Unraveling motion in proteins by combining NMR relaxometry and molecular dynamics simulations: A case study on ubiquitin.

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields.

DASH: Dynamic Attention-Based Substructure Hierarchy for Partial Charge Assignment.

We present a robust and computationally efficient approach for assigning partial charges of atoms in molecules. The method is based on a hierarchical tree constructed from attention values extracted from a graph neural network (GNN), which was trained to predict atomic partial charges from accurate quantum-mechanical (QM) calculations. The resulting dynamic attention-based substructure hierarchy (DASH) approach provides fast assignment of partial charges with the same accuracy as the GNN itself, is software-independent, and can easily be integrated in existing parametrization pipelines, as shown for the Open force field (OpenFF). The implementation of the DASH workflow, the final DASH tree, and the training set are available as open source/open data from public repositories.

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation.

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from 15N relaxation and side-chain dynamics from 13C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.

Oral Diagnostic Methods for the Detection of Periodontal Disease.

Periodontitis is a common immune-inflammatory oral disease. Early detection plays an important role in its prevention and progression. Saliva is a reliable medium that mirrors periodontal health and is easily obtainable for identifying periodontal biomarkers in point-of-care diagnostics. The aim of this study is to evaluate the effectiveness of diagnostic salivary tests to determine periodontal status. Whole saliva (stimulated/unstimulated) from twenty healthy and twenty stage III grade B generalized periodontitis patients was tested for lactoferrin, alkaline phosphatase, calcium, density, osmolarity, pH, phosphate, buffer capacity, salivary flow rate and dynamic viscosity. A semi-quantitative urinary strip test was used to evaluate markers of inflammation in saliva (erythrocytes, leukocytes, urobilinogen, nitrite, glucose, bilirubin, and ketones), clinical periodontal parameters and pathogenic bacteria. Concentrations of lactoferrin, hemoglobin, and leukocytes were found to be significantly higher in the stimulated and unstimulated saliva in periodontitis patients compared to healthy patients, whereas alkaline phosphatase levels were higher in unstimulated saliva of periodontitis patients (p < 0.05). Periodontal biomarker analysis using test strips may be considered rapid and easy tool for distinguishing between periodontitis and healthy patients. The increase in lactoferrin, hemoglobin, and leucocytes-determined by strip tests-may provide a non-invasive method of periodontal diagnosis.

Density artefacts at interfaces caused by multiple time-step effects in molecular dynamics simulations.

Background: Molecular dynamics (MD) simulations have become an important tool to provide insight into molecular processes involving biomolecules such as proteins, DNA, carbohydrates and membranes. As these processes cover a wide range of time scales, multiple time-step integration methods are often employed to increase the speed of MD simulations. For example, in the twin-range (TR) scheme, the nonbonded forces within the long-range cutoff are split into a short-range contribution updated every time step (inner time step) and a less frequently updated mid-range contribution (outer time step). The presence of different time steps can, however, cause numerical artefacts. Methods: The effects of multiple time-step algorithms at interfaces between polar and apolar media are investigated with MD simulations. Such interfaces occur with biological membranes or proteins in solution. Results: In this work, it is shown that the TR splitting of the nonbonded forces leads to artificial density increases at interfaces. The presence of the observed artefacts was found to be independent of the interface shape and the thermostatting method used. It is further shown that integration with an impulse-wise reversible reference system propagation algorithm (RESPA) only shifts the occurrence of density artefacts towards larger outer time steps. Using a single-range (SR) treatment of the nonbonded interactions, on the other hand, resolves the density issue for pairlist-update periods of up to 40 fs. Conclusion: A SR scheme avoids numerical artefacts and offers an interesting alternative to TR RESPA with respect to performance optimization.

[What influences end-of-life decisions? Results of a representative German survey]. / Was beeinflusst Entscheidungen am Lebensende? Ergebnisse einer repräsentativen Umfrage in Deutschland.

BACKGROUND: Because of demographic changes of an aging society, palliative care is becoming increasingly important. It is therefore necessary to evaluate preferences at the end of life at an early stage to meet the needs and requests of future patients. OBJECTIVES: The aim of this study was to find out preferences in a theoretical scenario ("If you developed a serious medical condition such as cancer and you had less than a year to live…") regarding the desired involvement in decision-making at the end of life and the preferred place of death. MATERIALS AND METHODS: As part of the international PRISMA project, a representative telephone survey was carried out in Germany. RESULTS: A total of 1,363 Germans took part in the survey (response rate 29.0 %, 47.1 ± 15.7 years, 42 % male). 90.8 % wanted to make their own decisions with regard to end-of-life care, which was most important for people with higher education. 83.3 % wanted to predetermine decisions by means of an advance directive in case they are no longer able to make them at the time. This was again more important for individuals with higher education and for older subjects (≥ 65 years). The preferred place of death was their own home (63.3 %), and the least preferred place for death was in hospital in 48.2 %. In particular, women did not want to die in a hospital. CONCLUSION: These results should be considered when planning health care structures to meet the wishes of people at the end of their life, in particular to strengthen the importance of patient provision and the desire for their own home to be the preferred place to die.

Validity of the 3D VECTRA photogrammetric surface imaging system for cranio-maxillofacial anthropometric measurements.

PURPOSE: The use of three-dimensional (3D) photography for anthropometric measurements is of increasing interest, especially in the cranio-maxillofacial field. Before standard implementation, accurate determination of the precision and accuracy of each system is mandatory. METHODS: A mannequin head was labelled with 52 landmarks, and 28 three-dimensional images were taken using a commercially available five-pod 3D photosystem (3D VECTRA; Canfield, Fairfield, NJ) in different head positions. Distances between the landmarks were measured manually using a conventional calliper and compared with the digitally calculated distances acquired from labelling by two independent observers. The experimental set-up accounted for clinical circumstances by varying the positioning (vertical, horizontal, sagittal) of the phantom. RESULTS: In the entire calliper measurement data set (n = 410), a significant difference (p = 0.02) between the directly measured and corresponding virtually calculated distances was found. The mean aberration between both modalities covering all data was 7.96 mm. No differences (p = 0.94) between the two groups were found using a cut-off of 10 % (leaving n = 369 distances) due to considerable errors in direct measurements and the necessary manual data translation. The mean diversity of both measurement modalities after cut-off was 1.33 mm (maximum, 6.70 mm). Inter-observer analysis of all 1,326 distances showed no difference (p = 0.99; maximal difference, 0.58 mm) in the digital measurements. CONCLUSION: The precision and accuracy of this five-pod 3D photosystem suggests its suitability for clinical applications, particularly anthropometric studies. Three-hundred-and-sixty degree surface-contour mapping of the craniofacial region within milliseconds is particularly useful in paediatric patients. Proper patient positioning is essential for high-quality imaging.

Interleukin 6 stimulates endothelial binding and transport of high-density lipoprotein through induction of endothelial lipase.

OBJECTIVE: In the reverse cholesterol transport pathway, high-density lipoprotein (HDL) passes the endothelial cell barrier by mechanisms involving the scavenger receptor class B type I and the ATP-binding cassette G1. However, little is known on how inflammation influences this transendothelial transport. APPROACH AND RESULTS: On stimulation with interleukin-6, cultivated primary endothelial cells showed increased binding and transport of (125)I-HDL without changing the expression of scavenger receptor class B type I and ATP-binding cassette G1. Therefore, we analyzed the involvement of endothelial lipase (EL), a known HDL-binding protein expressed by endothelial cells. Here, we show an increased EL expression after interleukin-6 stimulation. Moreover, using pharmacological inhibitors or RNA interference against EL, we demonstrated its participation in HDL binding and transport through the endothelium. Furthermore, adenovirus-mediated transfection of endothelial cells with either catalytically active or nonactive EL revealed that EL facilitates the endothelial binding and transport by both bridging and lipolysis of HDL. EL was also found responsible for the reduction of HDL particle size occurring during the specific transport through a monolayer of endothelial cells. Finally, pharmacological inhibition of EL reversed the inducing effect of interleukin-6 on HDL binding and transport. CONCLUSIONS: Interleukin-6 stimulates the translocation of HDL through the endothelium, the first step in reverse cholesterol transport pathway, by enhancing EL expression. In addition, we demonstrated the role of EL in the transendothelial transport of HDL.

Improving oral hygiene skills by computer-based training: a randomized controlled comparison of the modified Bass and the Fones techniques.

BACKGROUND: Gingivitis and other plaque-associated diseases have a high prevalence in western communities even though the majority of adults report daily oral hygiene. This indicates a lack of oral hygiene skills. Currently, there is no clear evidence as to which brushing technique would bring about the best oral hygiene skills. While the modified Bass technique is often recommended by dentists and in textbooks, the Fones technique is often recommended in patient brochures. Still, standardized comparisons of the effectiveness of teaching these techniques are lacking. METHODOLOGY/PRINCIPAL FINDINGS: In a final sample of n = 56 students, this multidisciplinary, randomized, examiner-blinded, controlled study compared the effects of parallel and standardized interactive computer presentations teaching either the Fones or the modified Bass technique. A control group was taught the basics of tooth brushing alone. Oral hygiene skills (remaining plaque after thorough oral hygiene) and gingivitis were assessed at baseline and 6, 12, and 28 weeks after the intervention. We found a significant group×time interaction for gingivitis (F(4/102) = 3.267; p = 0.016; ε = 0.957; η(2) = 0.114) and a significant main effect of group for oral hygiene skills (F(2/51) = 7.088; p = 0.002; η(2) = 0.218). Fones was superior to Bass; Bass did not differ from the control group. Group differences were most prominent after 6 and 12 weeks. CONCLUSIONS/SIGNIFICANCE: The present trial indicates an advantage of teaching the Fones as compared to the modified Bass technique with respect to oral hygiene skills and gingivitis. Future studies are needed to analyze whether the disadvantage of teaching the Bass technique observed here is restricted to the teaching method employed. TRIAL REGISTRATION: German Clinical Trials Register DRKS00003488.

High-density lipoprotein transport through aortic endothelial cells involves scavenger receptor BI and ATP-binding cassette transporter G1.

Cholesterol efflux from macrophage foam cells is a rate-limiting step in reverse cholesterol transport. In this process cholesterol acceptors like high-density lipoproteins (HDL) and apolipoprotein (apo)A-I must cross the endothelium to get access to the donor cells in the arterial intima. Previously, we have shown that apoA-I passes a monolayer of aortic endothelial cells (ECs) from the apical to the basolateral side by transcytosis, which is modulated by the ATP-binding cassette transporter (ABC)A1. Here, we analyzed the interaction of mature HDL with ECs. ECs bind HDL in a specific and saturable manner. Both cell surface biotinylation experiments and immunofluorescence microscopy of HDL recovered approximately 30% of the cell-associated HDL intracellularly. Cultivated on inserts ECs bind, internalize, and translocate HDL from the apical to the basolateral compartment in a specific and temperature-dependent manner. The size of the translocated particle was reduced, but its protein moiety remained intact. Using RNA interference, we investigated the impact of SR-BI, ABCA1, and ABCG1 on binding, internalization, and transcytosis of HDL by ECs. HDL binding was reduced by 50% and 30% after silencing of SR-BI and ABCG1, respectively, but not at all after diminishing ABCA1 expression. Knock down of SR-BI and, even more so, ABCG1 reduced HDL transcytosis but did not affect inulin permeability. Cosilencing of both proteins did not further reduce HDL binding, internalization, or transport. In conclusion, ECs transcytose HDL by mechanisms that involve either SR-BI or ABCG1 but not ABCA1.