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SARS-CoV-2 infections led to a worldwide pandemic in 2020. As of 2024, therapeutics against SARS-CoV-2 have continued to be desirable. NSP14 is a dual-function methyltransferase (MTase) and exonuclease (ExoN) with key roles in SARS-CoV-2 genome propagation and host immune system evasion. In this work, we developed high-throughput screening (HTS) assays for NSP14 MTase and ExoN activities. We screened both activities against a collection of 40,664 compounds. A total of 1677 initial hit compounds were identified, cherrypicked, counterscreened for assay interference, and screened for off-target selectivity. We identified 396 and 174 high-quality hits against the MTase and ExoN activities, respectively. Along with inhibitors for individual activities, we identified dual-activity inhibitors, including a novel inhibitor that is not competitive with any substrate and interacts with a putative allosteric binding site. This study represents the largest published screen of SARS-CoV-2 NSP14 MTase and ExoN activities to date and culminates in a pipeline for the NSP14 drug discovery. more...

Mutations in RAS and PI3Kα are major drivers of human cancer. Their interaction plays a crucial role in activating PI3Kα and amplifying the PI3K-AKT-mTOR pathway. Disrupting RAS-PI3Kα interaction enhances survival in lung and skin cancer models and reduces tumor growth and angiogenesis, although the structural details of this interaction remain unclear. Here, we present structures of KRAS, RRAS2, and MRAS bound to the catalytic subunit (p110α) of PI3Kα, elucidating the interaction interfaces and local conformational changes upon complex formation. Structural and mutational analyses highlighted key residues in RAS and PI3Kα impacting binding affinity and revealed isoform-specific differences at the interaction interface in RAS and PI3K isoforms, providing a rationale for their differential affinities. Notably, in the RAS-p110α complex structures, RAS interaction with p110α is limited to the RAS-binding domain and does not involve the kinase domain. This study underscores the pivotal role of the RAS-PI3Kα interaction in PI3Kα activation and provides a blueprint for designing PI3Kα isoform-specific inhibitors to disrupt this interaction., Competing Interests: Competing interests: All authors, except F.M., have no competing interests. F.M. is a consultant for Ideaya Biosciences, Kura Oncology, Leidos Biomedical Research, Pfizer, Daiichi Sankyo, Amgen, PMV Pharma, OPNA-IO, and Quanta Therapeutics, has received research grants from Boehringer-Ingelheim, and is a consultant for and cofounder of BridgeBio Pharma., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.) more...

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the β and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases., Competing Interests: Declaration of Competing interest The authors declare they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.) more...

Tobacco-etch-virus (TEV) protease is the workhorse of many laboratories in which protein expression is the linchpin of downstream experiments. TEV protease is remarkable in its sequence specificity as the cleavage sequence rarely appears in higher organisms and its ability to cleave fusion tag proteins from proteins of interest. Herein we report work done on large-scale production of TEV protease using different promotors, media, fusion tags, and expression platforms. During our work we detected post-translational modification (gluconoylation and phosphogluconoylation) of TEV protease and the subsequent effects this has on the purity of the protein. Subsequently we made our pgl plus bacteria that negates these modifications and their effects. We also introduce a GFP-based assay for measurement of activity and ultimately a new set of protocols for producing 400-500 mg/L TEV protease., Competing Interests: Conflicts of interest The authors declare they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.) more...

Humanity is facing a polycrisis, including climate change, loss of biodiversity, and economic inequalities. This commentary examines the role of physical activity (PA) research in the context of climate change, highlighting 3 perspectives: PA as a victim, as a perpetrator, and as part of the solution. The first perspective conceptualizes PA as a victim of climate change, as heat waves, extreme weather events, and high ozone levels pose a threat to health-enhancing PA and sports; adaptation strategies are needed and potentially lifesaving. The second perspective describes PA as a perpetrator to climate change due to the significant carbon footprint of some forms of PA and sport. The third perspective focuses on PA as a solution to climate change, as some forms of PA-such as active transport and urban gardening-have both individual and planetary health benefits. In addition, PA provides health benefits in extreme situations by reducing stress and maintaining health in times of crisis. Each of these 3 perspectives can guide future research in the field of PA and health. more...

The diversity of chemical and structural attributes of proteins makes it inherently difficult to produce a wide range of proteins in a single recombinant protein production system. The nature of the target proteins themselves, along with cost, ease of use, and speed, are typically cited as major factors to consider in production. Despite a wide variety of alternative expression systems, most recombinant proteins for research and therapeutics are produced in a limited number of systems: Escherichia coli, yeast, insect cells, and the mammalian cell lines HEK293 and CHO. Recent interest in Vibrio natriegens as a new bacterial recombinant protein expression host is due in part to its short doubling time of ≤ 10 min but also stems from the promise of compatibility with techniques and genetic systems developed for E. coli. We successfully incorporated V. natriegens as an additional bacterial expression system for recombinant protein production and report improvements to published protocols as well as new protocols that expand the versatility of the system. While not all proteins benefit from production in V. natriegens, we successfully produced several proteins that were difficult or impossible to produce in E. coli. We also show that in some cases, the increased yield is due to higher levels of properly folded protein. Additionally, we were able to adapt our enhanced isotope incorporation methods for use with V. natriegens. Taken together, these observations and improvements allowed production of proteins for structural biology, biochemistry, assay development, and structure-based drug design in V. natriegens that were impossible and/or unaffordable to produce in E. coli., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.) more...

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment., Competing Interests: Declaration of Competing interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.) more...

Development of new targeted inhibitors for oncogenic KRAS mutants may benefit from insight into how a given mutation influences the accessibility of protein residues and how compounds interact with mutant or wild-type KRAS proteins. Targeted proteomic analysis, a key validation step in the KRAS inhibitor development process, typically involves both intact mass- and peptide-based methods to confirm compound localization or quantify binding. However, these methods may not always provide a clear picture of the compound binding affinity for KRAS, how specific the compound is to the target KRAS residue, and how experimental conditions may impact these factors. To address this, we have developed a novel top-down proteomic assay to evaluate in vitro KRAS4B-compound engagement while assessing relative quantitation in parallel. We present two applications to demonstrate the capabilities of our assay: maleimide-biotin labeling of a KRAS4B G12D cysteine mutant panel and treatment of three KRAS4B proteins (WT, G12C, and G13C) with small molecule compounds. Our results show the time- or concentration-dependence of KRAS4B-compound engagement in context of the intact protein molecule while directly mapping the compound binding site. more...

Background: Accurate and fast measurement of physical activity is important for surveillance. Even though many physical activity questionnaires (PAQ) are currently used in research, it is unclear which of them is the most reliable, valid, and easy to use. This systematic review aimed to identify existing brief PAQs, describe and compare their measurement properties, and assess their level of readability., Methods: We performed a systematic review based on the PRISMA statement. Literature searches were conducted in six scientific databases. Articles were included if they evaluated validity and/or reliability of brief (i.e., with a maximum of three questions) physical activity or exercise questionnaires intended for healthy adults. Due to the heterogeneity of studies, data were summarized narratively. The level of readability was calculated according to the Flesch-Kincaid formula., Results: In total, 35 articles published in English or Spanish were included, evaluating 32 distinct brief PAQs. The studies indicated moderate to good levels of reliability for the PAQs. However, the majority of results showed weak validity when validated against device-based measurements and demonstrated weak to moderate validity when validated against other PAQs. Most of the assessed PAQs met the criterion of being "short," allowing respondents to complete them in less than one minute either by themselves or with an interviewer. However, only 17 questionnaires had a readability level that indicates that the PAQ is easy to understand for the majority of the population., Conclusions: This review identified a variety of brief PAQs, but most of them were evaluated in only a single study. Validity and reliability of short and long questionnaires are found to be at a comparable level, short PAQs can be recommended for use in surveillance systems. However, the methods used to assess measurement properties varied widely across studies, limiting the comparability between different PAQs and making it challenging to identify a single tool as the most suitable. None of the evaluated brief PAQs allowed for the measurement of whether a person fulfills current WHO physical activity guidelines. Future development or adaptation of PAQs should prioritize readability as an important factor to enhance their usability., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tcymbal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.) more...

KRAS , the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a's thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably, KRAS4a exhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approaching KRAS4b in the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells. more...

We present a novel method to determine engagement and specificity of KRAS4B-targeting compounds in vitro. By employing top-down mass spectrometry (MS), which analyzes intact and modified protein molecules (proteoforms), we can directly visualize and confidently characterize each KRAS4B species within compound-treated samples. Moreover, by employing targeted MS2 fragmentation, we can precisely localize each compound molecule to a specific residue on a given KRAS4B proteoform. This method allows us to comprehensively evaluate compound specificity, clearly detect nonspecific binding events, and determine the order and frequency with which they occur. We provide two proof-of-concept examples of our method employing publicly available compounds, along with detailed protocols for sample preparation, top-down MS data acquisition, targeted proteoform MS2 fragmentation, and analysis of the resulting data. Our results demonstrate the concentration dependence of KRAS4B-compound engagement and highlight the ability of top-down MS to directly map compound binding location(s) without disrupting the KRAS4B primary structure. Our hope is that this novel method may help accelerate the identification of new successful targeted inhibitors for KRAS4B and other RAS isoforms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) more...

Background: Even though the importance of physical activity policy monitoring has increased in the last decade, there is a lack of understanding what different approaches exist and which methodology they employ. In order to address this research gap, this review attempts to map existing approaches of physical activity policy monitoring and to analyse methodological aspects, especially with regards to the roles of governments and researchers., Methods: A systematic search was conducted in five scientific databases (PubMed, Scopus, SportDiscus, Psycinfo, Web of Knowledge) in July 2021, and the identified records were screened independently by two reviewers. Records were included if they (a) focused on the monitoring of public policies to promote PA, (b) allowed to compare policies across time, across nations/regions or across policy sectors, and (c) were written in English, German or Russian. During full text analysis, information on methodological aspects was extracted and studies were categorized based on the level of government involvement., Results: The search yielded in a total of 112 studies. 86 of these studies (76.8%) followed a research-driven approach (little or no government involvement) while only two studies (1.8%) were based on a government-driven approach (led by governments). The remaining 24 studies (21.4%) were based on a co-production approach (strong collaboration between researchers and governments). All in all, 18 different tools for physical activity policy monitoring were identified; key examples are the Report Cards on Physical Activity for Children and Youth (research-driven approach), the HEPA Monitoring Framework (government-driven approach) and the HEPA Policy Audit Tool (co-production approach)., Conclusions: The level of government involvement in policy monitoring differs significantly, and research-driven, government-driven and co-production approaches can be distinguished. These approaches have different strengths and weaknesses, and can be linked to distinct theories of change and models on research-policy relations. Increasing awareness on the implications of these approaches is key to improve the understanding and further development of physical activity policy monitoring., (© 2023. The Author(s).) more...

Introduction: While there are several approaches to collect basic information on physical activity (PA) promotion policies, some governments require more in-depth overviews on the situation in their country. In Germany, the Federal Ministry of Health expressed its interest in collecting detailed data on target group specific PA promotion, as relevant competences are distributed across a wide range of political levels and sectors. This study describes the development of a policy brief on physical activity promotion for children and adolescents in Germany. In particular, it addresses two major gaps in the current literature by systematically assessing good practice examples and "routine practices," i.e., PA promotion activities already taking place on large scale and regular basis., Materials and Methods: Based on relevant national and international guidelines, the TARGET:PA tool was co-produced by researchers and ministry officials. It includes (1) PA recommendations, (2) national prevalence rates, (3) recommendations for PA promotion, and data on national (4) routine practices, (5) good practice projects and (6) policies. Data were collected for children and adolescents in Germany using desk research, semi-structured interviews and secondary data analysis., Results: A policy brief and scientific background document were developed. Results showed that 46% of the 4-5-year-olds fulfil WHO recommendations but only 15% of the 11-17-year-olds, and that girls are less active than boys. Currently, in Germany no valid data are available on the PA behaviour of children under the age of three. An overview of routine practices for PA promotion for children and adolescents was compiled, and experts were asked to critically assess their effectiveness, reach and durability. Overall, 339 target group specific projects for PA promotion were found, with 22 classified as examples of good practice. National PA policies for children and adolescents were identified across different sectors and settings., Conclusion: The study provides a comprehensive overview of the current status of PA promotion for children and adolescents in Germany. The co-production of the policy brief was a strength of the study, as it allowed researchers to take the needs of ministry officials into account, and as it supported the immediate uptake of results in the policymaking process. Future studies should test the applicability of the TARGET:PA tool to different target groups and countries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Messing, Gelius, Abu-Omar, Marzi, Beck, Geidl, Grüne, Tcymbal, Reimers and Pfeifer.) more...

RIT1 is a RAS guanosine triphosphatase (GTPase) that regulates different aspects of signal transduction and is mutated in lung cancer, leukemia, and in the germline of individuals with Noonan syndrome. Pathogenic RIT1 proteins promote mitogen-activated protein kinase (MAPK) hyperactivation; however, this mechanism remains poorly understood. Here, we show that RAF kinases are direct effectors of membrane-bound mutant RIT1 necessary for MAPK activation. We identify critical residues in RIT1 that facilitate interaction with membrane lipids and show that these are necessary for association with RAF kinases and MAPK activation. Although mutant RIT1 binds to RAF kinases directly, it fails to activate MAPK signaling in the absence of classical RAS proteins. Consistent with aberrant RAF/MAPK activation as a driver of disease, we show that pathway inhibition alleviates cardiac hypertrophy in a mouse model of RIT1 mutant Noonan syndrome. These data shed light on the function of pathogenic RIT1 and identify avenues for therapeutic intervention. more...

Recombinant mammalian proteins are routinely produced in E. coli and thus lack post-translational modifications. KRAS4b is processed at both the N- and C-terminus, resulting in an acetylation of the N-terminus (at Thr, after aminopeptidase removal of the original N-term Met) and farnesylation/carboxymethylation of the C-terminal Cys (after proteolytic cleavage of the original C-terminal three amino acids, Val-Iso-Met). Processing of KRAS enables it to associate with the plasma membrane and fulfill its function in cell signaling. We describe here the production of recombinant KRAS4b from our modified baculovirus/insect cell expression system that accurately incorporates these in vivo modifications to allow experiments that anchor KRAS4b to membrane mimetics (e.g., nanodiscs and liposomes). more...

Introduction: Regular physical activity provides many health benefits to older adults. As a well-known form of physical activity, cycling can be an appropriate means for older people to meet WHO recommendations and to improve their health. In addition, cycling can help to protect the environment and reduce greenhouse gas emissions. The primary aim of this scoping review is to identify the currently available scientific evidence and gaps of research in this field., Methods: A systematic search in seven databases resulted in 7,192 studies. After the exclusion of duplicates, studies were screened by two independent reviewers in a two-stage process. Based on previously defined inclusion criteria, 123 studies were included. Data extraction was based on a descriptive analytical method, and seven categories for the main topics of studies were developed. Data were extracted by three reviewers to analyze different characteristics of included articles such as age range, study design, data type, gender, type of bicycle, and country of origin., Results: The included studies covered the following topics: (1) traffic safety, (2) cycling as physical activity or for transport, (3) health benefits, (4) environmental factors, (5) facilitators and barriers, (6) application of technology and (7) promotion of cycling. Results show that the majority of studies were performed in both younger (60-79 years) and older (80+ years) adults. Most studies had an observational study design, used conventional bicycles, and were based on quantitative methods. Researchers from the United States, Netherlands, and Japan published the highest number of studies related to cycling., Discussion: Traffic safety was the most prevalent focus of the included studies. Gaps were identified with regard to studies focusing on the promotion of cycling, application of technology, as well as facilitators and barriers of cycling. While research on traffic safety should continue to be a high priority for public health, potentially more research should focus on how to get older people to bicycle more. This is warranted by the proven individual and planetary health benefits of cycling and the urgency of combating climate change., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Kardan, Akter, Iqbal, Tcymbal, Messing, Gelius and Abu-Omar.) more...

We present the results of a large, US$8.9 million campaign-wide field experiment, conducted among 2 million moderate- and low-information persuadable voters in five battleground states during the 2020 US presidential election. Treatment group participants were exposed to an 8-month-long advertising programme delivered via social media, designed to persuade people to vote against Donald Trump and for Joe Biden. We found no evidence that the programme increased or decreased turnout on average. We found evidence of differential turnout effects by modelled level of Trump support: the campaign increased voting among Biden leaners by 0.4 percentage points (s.e. = 0.2 pp) and decreased voting among Trump leaners by 0.3 percentage points (s.e. = 0.3 pp) for a difference in conditional average treatment effects of 0.7 points (t 1,035,571  = -2.09; P = 0.036; [Formula: see text] points; 95% confidence interval = -0.014 to 0). An important but exploratory finding is that the strongest differential effects appear in early voting data, which may inform future work on early campaigning in a post-COVID electoral environment. Our results indicate that differential mobilization effects of even large digital advertising campaigns in presidential elections are likely to be modest., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.) more...

Objectives: Evidence-based strategies to reduce loneliness in later life are needed because loneliness impacts all domains of health, functioning, and quality of life. Volunteering is a promising strategy, as a large literature of observational studies documents associations between volunteering and better health and well-being. However, relatively few studies have used randomized controlled trials (RCTs) to examine benefits of volunteering, and none have examined loneliness. The primary objective of the Helping Older People Engage (HOPE) study is to examine the social-emotional benefits of a social volunteering program for lonely older adults. This manuscript describes the rationale and design of the trial., Methods: We are randomly assigning adults aged 60 or older (up to 300) who report loneliness to 12 months of either AmeriCorps Seniors volunteering program or an active control (self-guided life review). Co-primary outcomes are assessed via self-report-loneliness (UCLA Loneliness Scale) and quality of life (WHOQOL-Bref). Enrollment was completed in May 2022 and follow-up assessments will continue through May 2023, with completion of primary outcomes soon thereafter., Conclusions: Since older adults who report loneliness are less likely to actively seek out volunteering opportunities, if results support efficacy of volunteering for reducing loneliness, dissemination and scaling up efforts may involve connecting primary care patients who are lonely with AmeriCorps Seniors through aging services agencies.This RCT is registered at clinicaltrials.gov (NCT03343483)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.) more...

Background: The Policy Evaluation Network proposes a consolidated approach to measure comparable health indicators across European health surveillance systems to evaluate effectiveness of policy action., Methods: In a stepwise approach, questionnaire items used by the systems for measuring diet and physical activity data to describe health indicators were identified based on their validity, reliability, and suitability to monitor achievement of health recommendations. They were collated to unified questionnaire modules and discussed bilaterally with representatives of these systems to explore barriers and facilitators for implementation. Also, establishment of a methodological competence platform was proposed, in which the surveillance and monitoring systems agree on the priorities and common quality standards for the harmonization process and to coordinate the integration of questionnaire modules into existing systems., Results: In total, seven questionnaire modules were developed, of which two diet and two physical activity modules were proposed for implementation. Each module allows measurement of data reflecting only partial aspects of national and WHO recommendations related to diet and physical activity. Main barriers were the requirements of systems to monitor temporal trends and to minimize costs. Main facilitator for implementation was the systems' use of questionnaire items that were comparable to the unified modules. Representatives agreed to participate in a methodological competence platform., Conclusion: We successfully took first steps in the realization of the roadmap towards a harmonization of European surveillance by introducing unified questionnaire modules allowing the collection of comparable health indicators and by initiating the establishment of a competence platform to guide this process., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.) more...

Background: This review of reviews aimed to: (1) summarize the evidence from published reviews on the effectiveness of mass-media campaigns to promote physical activity (PA) or PA-related determinants (intermediate psychological and proximal outcomes) and (2) to identify policy-relevant recommendations related to successful PA campaigns., Methods: An extensive literature search was performed on 1 March 2021. Reviews that evaluated the impact of campaigns on distal (e.g. PA) and/or proximal outcomes of PA (awareness, knowledge, etc.) and that targeted the general population or subsets were included. Quality of reviews was assessed using the AMSTAR-2 tool. Policy-relevant recommendations were systematically derived and synthesized and formulated as good practice statements. A protocol was registered beforehand (ID: CRD42021249184)., Results: A total of 1915 studies were identified, of which 22 reviews were included. The most consistent evidence was found for the effectiveness of mass-media campaigns on proximal outcomes, while the evidence for distal outcomes was mixed. Good practice statements were derived: (1) to achieve behaviour change, mass-media is an important component of larger, multilevel and multicomponent strategies; (2) mass-media strategies should be coordinated and aligned at local- and national-level and be sustained, monitored and resourced at these levels and (3) media should be tailored to reduce socioeconomic inequalities., Conclusions: Mass-media can play an important role in the promotion of PA. In general, evidence was more inconsistent for effectiveness on distal outcomes than for proximal outcomes. Policy-relevant recommendations include that mass-media strategies should be resourced, coordinated, aligned, sustained, monitored and evaluated on the local and national level., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.) more...

Background: A multifaceted response, including government action, is essential to improve population levels of physical activity (PA). This article describes the development process of the 'Physical Activity Environment Policy Index' (PA-EPI) monitoring framework, a tool to assess government policies and actions for creating a healthy PA environment., Methods: An iterative process was undertaken. This involved a review of policy documents from authoritative organizations, a PA policy audit of four European countries, and a systematic review of scientific literature. This was followed by an online consultation with academic experts (N = 101; 20 countries, 72% response rate), and policymakers (N = 40, 4 EU countries). During this process, consensus workshops were conducted, where quantitative and qualitative data, alongside theoretical and pragmatic considerations, were used to inform PA-EPI development., Results: The PA-EPI is conceptualized as a two-component 'policy' and 'infrastructure support' framework. The two-components comprise eight policy and seven infrastructure support domains. The policy domains are education, transport, urban design, healthcare, public education (including mass media), sport-for-all, workplaces and community. The infrastructure support domains are leadership, governance, monitoring and intelligence, funding and resources, platforms for interaction, workforce development and health-in-all-policies. Forty-five 'good practice statements' or indicators of ideal good practice within each domain conclude the PA-EPI. A potential eight-step process for conducting the PA-EPI is described., Conclusions: Once pre-tested and piloted in several countries of various sizes and income levels, the PA-EPI good practice statements will evolve into benchmarks established by governments at the forefront of creating and implementing policies to address inactivity., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.) more...

Background: In 2016, the first evidence-based National Recommendations for Physical Activity and Physical Activity Promotion were published in Germany. These recommendations are primarily intended for experts, decision makers and stakeholders., Objectives: This study aims to describe the development of dissemination strategies for these recommendations., Process: To facilitate the co-production of knowledge between practitioners, decision makers, and researchers, a participatory approach was applied. This approach involved the development of target group-specific strategies for disseminating the recommendations. This was achieved in two workshops and one working group phase; 92 professionals and decision makers participated in the process., Results: The working groups developed specific dissemination strategies that were grouped into the following strategy types: (1) inform multipliers, (2) activate multipliers, (3) use existing and develop new networks, (4) initiate policy change., Conclusion: The participatory approach adopted in this project was successful in developing dissemination strategies and is unique from an international perspective. To improve the evaluation of such co-production processes, future research should determine and operationalize appropriate indicators., Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.) more...

SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in multiple KRAS cancer cell lines. SHOC2 forms a heterotrimeric complex with MRAS and PP1C that is essential for regulating RAF and MAPK-pathway activation by dephosphorylating a specific phosphoserine on RAF kinases. Here we present the high-resolution crystal structure of the SHOC2-MRAS-PP1C (SMP) complex and apo-SHOC2. Our structures reveal that SHOC2, MRAS, and PP1C form a stable ternary complex in which all three proteins synergistically interact with each other. Our results show that dephosphorylation of RAF substrates by PP1C is enhanced upon interacting with SHOC2 and MRAS. The SMP complex forms only when MRAS is in an active state and is dependent on SHOC2 functioning as a scaffolding protein in the complex by bringing PP1C and MRAS together. Our results provide structural insights into the role of the SMP complex in RAF activation and how mutations found in Noonan syndrome enhance complex formation, and reveal new avenues for therapeutic interventions., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.) more...

Background: Policy is an important element of influencing individual health-related behaviours associated to major risk factors for non-communicable diseases (NCDs) such as smoking, alcohol consumption, unhealthy eating and physical inactivity. However, our understanding of the specific measures recommended in NCD prevention policy-making remains limited. This study analysed recent World Health Organization (WHO) documents to identify common policy instruments suggested for national NCD prevention policy and to assess similarities and differences between policies targeting different health-related behaviours., Methods: Evert Vedung's typology of policy instruments, which differentiates between regulatory, economic/ fiscal and soft instruments, served as a basis for this analysis. A systematic search on WHO websites was conducted to identify documents relating to tobacco, alcohol, nutrition and physical activity. The staff of the respective units at the WHO Regional Office for Europe conducted an expert validation of these documents. The resulting documents were systematically searched for policy instruments. A word frequency analysis was conducted to estimate the use of individual instruments in the different policy fields, followed by an additional in-depth coding and content analysis by two independent reviewers., Results: Across all health-related behaviours, the following policy instruments were suggested most frequently in WHO guidance documents: laws, regulations, standards, taxes, prices, campaigns, recommendations, partnerships and coordination. The analysis showed that regulatory and economic/fiscal policy instruments are mainly applied in tobacco and alcohol policy, while soft instruments dominate in the fields of nutrition and especially physical activity., Conclusion: The study confirms perceived differences regarding recommended policy instruments in the different policy fields and supports arguments that "harder" instruments still appear to be underutilized in nutrition and physical activity. However, more comprehensive research is needed, especially with respect to actual instrument use and effectiveness in national-level NCD prevention policy., (© 2022 The Author(s); Published by Kerman University of Medical Sciences This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.) more...

Background: Fighting the climate crisis is the greatest challenge of our time and will touch all aspects of people's lives. In this context, the United Nations (UN) have called on the sport sector to reduce its negative impacts on the environment and show 'climate leadership'. While some efforts have already been made with regards to mega sport events, there is still a dearth of approaches on limiting the climate impact of recreational sport and exercise programmes., Methods: Based on the UN-Framework 'Sports for Climate Action', literature reviews and additional desk research, a checklist to support local level stakeholders in providing climate-friendly sport and exercise programmes was developed., Results: The proposed checklist consists of five dimensions that need to be considered when designing and offering a climate-friendly sport and exercise programme: (i) active transport to exercise programmes, (ii) the carbon footprint of different types of exercises, (iii) low carbon sport clothing and equipment, (iv) instructors as champions for climate action and (v) advertising and communication. These five dimensions result in a 16-item checklist that supports the planning, advertising, implementation and evaluation of climate-friendly sport and exercise programmes., Conclusions: The proposed checklist intends to facilitate the development of climate-friendly sport and exercise programmes. However, additional work is needed to test the implementation of the checklist at the local level. While the sport sector can make its own contributions to reduce its climate impact, intersectoral action is needed to improve infrastructure for active transport and to build sustainable sport facilities., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.) more...

Background: An analysis of currently existing partnerships and cross-country collaboration for physical activity (PA) promotion is valuable for understanding how such partnerships operate, and how they impact national PA promotion efforts. This study aimed to outline the formation and development of the European Union's (EU) Physical Activity Focal Points Network, to evaluate its outputs and benefits and to describe its potential and challenges., Methods: A mixed methods approach were employed, including document analysis, semi-structured interviews with key officials and an online evaluation survey with the focal points., Results: The network was founded in 2014. Its main task is to coordinate the national collection of information for the EU's Health-Enhancing Physical Activity (HEPA) Monitoring Framework. Besides collecting data, focal points usually meet twice a year to share best practices and plan activities for the promotion of PA within the EU. The results of the evaluation survey show that participation in the network helped members specify goals for PA promotion, gain more knowledge regarding how to promote PA, identify more opportunities to promote PA in their country and to join a collaborative project with other countries., Conclusions: The study shows that the EU Physical Activity Focal Points Network may serve as an example of successful cross-country collaboration in PA promotion. The network has been able to make a contribution to monitoring the implementation of the EU Council Recommendation on HEPA across sectors in particular and of PA promotion in the EU in general., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Public Health Association.) more...

Insufficient physical activity (PA) is one of major risk factors for serious diseases and premature mortality worldwide. Public policies to enhance PA across society are recognized as an effective tool against the problem. This paper presents the results of a comprehensive assessment of national-level PA policy approach in Poland. A standardized survey of Word Health Organization named the Health-Enhancing Physical Activity Policy Audit Tool (HEPA PAT) was used for data collection. Content analysis and strengths, weaknesses, opportunities, and threats analysis (SWOT) were used to characterize various PA policy aspects, to appraise the current situation, and accommodate organizational and environmental factors that it is influenced by. The results show that the national PA policy approach has been constantly developing in Poland, but there is room for improvement in a number of areas. The most important weaknesses are the lack of clear leadership, no mechanisms in place to coordinate efforts undertaken at different levels, and lack of collaboration across different levels of government and across different sectors of economy. Providing an umbrella covering all PA promotion policies and activities is, therefore, a key issue to be addressed. The country should seize the opportunity coming from an increasing awareness of a healthy lifestyle among Polish society. more...

KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31 P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-μs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective. more...

Introduction: Over 40 million deaths annually are due to noncommunicable diseases, 15 million of these are premature deaths and physical inactivity contributes an estimated 9% to this figure. Global responses have included the Sustainable Development Goals (SDGs) and the Global Action Plan on Physical Activity (GAPPA). Both point to policy action on physical activity (PA) to address change, yet the impact of policy on PA outcomes is unknown.  The protocol described outlines the methodology for systematic literature reviews that will be undertaken by the Policy Evaluation Network (PEN) to address this knowledge gap. Methods: The seven best investments for promotion of population PA identified in the Toronto Charter highlighted seven policy domains (schools, transport, urban design, primary health care systems, public education, community-wide programmes and sport) which will form the basis of these PEN reviews. Seven individual scientific literature searches across six electronic databases will be conducted. Each will use the key concepts of policy, PA, evaluation and a distinct concept for each of the seven policy domains. This will be supplemented with a search of the reference list of included articles. Methodological quality will be assessed and overall effectiveness for each included study will be described according to pre-determined criteria. Conclusions: Each review will provide policy makers with a list of policy statements and corresponding actions which the evidence has determined impact on PA directly or indirectly. By collating the evidence, and demonstrating the depth of the science base which informs these policy recommendations, each review will provide guidance to policymakers to use evidence-based or evidence-informed policies to achieve the 15% relative reduction in physical inactivity as defined by GAPPA. Registration:  PROSPERO CRD42020156630 (10/07/2020)., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Volf K et al.) more...

Background: Physical inactivity is a key determinant of noncommunicable diseases. Therefore, the World Health Organization (WHO) as well as researchers worldwide have developed different tools to monitor and audit policies to promote physical activity. However, these tools have so far not been used to systematically collect and analyse data on physical activity promoting policies in Germany., Aim: This study aims to provide a systematic overview of policies to promote physical activity in Germany., Methods: The study was conducted as part of the Policy Evaluation Network ( www.jpi-pen.eu ). Data from the European Union Physical Activity Monitoring Framework, desk research, and an expert survey were utilised and collected with the WHO's health-enhancing physical activity (HEPA) policy audit tool (PAT)., Results: The results highlight the wide range of relevant stakeholders and provide an overview of current policies as well as surveillance, evaluation, and funding. Significant accomplishments and persistent challenges are identified., Discussion: An international comparison shows that, in contrast to Germany, other countries have formulated measurable goals for physical activity promotion on a national level. However, Germany is among a minority of countries with specific physical activity recommendations for adults with noncommunicable diseases. Further structural development of physical activity promotion in Germany is necessary., (© 2021. The Author(s).) more...

Public awareness of the importance of physical activity has increased due to the many lockdowns imposed during the COVID-19 pandemic. This has brought more widespread attention to a question previously confined primarily to parts of the physical activity promotion community: Do humans have a right to be active? While the public health benefits of physical activity are undisputed, up to now no clear understanding has emerged as to whether physical activity represents a human right. Even though the right to physical activity is not explicitly recognized in international human rights treaties, it seems possible to derive it from well-established human rights such as the right to health, the right to rest and leisure, the right to education, and the principle of nondiscrimination. This paper shows how a right to physical activity could be derived from international human rights treaties, how the attributes of such a right could be defined, and which state obligations would be associated with it. Given that the current human rights discourse in this field focuses mainly on the interconnections between sport and human rights, we would like to argue that there is added value in a debate about physical activity as a human right., Competing Interests: Competing interests: None declared., (Copyright © 2021 Messing, Krennerich, Abu-Omar, Ferschl, and Gelius.) more...

Background: The COVID-19 pandemic is a major challenge for societies and governments around the world that severely affects all aspects of health promotion. This study assesses the potential influence of the first wave of the pandemic on national physical activity promotion policy in the European Union (EU)., Methods: Data were collected using an online survey among members of the EU Physical Activity Focal Point Network, which consists of government officials from all EU member states., Results: The COVID-19 pandemic has significantly affected physical activity promotion across the EU. In particular, experts indicated that it has negatively impacted opportunities for physical activity in their countries. There have, however, been positive effects of the crisis on public awareness of physical activity. While almost all countries were able to issue physical activity recommendations during quarantine, opinions varied regarding the overall impact of the pandemic on governmental capacities for physical activity promotion and policy., Conclusions: This study shows that the COVID-19 crisis has had both negative and positive effects on physical activity promotion in the EU. The positive experiences reported by some members of the Focal Point Network may assist other countries in identifying potential policy windows and strategies for the ongoing pandemic. more...

RAS proteins are molecular switches that interact with effector proteins when bound to guanosine triphosphate, stimulating downstream signaling in response to multiple stimuli. Although several canonical downstream effectors have been extensively studied and tested as potential targets for RAS-driven cancers, many of these remain poorly characterized. In this study, we undertook a biochemical and structural approach to further study the role of Sin1 as a RAS effector. Sin1 interacted predominantly with KRAS isoform 4A in cells through an atypical RAS-binding domain that we have characterized by X-ray crystallography. Despite the essential role of Sin1 in the assembly and activity of mTORC2, we find that the interaction with RAS is not required for these functions. Cells and mice expressing a mutant of Sin1 that is unable to bind RAS are proficient for activation and assembly of mTORC2. Our results suggest that Sin1 is a bona fide RAS effector that regulates downstream signaling in an mTORC2-independent manner., Competing Interests: Competing interest statement: F.M. is a consultant for Ideaya Biosciences, Kura Oncology, Leidos Biomedical Research, Pfizer, Daiichi Sankyo, Amgen, PMV Pharma, OPNA-IO, and Quanta Therapeutics and has received research grants from Boehringer-Ingelheim and is a consultant for and cofounder of BridgeBio Pharma. P.C. is a cofounder and advisory board member of Venthera. more...

Objectives: To determine whether peer companionship delivered by an aging services agency to socially-disconnected older adult primary care patients was associated with improvement in suicidal ideation depression, anxiety, and psychological connectedness., Design: Pragmatic, nonblinded, parallel-group, randomized controlled trial comparing peer companionship, The Senior Connection (TSC), to care-as-usual (CAU)., Setting: Lifespan, a nonmedical, community-based aging services agency., Participants: Adult primary care patients ages 60 years or older who endorsed feelings of loneliness or being a burden on others., Intervention: TSC was delivered by Lifespan volunteers who provided supportive visits and phone calls in the subjects' homes. CAU involved no peer companion assignment., Measurements: The primary outcome was suicidal ideation assessed by the Geriatric Suicide Ideation Scale; secondary outcomes were depression, anxiety, and feelings of belonging and being a burden on others. Data were collected at baseline, 3-, 6-, and 12-months., Results: Subjects (55% female) had a mean age of 71 years. There was no difference between groups in change in suicidal ideation or belonging. Subjects randomized to TSC had greater reduction in depression (PHQ-9; 2.33 point reduction for TSC versus 1.32 for CAU, p = 0.05), anxiety (GAD-7; TSC 1.52 versus CAU 0.28, p = 0.03), and perceived burden on others (INQ; 0.46 TSC versus 0.09 CAU, p <0.01)., Conclusions: TSC was superior to CAU for improving depression, anxiety, and perceived burden, but not suicidal ideation. Although effect sizes were small, the low-cost and nationwide availability of peer companionship justify further examination of its effectiveness and scalability in improving mental health outcomes of socially disconnected older adults., (Copyright © 2020. Published by Elsevier Inc.) more...

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates in the United States and elsewhere. To address this, we analyzed seropositivity in 9089 adults in the United States who had not been diagnosed previously with COVID-19. Individuals with characteristics that reflected the U.S. population ( n = 27,716) were selected by quota sampling from 462,949 volunteers. Enrolled participants ( n = 11,382) provided medical, geographic, demographic, and socioeconomic information and dried blood samples. Survey questions coincident with the Behavioral Risk Factor Surveillance System survey, a large probability-based national survey, were used to adjust for selection bias. Most blood samples (88.7%) were collected between 10 May and 31 July 2020 and were processed using ELISA to measure seropositivity (IgG and IgM antibodies against SARS-CoV-2 spike protein and the spike protein receptor binding domain). The overall weighted undiagnosed seropositivity estimate was 4.6% (95% CI, 2.6 to 6.5%), with race, age, sex, ethnicity, and urban/rural subgroup estimates ranging from 1.1% to 14.2%. The highest seropositivity estimates were in African American participants; younger, female, and Hispanic participants; and residents of urban centers. These data indicate that there were 4.8 undiagnosed SARS-CoV-2 infections for every diagnosed case of COVID-19, and an estimated 16.8 million infections were undiagnosed by mid-July 2020 in the United States., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).) more...

Study Objectives: We examined how telemedicine evaluation compares to face-to-face evaluation in identifying risk for sleep-disordered breathing., Methods: This was a randomized interrater reliability study of 90 participants referred to a university sleep center. Participants were evaluated by a clinician investigator seeing the patient in-person, then randomized to a second clinician investigator who performed a patient evaluation online via audio-video conferencing. The primary comparator was pretest probability for obstructive sleep apnea., Results: The primary outcome comparing pretest probability for obstructive sleep apnea showed a weighted kappa value of 0.414 (standard error 0.090, P = .002), suggesting moderate agreement between the 2 raters. Kappa values of our secondary outcomes varied widely, but the kappa values were lower for physical exam findings compared to historical elements., Conclusions: Evaluation for pretest probability for obstructive sleep apnea via telemedicine has a moderate interrater correlation with in-person assessment. A low degree of interrater reliability for physical exam elements suggests telemedicine assessment for obstructive sleep apnea could be hampered by a suboptimal physical exam. Employing standardized scales for obstructive sleep apnea when performing telemedicine evaluations may help with risk-stratification and ultimately lead to more tailored clinical management., (© 2021 American Academy of Sleep Medicine.) more...

Background: Despite the well-established health benefits of physical activity (PA) for young people (aged 4-19 years), most do not meet PA guidelines. Policies that support PA in schools may be promising, but their impact on PA behavior is poorly understood. The aim of this systematic review was to ascertain the level and type of evidence reported in the international scientific literature for policies within the school setting that contribute directly or indirectly to increasing PA., Methods: This systematic review is compliant with Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines. Six databases were searched using key concepts of policy, school, evaluation, and PA. Following title and abstract screening of 2323 studies, 25 progressed to data synthesis. Methodological quality was assessed using standardized tools, and the strength of the evidence of policy impact was described based on pre-determined codes: positive, negative, inconclusive, or untested statistically., Results: Evidence emerged for 9 policy areas that had a direct or indirect effect on PA within the school setting. These were whole school PA policy, physical education, sport/extracurricular PA, classroom-based PA, active breaks/recess, physical environment, shared use agreements, active school transport, and surveillance. The bulk of the evidence was significantly positive (54%), 27% was inconclusive, 9% was significantly negative, and 11% was untested (due to rounding, some numbers add to 99% or 101%). Frequency of evidence was highest in the primary setting (41%), 34% in the secondary setting, and 24% in primary/secondary combined school settings. By policy area, frequency of evidence was highest for sport/extracurricular PA (35%), 17% for physical education, and 12% for whole school PA policy, with evidence for shared use agreements between schools and local communities rarely reported (2%). Comparing relative strength of evidence, the evidence for shared use agreements, though sparse, was 100% positive, while 60% of the evidence for whole school PA policy, 59% of the evidence for sport/extracurricular PA, 57% of the evidence for physical education, 50% of the evidence for PA in classroom, and 50% of the evidence for active breaks/recess were positive., Conclusion: The current evidence base supports the effectiveness of PA policy actions within the school setting but cautions against a "one-size-fits-all" approach and emphasizes the need to examine policy implementation to maximize translation into practice. Greater clarity regarding terminology, measurement, and methods for evaluation of policy interventions is needed., (Copyright © 2021. Production and hosting by Elsevier B.V.) more...