with electric contacts at the tip. In this study, we determined the amplitude of the uniform electric fields induced in body tissues that cause a local increase in the tissue temperature by 1 °C in the presence of this generic implant for a wide range of frequencies and wire lengths. The field amplitudes were compared to the basic restrictions of the current exposure guidelines for both occupational and uncontrolled exposure. Results showed that a 1 °C temperature increase in the tissues around the tips of the generic implant can be reached for field strengths much smaller than 1% of those in the basic restrictions. The simulated results were validated by experimental evaluations. The impact of perfusion was investigated and was found to lead to a reduction in the local temperature peak by only 1.6-3 times. Additional simulations inside an inhomogeneous anatomical model were performed to ascertain whether similar heating as in the generic model was observed. The significant temperature elevations due to the presence of a generic implant indicate that demonstrating compliance with the basic restrictions might not be sufficient for persons with implants. Special considerations may be required, especially in the case of novel, emerging technologies that feature strong near-fields at frequencies below 10 MHz (e.g., wireless power-transfer systems).     

Influence of GLP-1 on Myocardial Glucose Metabolism in Healthy Men during Normo- or Hypoglycemia

Background and Aims

Glucagon-like peptide-1 (GLP-1) may provide beneficial cardiovascular effects, possibly due to enhanced myocardial energetic efficiency by increasing myocardial glucose uptake (MGU). We assessed the effects of GLP-1 on MGU in healthy subjects during normo- and hypoglycemia.

Materials and Methods

We included eighteen healthy men in two randomized, double-blinded, placebo-controlled cross-over studies. MGU was assessed with GLP-1 or saline infusion during pituitary-pancreatic normo- (plasma glucose (PG): 4.5 mM, n = 10) and hypoglycemic clamps (PG: 3.0 mM, n = 8) by positron emission tomography with ¹⁸fluoro-deoxy-glucose (¹⁸F-FDG) as tracer.

Results

In the normoglycemia study mean (± SD) age was 25±3 years, and BMI was 22.6±0.6 kg/m² and in the hypoglycemia study the mean age was 23±2 years with a mean body mass index of 23±2 kg/m². GLP-1 did not change MGU during normoglycemia (mean (+/− SD) 0.15+/−0.04 and 0.16+/−0.03 µmol/g/min, P = 0.46) or during hypoglycemia (0.16+/−0.03 and 0.13+/−0.04 µmol/g/min, P = 0.14). However, the effect of GLP-1 on MGU was negatively correlated to baseline MGU both during normo- and hypoglycemia, (P = 0.006, r² = 0.64 and P = 0.018, r² = 0.64, respectively) and changes in MGU correlated positively with the level of insulin resistance (HOMA 2IR) during hypoglycemia, P = 0.04, r² = 0.54. GLP-1 mediated an increase in circulating glucagon levels at PG levels below 3.5 mM and increased glucose infusion rates during the hypoglycemia study. No differences in other circulating hormones or metabolites were found.

Conclusions

While GLP-1 does not affect overall MGU, GLP-1 induces changes in MGU dependent on baseline MGU such that GLP-1 increases MGU in subjects with low baseline MGU and decreases MGU in subjects with high baseline MGU. GLP-1 preserves MGU during hypoglycemia in insulin resistant subjects.ClinicalTrials.gov registration numbers: {"type":"clinical-trial","attrs":{"text":"NCT00418288","term_id":"NCT00418288"}}NCT00418288: (hypoglycemia) and {"type":"clinical-trial","attrs":{"text":"NCT00256256","term_id":"NCT00256256"}}NCT00256256: (normoglycemia).     

Uranyl photoprobing of nonbent A/T- and bent A-tracts. A difference of flexibility?

In this study, we have systematically compared the uranyl photocleavage of a range of bent A-tracts and nonbent TA-tracts as well as interrupted A-tracts. We demonstrate that uranyl photocleavage of A-tracts and TA-tracts is almost identical, indicating a very similar minor groove conformation. Furthermore, a 10 base pair A-tract is divided into two independent tracts by an intervening TA or GC step. Uranyl probing also clearly distinguishes the bent A4T4 and the nonbent T4A4 sequences as adopting different structures, and our interpretation of the data is consistent with a structure for the bent A4T4 sequence that resembles a continuous A-tract, whereas the nonbent T4A4 sequences are closer to two independent and opposite A-tracts that cancel each other in terms of macroscopic bending. Finally, we also note that even single TA and TAT steps are highly sensitive to uranyl photocleavage and propose that in addition to average minor groove width, uranyl also senses DNA helix flexibility/deformability. Thus, the structural difference of TA-tracts and A-tracts may to a large extent reflect a difference in flexibility, and DNA curvature may consequently require a rigid narrow minor groove conformation that creates distinct A-tract-B-DNA junctions as the predominant cause of the bending.     

Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis

Background/Methods

The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.

Results

Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.

Conclusion

The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.