133 research outputs found
A FRET-based method for monitoring structural transitions in protein self-organization
Proteins assemble into a variety of dynamic and functional structures. Their structural transitions are often challenging to distinguish inside cells, particularly with a high spatiotemporal resolution. Here, we present a fluorescence resonance energy transfer (FRET)-based method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor self-assembly of three proteins. We show that the mutant Huntingtin exon1 (mHttex1) first forms less-ordered assemblies, which develop into fibril-like aggregates, and demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1. We also monitor the structural changes in fused in sarcoma (FUS) condensates. This method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation
Combining Individual-Level Discrete Choice Experiment Estimates and Costs to Inform Health Care Management Decisions about Customized Care: The Case of Follow-Up Strategies after Breast Cancer Treatment
AbstractObjectiveCustomized care can be beneficial for patients when preferences for health care programs are heterogeneous. Yet, there is little guidance on how individual-specific preferences and cost data can be combined to inform health care decisions about customized care. Therefore, we propose a discrete choice experiment–based approach that illustrates how to analyze the cost-effectiveness of customized (and noncustomized) care programs to provide information for hospital managers.MethodsWe exploit the fact that choice models make it possible to determine whether preference heterogeneity exists and to obtain individual-specific parameter estimates. We present an approach of how to combine these individual-specific parameter estimates from a random parameter model (mixed logit model) with cost data to analyze the cost-effectiveness of customized care and demonstrate our method in the case of follow-up after breast cancer treatment.ResultsWe found that there is significant preference heterogeneity for all except two attributes of breast cancer treatment follow-up and that the fully customized care program leads to higher utility and lower costs than the current standardized program. Compared with the single alternative program, the fully customized care program has increased benefits and higher costs. Thus, it is necessary for health care decision makers to judge whether the use of resources for customized care is cost-effective.ConclusionsDecision makers should consider using the results obtained from our methodological approach when they consider implementing customized health care programs, because it may help to find ways to save costs and increase patient satisfaction
Erratum
We acknowledge cofunding of the research from the EU ITN project SynCrop (project number 764591), appointment of Luca Mantovanelli
A physicochemical roadmap of yeast replicative aging
Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding, are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show the macromolecular crowding changes less in longer-lived cells in contrast to shorter-lived cells. While the average pH and crowding levels change only modestly with aging, we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively-aged yeast cells
A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast
Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells
A physicochemical perspective of aging from single-cell analysis of ph, macromolecular and organellar crowding in yeast
Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells. © 2020, eLife Sciences Publications Ltd. All rights reserved
Evaluating the Age-Based Recommendations for Long-Term Follow-Up in Breast Cancer
Background: After 5 years of annual follow-up following breast cancer, Dutch guidelines are age based: annual follow-up for women <60 years, 60–75 years biennial, and none for >75 years. We determined how the risk of recurrence corresponds to these consensus-based recommendations and to the risk of primary breast cancer in the general screening population. Subjects, Materials, and Methods: Women with early-stage breast cancer in 2003/2005 were selected from the Netherlands Cancer Registry (n = 18,568). Cumulative incidence functions were estimated for follow-up years 5–10 for locoregional recurrences (LRRs) and second primary tumors (SPs). Risks were compared with the screening population without history of breast cancer. Alternative cutoffs for age were determined by log-rank tests. Results: The cumulative risk for LRR/SP was lower in women <60 years (5.9%, 95% confidence interval [CI] 5.3–6.6) who are under annual follow-up than for women 60–75 (6.3%, 95% CI 5.6–7.1) receiving biennial visits. All risks were higher than the 5-year risk of a primary tumor in the screening population (ranging from 1.4% to 1.9%). Age cutoffs <50, 50–69, and > 69 revealed better risk differentiation and would provide more risk-based schedules. Still, other factors, including systemic treatments, had an even greater impact on recurrence risks. Conclusion: The current consensus-based recommendations use suboptimal age cutoffs. The proposed alternative cutoffs will lead to a more balanced risk-based follow-up and thereby more efficient allocation of resources. However, more factors should be taken into account for truly individualizing follow-up based on risk for recurrence. Implications for Practice: The current age-based recommendations for breast cancer follow-up after 5 years are suboptimal and do not reflect the actual risk of recurrent disease. This results in situations in which women with higher risks actually receive less follow-up than those with a lower risk of recurrence. Alternative cutoffs could be a start toward risk-based follow-up and thereby more efficient allocation of resources. However, age, or any single risk factor, is not able to capture the risk differences and therefore is not sufficient for determining follow-up. More risk factors should be taken into account for truly individualizing follow-up based on the risk for recurrence.</p
Clinical Trial Strategies to Compare Protons With Photons
The favorable beam properties of protons can be translated into clinical benefits by target dose escalation to improve local control without enhancing unacceptable radiation toxicity or to spare normal tissues to prevent radiation-induced side effects without jeopardizing local tumor control. For the clinical validation of the added value of protons to improve local control, randomized controlled trials are required. For the clinical validation of the added value of protons to prevent side effects, both model-based validation or randomized controlled trials can be used. Model-based patient selection for proton therapy is crucial, independent of the validation approach. Combining these approaches in rapid learning health care systems is expected to yield the most efficient and scientifically sound way to continuously improve patient selection and the therapeutic window, eventually leading to more cancer survivors with better quality of life. (C) 2018 The Authors. Published by Elsevier Inc
The influence of fluorescent protein maturation on FRET measurements in living cells
Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet identifying and preventing potential artifacts are needed to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression both in prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster maturing FRET donors. We developed an improved version of the donor mTurquoise2, which matures faster than the parental protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically-encoded FRET sensors
National Protocol for Model-Based Selection for Proton Therapy in Head and Neck Cancer
In the Netherlands, the model-based approach is used to identify patients with head and neck cancer who may benefit most from proton therapy in terms of prevention of late radiation-induced side effects in comparison with photon therapy. To this purpose, a National Indication Protocol Proton therapy for Head and Neck Cancer patients (NIPP-HNC) was developed, which has been approved by the health care authorities. When patients qualify according to the guidelines of the NIPP-HNC, proton therapy is fully reimbursed. This article describes the procedures that were followed to develop this NIPP-HNC and provides all necessary information to introduce model-based selection for patients with head and neck cancer into routine clinical practice.</p
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