81 to 0.90, indicating good reliability. Intramethod reliability estimated by ICCa(3,1) was 0.86 with a 95% confidence interval of 0.81 to 0.90, also indicating good reliability. Bland-Altman analysis showed a difference of 0.175 (0.49), and limits of agreement ranged from -0.79 to 1.14. CONCLUSIONS The pain level module on the app is highly reliable and interchangeable with the paper VAS version. This tool could potentially help clinicians and researchers precisely assess pain in a simple, economic way with the use of a ubiquitous technology. ©Carles Escalona-Marfil, Andrea Coda, Jorge Ruiz-Moreno, Lluís Miquel Riu-Gispert, Xavier Gironès. Originally published in the Journal of Medical Internet Research (http//www.jmir.org), 12.02.2020.BACKGROUND Defining the transition from relapsing-remitting multiple sclerosis (RRMS) to secondary progressive multiple sclerosis (SPMS) can be challenging and delayed. A digital tool (MSProDiscuss) was developed to facilitate physician-patient discussion in evaluating early, subtle signs of multiple sclerosis (MS) disease progression representing this transition. OBJECTIVE This study aimed to determine cut-off values and corresponding sensitivity and specificity for predefined scoring algorithms, with or without including Expanded Disability Status Scale (EDSS) scores, to differentiate between RRMS and SPMS patients and to evaluate psychometric properties. METHODS Experienced neurologists completed the tool for patients with confirmed RRMS or SPMS and those suspected to be transitioning to SPMS. In addition to age and EDSS score, each patient’s current disease status (disease activity, symptoms, and its impacts on daily life) was collected while completing the draft tool. Receiver operating characteristic (Rclass correlation coefficient=.95). CONCLUSIONS The MSProDiscuss tool differentiated RRMS patients from SPMS patients with high sensitivity and specificity. In clinical practice, it may be a useful tool to evaluate early, subtle signs of MS disease progression indicating the evolution of RRMS to SPMS. MSProDiscuss will help assess the current level of progression in an individual patient and facilitate a more informed physician-patient discussion. ©Tjalf Ziemssen, Daniela Piani-Meier, Bryan Bennett, Chloe Johnson, Katie Tinsley, Andrew Trigg, Thomas Hach, Frank Dahlke, Davorka Tomic, Chloe Tolley, Mark S Freedman. Originally published in the Journal of Medical Internet Research (http//www.jmir.org), 12.02.2020.BACKGROUND It is not well established whether a virtual multidisciplinary care program for persons with advanced chronic kidney disease (CKD) can improve their knowledge about their disease, increase their interest in home dialysis therapies, and result in more planned outpatient (versus inpatient) dialysis starts. OBJECTIVE We aimed to evaluate the feasibility and preliminary associations of program participation with disease knowledge, home dialysis modality preference, and outpatient dialysis initiation among persons with advanced CKD in a community-based nephrology practice. METHODS In a matched prospective cohort, we enrolled adults aged 18 to 85 years with at least two estimated glomerular filtration rates (eGFRs) of less than 30 mL/min/1.73 m2 into the Cricket Health program and compared them with controls receiving care at the same clinic, matched on age, gender, eGFR, and presence of heart failure and diabetes. The intervention included online education materials, a virtual multidisciplinary team (nung versus 22% (2/9) of control patients (hazard ratio 6.89, 95% CI 1.46-32.66). Compared to before participation, patients who completed the program had higher disease knowledge levels (mean 52%, SD 29% versus mean 94%, SD 14% of questions correct on knowledge-based survey, P less then .001) and were more likely to choose a home modality as their first dialysis choice (36%, 7/22 versus 68%, 15/22, P=.047) after program completion. CONCLUSIONS The Cricket Health program can improve patient knowledge about CKD and increase interest in home dialysis modalities, and may increase the proportion of dialysis starts in the outpatient setting. ©Paulina Kaiser, Olivia Pipitone, Anthony Franklin, Dixie R Jackson, Elizabeth A Moore, Christopher R Dubuque, Carmen A Peralta, Anthony C De Mory. Originally published in the Journal of Medical Internet Research (http//www.jmir.org), 12.02.2020.Stem Cell Reports frequently receives manuscripts dealing with the topic of cancer stem cells. Many of the submissions on this topic have major shortcomings in their content or limits to the conclusions that can be drawn from the results presented. The purpose of this Commentary is to highlight some of the underlying issues so that authors can enhance the strength of their research contributions. Populations of genetically identical cells generally show a large variability in cell phenotypes, which is typically associated with the stochastic nature of gene expression processes. It is widely believed that a significant source of such randomness is transcriptional bursting, which is when periods of active production of RNA molecules alternate with periods of RNA degradation. However, the molecular mechanisms of such strong fluctuations remain unclear. Recent studies suggest that DNA supercoiling, which happens during transcription, might be directly related to the bursting behavior. Stimulated by these observations, we developed a stochastic mechanochemical model of supercoiling-induced transcriptional bursting in which the RNA synthesis leads to the buildup of torsion in DNA. This slows down the RNA production until it is bound by the enzyme gyrase to DNA, which releases the stress and allows for the RNA synthesis to restart with the original rate. Using a thermodynamically consistent coupling between mechanical and chemical processes, the dynamic properties of transcription are explicitly evaluated. Selleck PDD00017273 In addition, a first-passage method to evaluate the dynamics of transcription is developed. Theoretical analysis shows that transcriptional bursting is observed when both the supercoiling and the mechanical stress release due to gyrase are present in the system. It is also found that the overall RNA production rate is not constant and depends on the number of previously synthesized RNA molecules. A comparison with experimental data on bacteria allows us to evaluate the energetic cost of supercoiling during transcription. It is argued that the relatively weak mechanochemical coupling might allow transcription to be regulated most effectively. The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pretransition at 72°C with an unfolding enthalpy of ΔHcal ∼200-300 kcal/mol and a main transition at 85°C with an enthalpy of ∼900-1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analyzed with the multistate Zimm-Bragg theory. For the investigated mAb, unfolding is characterized by a cooperativity parameter σ ∼6 × 10-5 and a Gibbs free energy of unfolding of gnu ∼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, ν∼220 ± 50 amino acids are involved in the pretransition and ν∼850 ± 30 in the main transition, accounting for ∼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔHcal and lowers the midpoint temperature Tm. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25°C are predicted to be 2-3 M for the pretransition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCl molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pretransition and 0.55 for the main transition. The pretransition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody. Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates the exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated the effect, using NMR spectroscopy, of substituting key charged Arg, Lys, Glu, and Asp residues by Gly or His. Results indicate that such mutations result in the transient breaking of the helix at the site of mutation but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH stability. Whereas a single Gly substitution caused transient breaks ∼20% of the time, two consecutive Gly substitutions break the helix ∼65% of the time. NMR relaxation measurements indicate that the exchange rate between an intact and a broken helix is fast (>300,000 s-1) and that for the wild-type sequence, the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, in which the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines. Published by Elsevier Inc.Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM. Published by Elsevier Inc.