All phosphagen kinases contain a conserved cysteine residue which has been shown by crystallographic studies, on both creatine kinase and arginine kinase, to be located in the active site. There are conflicting reports as to whether this cysteine is essential for catalysis. In this study we have used site-directed mutagenesis to replace Cys282 of human muscle creatine kinase with serine and methionine. In addition, we have replaced Cys282, conserved across all creatine kinases, with alanine. No activity was found with the C282M mutant. The C282S mutant showed significant, albeit greatly reduced, activity in both the forward (creatine phosphorylation) and reverse (MgADP phosphorylation) reactions. The K(m) for creatine was increased approximately 10-fold, but the K(m) for phosphocreatine was relatively unaffected. The V and V/K pH-profiles for the wild-type enzyme were similar to those reported for rabbit muscle creatine kinase, the most widely studied creatine kinase isozyme. However, the V/K(creatine) profile for the C282S mutant was missing a pK of 5.4. This suggests that Cys282 exists as the thiolate anion, and is necessary for the optimal binding of creatine. The low pK of Cys282 was also determined spectrophotometrically and found to be 5.6 +/- 0.1. The S284A mutant was found to have reduced catalytic activity, as well as a 15-fold increase in K(m) for creatine. The pK(a) of Cys282 in this mutant was found to be 6.7 +/- 0.1, indicating that H-bonding to Ser284 is an important, but not the sole, factor contributing to the unusually low pK(a) of Cys282.

Disease modelling has had considerable policy impact during the ongoing COVID-19 pandemic, and it is increasingly acknowledged that combining multiple models can improve the reliability of outputs. Here we report insights from ten weeks of collaborative short-term forecasting of COVID-19 in Germany and Poland (12 October–19 December 2020). The study period covers the onset of the second wave in both countries, with tightening non-pharmaceutical interventions (NPIs) and subsequently a decay (Poland) or plateau and renewed increase (Germany) in reported cases. Thirteen independent teams provided probabilistic real-time forecasts of COVID-19 cases and deaths. These were reported for lead times of one to four weeks, with evaluation focused on one- and two-week horizons, which are less affected by changing NPIs. Heterogeneity between forecasts was considerable both in terms of point predictions and forecast spread. Ensemble forecasts showed good relative performance, in particular in terms of coverage, but did not clearly dominate single-model predictions. The study was preregistered and will be followed up in future phases of the pandemic.

BackgroundIn order to better understand the perspectives of patients and physicians regarding the treatment and management of rheumatoid arthritis (RA), we present and compare results from a patient-based and a physician-based survey developed by the RA NarRAtive advisory panel.MethodsThe RA NarRAtive initiative is directed by a global advisory panel of 39 healthcare providers and patient organization leaders from 17 countries. A survey of patients self-reporting a diagnosis of RA and a physician-based survey, designed by the advisory panel, were fielded online by Harris Poll from September 2014 to April 2016, and from August 2015 to October 2015, respectively.ResultsWe present findings from 1805 patients whose RA was primarily managed by a rheumatologist, and 1736 physicians managing patients with RA. Results confirmed that RA carries a substantial disease burden; half of the patients surveyed reported stopping participation in certain activities as a result of their disease. While 90% of physicians were satisfied with their communications with their patients regarding RA treatment, 61% of patients felt uncomfortable raising concerns or fears with their physician. Of the patients providing responses, 52% felt that improved dialogue/discussion would optimize their RA management, and 68% of physicians wished that they and their patients talked more about their RA goals and treatment. Overall, 88% of physicians agreed that patients involved in making treatment decisions tend to be more satisfied with their treatment experience.ConclusionThe results of these surveys highlight the impact of RA on patients, and a discrepancy between patient and physician views on communication. Further research, focused on improving patient–physician dialogue, shared goal-setting, and treatment planning, is needed.Electronic supplementary materialThe online version of this article (10.1186/s12955-018-1035-3) contains supplementary material, which is available to authorized users.

The individual enantiomers 8 and 12 of the potent and highly selective racemic A1-adenosine antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine (ENX, 4) were synthesized utilizing asymmetric Diels-Alder cycloadditions for the construction of the norbornane moieties. The absolute configuration of 12 was determined by X-ray crystallography of the 4-bromobenzoate 14, which was derived from the bridged secondary alcohol 13. The latter was obtained from 12 by an acid-catalyzed intramolecular rearrangement. The binding affinities of the enantiomers 8 and 12 and the racemate 4 at guinea pig, rat, and cloned human A1- and A2a-adenosine receptor subtypes were determined. The S-enantiomer 12 (CVT-124) appears to be one of the more potent and clearly the most A1-selective antagonist reported to date, with K1 values of 0.67 and 0.45 nM, respectively, at the rat and cloned human A1-receptors and with 1800-fold (rat) and 2400-fold (human) subtype selectivity. Both enantiomers, administered intravenously to saline-loaded rats, induced diuresis via antagonism of renal A1-adenosine receptors.