The aberrant differentiation of osteoclasts is a key feature of the pathogenesis of osteoporosis, which has a devastating impact on human health. While the effects of Orientin (Ori) on osteoporosis, particularly on RANKL‐stimulated osteoclast production and activation, remain still unclear, Ori has been found to display several biological activities, including antioxidant and anti‐inflammatory. In this work, we investigated the possible pathways through which Ori suppressed RANKL‐induced osteoclast development and showed for the first time that it does so. The macrophages from the bone marrow (BMMs) were cultivated and then treated with Ori after being stimulated with RANKL. Then, TRAP‐positive multinucleated cells were counted, and F‐actin ring analysis was used to assess Ori's impact on mature osteoclast development. In addition, dihydroethidium (DHE) staining was used to evaluate the impact of Ori on RANKL‐induced reactive oxygen species (ROS). In addition, we performed western blotting and quantitative RT‐PCR analysis to investigate probable causes of these downregulation effects. We discovered that Ori inhibits the creation of osteoclasts, the gene and protein expressions unique to osteoclasts, and the ROS production. By activating Nrf2 and other ROS‐scavenging enzymes, Ori reduces intracellular ROS levels. The expression of the main transcription factor of osteoclast development, c‐Fos, was downregulated together with NFATc1, CTSK, and NFATc2, thanks to Ori's inhibition of RANKL‐induced NF‐κB. Consistent with its in vitro antiosteoclastogenic action, Ori therapy in the ovariectomized (OVX) rat model was also able to restore bone mass and improve microarchitecture in the distal femurs. Together, our results demonstrate that Ori is a flavonoid molecule with therapeutic promise for bone illnesses associated with osteoclasts, such as osteoporosis. Orientin suppresses osteoclastogenesis and ameliorates ovariectomy‐induced osteoporosis via suppressing ROS production. These processes are mediated, respectively, by osteoblasts and osteoclasts  and RANKL are essential for osteoclast differentiation. Osteoclast precursor cells have been shown to respond favorably to M‐CSF, which has been shown to increase their proliferation and survival. By attaching to its receptor, RANK, RANKL plays a critical role in regulating mature osteoclast development, function, and survival. Moreover, osteoclastogenesis relies on the transcriptional activity of NFATc1, cathepsin K (CTSK), and c‐Fos, all of which are triggered by the nuclear factor‐κB (NF‐κB) signaling pathways, which are mobilized in part thanks to RANK recruitment  and dimethyl sulfoxide (DMSO) were acquired from MedChemExpression company . Both RANKL and M‐CSF were purchased from Research and Development  and diluted as directed by the manufacturer. Gibco was used to get fetal bovine serum (FBS) and minimal essential medium . Sigma was used for the purchase of the Cell Counting Kit‐8 (CCK‐8) assay kit, TRAcP staining reagents, and Phalloidin‐FITC . Cell Signaling Technology  provided both the primary antibodies  and the secondary antibodies. Abcam  supplied the primary antibodies for Nox1, Rac1, n‐Nrf2, and Lamin B; p‐p65, p65, and IκB were purchased from Santa Cruz Biotechnology . No less than analytical‐ or cell‐culture‐grade reagents were employed in any of the studies.2.2Rat experiments were conducted in accordance with Wenzhou Medical University's guidelines for the care of experimental animals, and they were all approved by the institution's Animal Care and Welfare Committee (SYXK2020‐0004).2.33 BMMs each were planted with the cells to test for cytotoxicity. Cells were exposed to the specified Ori dosages  after 24/48 h after adhering to them over the previous night. Incubation of the cells in the CCK‐8 reagent for 2 h was performed. At 450 nm, a spectrophotometer  was chosen to measure all of the samples.C57BL/6J mouse bone marrow stromal cells (BMMs) were transplanted into α‐MEM‐coated culture flasks containing 100 U/mL penicillin and 100 μg/mL streptomycin, FBS (10%), and M‐CSF (50 ng/mL). One to three generationally passed cells were employed in the assays that followed. Ninety‐six‐well plates containing 5 × 102.43 cells/well, BMMs were grown on 96‐well plates overnight. To induce osteoclast differentiation, the cells were cultured in a complete MEM medium supplemented every other day with either 40 μM Ori or vehicle control (DMSO). The cells were cultured for 5 days before being fixed in 4% paraformaldehyde (PFA) and stained for TRAcP using a TRAcP staining kit  as per the manufacturer's instructions . The procedure included washing the cells with PBS, fixing them in 4% paraformaldehyde for 15 min, washing the cells three more times, using Triton X‐100 (0.1%) for 15 min to permeabilize the cells, and staining them with Rhodamine‐phalloidin in the dark for 1 h. Cells were treated with DAPI for 10 min to stain the nuclei. For image analysis, cytoskeletons stained with F‐actin were seen and measured using a Nikon fluorescent microscope.2.6After 3 days of RANKL stimulation, bovine bone slices were implanted with equal numbers of BMM‐derived preosteoclasts and treated with Ori (40 μM) for an additional 48 h. Then, sonication and mechanical agitation were used to dislodge cells that had stuck to the bone slices. Using ImageJ software, the bone resorption area was measured after resorption pits were seen using an SEM (FEI Quanta 250).2.7To extract RNA, cells from various treatment groups were lysed using RNAiso Plus  following the manufacturer's requirements after being rinsed with cold PBS. After then, cDNA was produced by reverse‐transcription of the whole RNA. Using an RT‐PCR Detection System , quantitative real‐time PCR was carried out using the cDNA. The values were adjusted to reflect GAPDH levels. The genes' primer sequences are in appendix.2.8Different sets of cultured cells were given three 5‐min cold PBS washes between treatments. The cells were lysed for 20 min at 4°C in RIPA lysis buffer . The collected cell lysates were then centrifuged . Loading buffer was used to dissolve the acquired supernatants. Proteins from a 10‐μL sample were isolated on 10% SDS‐PAGE gels and transferred to PVDF membranes. . After blocking the membranes at room temperature for 2 h with 5% nonfat dry milk, they were washed twice for 10 min in TBST20. Primary antibodies were applied to the membranes and incubated at 4°C for 24 h, followed by incubation with secondary antibodies at room temperature for 2 h. An electrochemical luminescence reagent  was chosen to detect the protein bands. ImageJ was used to count the number of grays throughout each band.2.9DHE staining and MitoSox were performed to measure intracellular ROS and mitochondrial ROS, respectively. There was a 15‐min fixation in 4% formaldehyde followed by a PBS wash of the cells. The cells were incubated for 30 min at 37°C after the addition of DHE and MitoSox. After two rounds of PBS washing, fluorescence pictures were captured using a Nikon microscope. The level of fluorescence was measured.2.10https://www.rcsb.org/structure) provides crystal structure of the protein of interest (Nrf2 and p‐p65). The chemical structure of Orientin was obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).Protein database  are analyzed.2.11There are separate Ethics Committees for patients at Wenzhou Medical University's Yuying Children's Hospital and the Second Affiliated Hospital, both of which have given their blessing to the procedure for animal research. Fifteen female SD rats, aged 3 months and weighing 250 ± 20 g, were obtained from the Animal Center of the Chinese Academy of Science in Shanghai, China. The rats were divided into three groups, with five rats in each group: sham‐operated rats, OVX rats, and OVX rats treated with Ori (40 mg/kg). After 1 week of adaptive feeding, surgical removal of the ovaries was performed on both OVX rats and Ori‐treated OVX rats using a 10% chloral hydrate solution, whereas just one ovary was grown in the sham‐operated rat. PBS or Ori (40 mg/kg) was administered intraperitoneally every other day for 8 weeks, beginning 1 week after surgery. One day following their last dose, all of the rats in the study were put to sleep. Following their removal, the femurs were subjected to a 24‐h preservation period in 10% formalin. Subsequently, they underwent radiological examination through the use of micro‐computed tomography (micro‐CT) and histomorphometric bone analysis.2.12Using a high‐resolution micro‐CT scanner, three‐dimensional pictures of the distal femur were created . Using software in the micro‐CT workstation and three‐dimensional reconstructed pictures, the volume of interest's (VOI) microarchitectural characteristics were determined. Finally, quantitative measurements of characteristics relevant to the area of interest (ROI), such as the trabecular number , trabecular thickness , bone volume to tissue volume (BV/TV), and trabecular separation , were made to evaluate the quality of the bone.2.13Animals were doubly labeled before sacrifice by receiving 30 mg/kg of calcein. We performed the previously mentioned measurement of the mineralization apposition rate (MAR) , the levels of the bone resorption biomarkers TRAcP5 and β‐CTx were determined.2.16t‐test and ANOVA. One‐way ANOVA was applied for comparisons between more than two groups, while the Student's t‐test was used to assess differences between the two groups. Furthermore, two‐way ANOVA was employed to analyze the effects of various treatments. It was considered statistically significant if p < .05.Every piece of information offered comes from at least three different experiments. The data are presented as means ± standard error of the mean (SEM). Statistical significance was evaluated using Student's 33.1To determine the impact of Ori on cell viability Figure , various3.2To gain a deeper understanding of Ori's impact on osteoclast development and function, we utilized western blotting and quantitative polymerase chain reaction techniques. Specifically, we analyzed the expression levels of osteoclast‐specific genes such as NFATc1, c‐Fos, and CTSK at the protein and mRNA levels. Ori‐treated groups showed significant inhibition of RANKL‐induced osteoclast‐specific gene expression compared to the RANKL group Figure . Western3.3ROS plays a crucial role in the formation of osteoclasts in response to RNAKL stimulation, as evidenced by a growing body of research. We, therefore, investigated whether Ori can influence ROS production in BMMs. A DHE dye was used to detect intracellular ROS production, and Nikon microscopes were used to record DHE fluorescence. The Ori treatment group showed a significant reduction in the intensity of DHE fluorescence per positive cell, as compared to the RANKL treatment group. Figure . In addiFollowing this, we looked into how Ori influences NF‐κB/Nrf2 activation. We used molecular docking analysis to identify potential interactions between Ori and proteins in the NF‐κB/Nrf2 pathway. There are Ori binding sites in the Nrf2 and p‐p62 proteins Figure . Molecul3.4We created an ovariectomy‐induced osteoporosis rat model to investigate the potential therapeutic effects of Ori on the disease. The model and administration in OVX rats were as stated Figure . The rec3.5Furthermore, the suppressive impact of Ori on osteoclast formation was validated using H&E, Masson, and TRAcP staining, and Calcein fluorescence was used to determine Ori's impact in stimulating bone formation in vivo Figure . When co4The global community has identified osteoporosis as a major health issue. It is estimated that over 200 million individuals globally are currently living with osteoporosis ; resources ; writing – original draft (lead); writing – review and editing (lead). Xing Wang: Investigation ; supervision ; visualization . Ya‐Jing Pan: Formal analysis ; methodology ; project administration . Xiao‐Feng Shi: Data curation ; software . Lei Yang: Funding acquisition (lead). Yong‐Liang Lou: Conceptualization (lead).This work was supported by the National Natural Science Foundation of China (No. 82072310) and Traditional Chinese Medicine of Zhejiang Province Science and Technology plan project (No. 2023010569).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.


Ca. Methanoperedenaceae archaea mediates the anaerobic oxidation of methane (AOM) in different terrestrial environments. Using a newly developed high‐pressure laboratory incubation system, we investigated 214‐ and 249‐m deep groundwater samples at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Ca. Methanoperedenaceae archaea have been shown by genome‐resolved metagenomics, respectively. The groundwater samples amended with 13C‐labelled methane and amorphous Fe(III) were incubated at a pressure of 1.6 MPa. After 3–7 days of incubation, the AOM rate was 45.8 ± 19.8 nM/day in 214‐m groundwater. However, almost no activity was detected from 249‐m groundwater. Based on the results from 16S rRNA gene analysis, the abundance of Ca. Methanoperedenaceae archaea was high in the 214‐m deep groundwater sample, whereas Ca. Methanoperedenaceae archaea was undetected in the 249‐m deep groundwater sample. These results support the in situ AOM activity of Ca. Methanoperedenaceae archaea in the 214‐m deep subsurface borehole interval. Although the presence of Fe‐bearing phyllosilicates was demonstrated in the 214‐m deep groundwater, it needs to be determined whether Ca. Methanoperedenaceae archaea use the Fe‐bearing phyllosilicates as in situ electron acceptors by high‐pressure incubation amended with the Fe‐bearing phyllosilicates.The family  Candidatus (Ca.) Methanoperedenaceae , a metabolism performed by microorganisms, including anaerobic methanotrophic archaea (ANME) inhabiting deep seafloor sediments, has been long examined  and Mn(IV)  was constructed in northern Hokkaido, Japan. Our previous metagenomic study of groundwater from a 214‐m deep underground borehole has shown abundant 13C‐labelled methane . A borehole named 08‐E140‐C01 was drilled diagonally downward with a vertical angle of −45° from the 140‐m gallery at Horonobe URL . The groundwater sample obtained from an interval at an average depth of 214‐m below ground level (m b.g.l.) within the 08‐E140‐C01 borehole is referred to as ESB214. The other borehole, 09‐V250‐M02, was drilled horizontally from the 250‐m gallery. The groundwater sample obtained from an interval at a depth of 249‐m b.g.l. is referred to as VSB249. The temperature and hydrostatic pressure are 18–20°C Table  and arouMulti‐packer systems were used to collect groundwater from the target intervals  in DIC and alkalinity during incubation were measured to calculate the AOM rates. By dividing microbial cell densities in cultures at the beginning of the incubation by those in in the original Horonobe groundwater samples, the concentration factors were determined to be 7.2 and 3.2 times for ESB214 and VSB249, respectively. The concentration factors for ESB214 and VSB249 were not controlled to be the same, because the microbial cell densities were measured after the incubation experiments.For the rate measurements of AOM, a high‐pressure incubation system was newly developed to reproduce high‐pressure conditions. The system comprised a stainless vessel, a piston, a pressure gauge, valves, and gas lines Figure . The micAs a result, amorphous Fe(III) amendment using ESB214 showed relatively high AOM rates at 45.8 ± 19.8 nM/day  reducers and obligate anaerobes  reduction, amorphous Fe(III) might be favourable for Pseudomonas growth  amendment, the genus s Figure . The gend Figure . The genCa. Methanoperedenaceae. Thus, mineralogical characterization of the yellowish‐brown material was performed. Scanning electron microscopy equipped with energy dispersive spectrometry (SEM‐EDS) and x‐ray diffractometry (XRD) analyses were conducted to identify mineral phases in the yellowish‐brown material. The combination of XRD and SEM‐EDS analyses is necessary to identify a mineral phase, because XRD analysis provides no information on the chemical composition but the crystal structure. Instead, SEM‐EDS analysis provides no information on the crystal structure but the chemical composition of the material. The obtained EDS spectrum showed the elemental composition corresponding to a smectite mineral in association with a trace amount of iron  showed the AOM mediation in ESB214. Given the high concentration of total iron relative to that of Fe(II), Fe(III) might be present in ESB214 Table . As Fe for extracellular electron transfer. Remarkably, HGW‐Methanoperedenaceae‐1 and Kmv03 are equipped with 11 and 18 copies of MHCs, in which the maximum numbers of heme‐binding motifs (CXXCH) in a single MHC gene are >50 and 22, respectively. Many MHCs with high numbers of heme‐binding motifs are commonly found in bacterial groups, including the family Shewanellaceae and Geobacteraceae, for the electron transfer to solid metal oxides for their growth  are competitive with electron acceptors available in the incubation media. Additionally, some of the incubation conditions differ from in situ, namely, the absence of rock matrix, the lower concentration of nutrients originally contained in groundwater. Therefore, the obtained rates should be interpreted as the potential rates of the subsurface AOM.In this study, there was an attempt to measure AOM rates under high‐pressure conditions using a newly developed laboratory incubation system. The incubation was set to mimic in situ conditions as much as possible. A factor slightly different from the in situ counterparts was the cell densities, which were 7.2 and 3.2 times higher in the incubation media than those originally in ESB214 and VSB249, respectively. Although the higher cell density might enhance necromass degradation, this effect was reduced by keeping the incubation time short. Nevertheless, there is the potential that the AOM rates were underestimated, given that other Fe(III)‐reducing activities  was added to the incubation media prepared from ESB214. The Fe(III)‐dependent AOM of Ca. Methanoperedenaceae members. The average AOM rate of the Fe‐amended incubation experiment using ESB214 (45.8 ± 19.8 nM/day) is equivalent to the cell‐specific AOM rate of 0.25 ± 0.13 pM/day, which was calculated by dividing the rates with the Ca. Methanoperedenaceae cell number. In no‐amended or sulfate/nitrate amended systems, the AOM rates were much lower than that in the Fe‐amended system.In this study, we successfully quantified AOM rates mediated by the groundwater community, including the deep subsurface Ca. Methanoperedenaceae range 10–102 μM/day from near‐surface environment lineages  available in the borehole containing ESB214 to understand the factors controlling subsurface AOM activities.AOM activities measured by the incubation experiments are considered to reflect AOM activities in the boreholes. Nearly undetected AOM activity from the incubation experiments using VSB249 could be from the absence of Ca. Methanoperedenaceae members shown from the 16S rRNA gene amplicon analysis. In contrast, moderately high AOM activities measured by the incubation experiments might reflect the abundance of Ca. Methanoperedenaceae members  in the reducing conditions of ESB214, it is likely that the particulate components other than amorphous Fe(III) could be used as insoluble electron acceptors for AOM activities. Indeed, the presence of dioctahedral smectite associated with iron was verified using XRD and EDS analyses of ESB214 suspended particulates Figure . Thus, dCa. Methanoperedenaceae members from VSB249 might result from the lack of particulates. As expected, the AOM rate measurements of VSB249 showed the slowest AOM rates even with the amendment of amorphous Fe(III). This result highlights the importance of the particulates for the subsurface AOM activities.The importance of the particulates was indirectly evaluated for VSB249. The particulates were not visible on a 47‐mm diameter filter after filtering a large volume of the VSB249 groundwater. In this study, we did not detect any Ca. Methanoperedenaceae archaea can use Fe(III) in phyllosilicate minerals. Previously, the capability of Fe(III) reduction in smectite minerals has been shown for the bacterial groups with high numbers of heme‐rich MHC genes in their genomes, such as the genera Shewanella and Geobacter   reduction result in the abiotic reduction of U(VI) by biologically reduced iron . Without the amendments of electron acceptors, AOM activity was low but experimentally detectable. The AOM activity without the amended electron acceptors might be explained by the presence of Fe‐bearing phyllosilicates as suspended particles in the groundwater. Our future high‐pressure incubation experiments will be performed by using the Fe‐bearing phyllosilicates to determine in situ electron acceptors for subsurface AOM activity.In this study, a combination of high‐pressure incubation using Y.S. and H.N. designed the study. K.M., Y.S. and H.N. collected groundwater samples. H.N. and A.F. performed high‐pressure incubation experiments. T.I. and H.N. measured stable carbon isotopes. H.N. performed XRD and SEM‐EDS analyses. M.K., H.B., Y.A. and H.N. conducted 16S rRNA gene sequence analysis. H.N. and Y.S. co‐wrote the manuscript. All authors discussed the results and commented on the manuscript.The authors declare that there is no conflict of interest.Appendix S1: Supplementary InformationClick here for additional data file.


The population of older adults in rural areas is rising, and they experience higher rates of poverty and chronic illness, have poorer health behaviors, and experience different challenges than those in urban areas. This scoping review seeks to (1) map the state of the science of age‐friendly systems in rural areas regarding structural characteristics, processes for delivering age‐friendly practices, and outcomes of age‐friendly systems, (2) analyze strengths, weakness, opportunities, and threats of age‐friendly system implementation, and (3) make person, practice, and policy‐level recommendations to support active aging and development of age‐friendly communities.An international scoping review was conducted of articles that used age‐friendly framing, had a sample age of 45 years of age or older, self‐identified as rural, and reported empiric data. Searches were conducted in PubMed, CINAHL, AgeLine, PsychINFO, EMBASE, Scopus, and Academic Search Elite on October 26, 2021, and rerun March 10, 2023. Data were charted across three analytic layers: socioecological model, Donabedian's framework, and SWOT analysis.Results reveal limited data on outcomes relevant to organizations, such as return on investment or healthcare utilization. While the SWOT analysis revealed many strengths of age‐friendly systems, including their impact on persons' outcomes, it also revealed several weaknesses, threats, and gaps. Namely, age‐friendly systems have weaknesses due to reliance on trained volunteers and staff, communication, and teamwork. System‐level threats include community and health system barriers, and challenges in poor/developing areas.While age‐friendly systems in this review were heterogeneous, there is an opportunity to focus on unifying elements including the World Health Organization age‐friendly cities framework or 4Ms framework for age‐friendly care. Despite the many benefits of age‐friendly systems, we must acknowledge limitations of the evidence base, pursue opportunities to examine organizational metrics to support implementation and sustainability of age‐friendly systems, and leverage improvements in age‐friendliness at a community level. In 2006, the World Health Organization developed an action plan focused on improving the lives of older adults (age 65 and older) across the globe by identifying how communities could become “age‐friendly” and encourage active aging, the process of optimizing opportunities for health, participation, and security to enhance quality of life as people age.Examining age‐friendly systems, systems  which support active aging, engagement/participation, and well‐being of older persons and their caregivers, in rural areas is critically important. The National Rural Health Association reports that 18% of the United States of America rural populations (vs. 12% urban) are comprised of adults aged 65 and older.Developing an age‐friendly system requires local level engagement and support to assess needs and prioritize interventions that promote older adults' health and ability to age in place.2In this scoping review, we mapped the empiric literature on the rural age‐friendly ecosystem. We selected a scoping review over other types of reviews because it best aligns with the objective of summarizing the quantity and characteristics of the literature by design and other key features.2.1In keeping with Arksey and O′Malley methodology2.2In this second stage, the focus was on developing a decision plan for our literature search with an emphasis on being as comprehensive as possible. This began with a series of meetings between the team leader (H. B.) and a health sciences librarian with expertise in systematic searches to discuss the scope of the project and strategies available to answer our research question.2.2.1Comprehensive inclusion criteria were agreed upon and required that any accepted papers must use an “age‐friendly” framing or model. Progenitor terms such as “elder friendly” or associated terms such as “patient priorities care” were accepted. Additional criteria included human studies, sample age of 45 years of age or older (to assure only adult studies), papers self‐identified as rural and reported empiric data . Studies were excluded if not using age‐friendly framing; meta‐analyses and systematic reviews were also excluded.2.2.2The medical librarian conducted a series of test searches, which the team leader (H. B.) reviewed to determine the specific terms and databases to query. After this preparatory phase, searches were conducted in PubMed, CINAHL, AgeLine, PsychINFO, EMBASE, Scopus, and Academic Search Elite on October 26, 2021. The same searches were rerun on March 10, 2023 in the same databases to update the search. See Supporting Information: Table 2.32.3.1The title/abstract screening used a hybrid of human‐ and technology‐screening processes. After the team was trained on the inclusion and exclusion criteria and use of the spreadsheet for the title/abstract screen, four team members  screened 25% of the studies per member for exclusion criteria in the human screen. One team member (L. N.) then conducted a random 20% reliability check, which resulted in 93.5% (72/77) agreement. In the technology round, term identification  in the spreadsheet was used to identify studies meeting the inclusion criteria of empiric studies. When in doubt, a paper was retained for the next level of screening. For example, 25 papers that did not include an abstract were automatically advanced to full text screening if inclusion/exclusion criteria were not clear from the title. Inclusion criteria of age and rural were confirmed during the full text screening. A 100% reliability check was conducted on this set of papers and any discrepancies were justified during team meetings.2.3.2Ten team members  conducted a full text screening and data extraction (7−11 articles per team member). Once again, a 100% reliability check was conducted resulting in each paper being examined by at least two people. Effort was made to give each screener a new set of papers from their previous screen. The inclusion/exclusion criteria were applied again before any extraction took place. Forty papers met the protocol criteria and advanced to data extraction.2.42.4.1Using the study protocol, a second spreadsheet was developed which included the data elements necessary to answer our research question. The full team went through a hands‐on training session covering definitions for each data element, reviewing an example paper which had undergone full extraction, and then a practice group extraction. Five study domains  were assessed. Subsequent, one‐on‐one training with the team leader (H. B.) was held as needed. A final abstraction quality check was conducted by the team leader (H. B.) after all data forms were submitted.2.5Following Arksey and O′Malley's methodology,33.1N = 70) were conducted in five countries: the United States of America (n = 23), Canada (n = 13), China (n = 7), the Netherlands (n = 7), and Australia (n = 5) (Table n = 28), qualitative (n = 25), mixed methods (n = 8), and implementation (n = 8) studies. Units of analysis included older adults/adults (n = 41), clinicians/healthcare providers (n = 12), service  providers (n = 7), caregivers/family (n = 3), government agencies/employees (n = 3), age‐friendly leaders or committee members (n = 3), communities (n = 3), students (n = 1), and health systems (n = 3). Forty‐eight studies used the World Health Organization age‐friendly cities framework, 10 studies used the 4Ms framework for age‐friendly care, and 10 studies did not report a guiding framework. Specific information about study samples and purposes can be found in Table Most of the included studies  3.2.2individual rolesinterpersonal communication, collaboration, and partnershiporganizational workflow,environmental evaluationEvidence related to the process of delivering age‐friendly practices included a focus on: (1) 3.2.3Evidence related to outcomes of age‐friendly systems largely focused on improving individual and community health and related outcomes, including persons' quality of life,3.3person and system level  and organizational age‐friendly processes on persons' outcomes.Main strengths of age‐friendly systems identified at the 3.3.2person level were issues with volunteer burnoutsystem level were dependence on volunteersMain weaknesses of age‐friendly systems identified at the 3.3.3person level were existing sense of community,system level were integration of age‐friendly systems into existing structure and practices,Main opportunities for age‐friendly systems identified at the 3.3.4person level were differences in individual perceptions and acceptance of roles and importancesystem level were readiness for change,Main threats to age‐friendly systems identified at the 3.4Three key takeaways became clear from this qualitative synthesis. First, age‐friendly systems are heterogeneous, involving acute care to community settings and multiple frameworks. However, there are unifying elements, such as a focus on encouraging active aging and a few prominent models: the World Health Organization age‐friendly cities framework and the 4Ms framework for age‐friendly care. Articles in this review focused on age‐friendliness of a variety of systems in rural areas, including hospitals, hospital units, cities, communities, community service agencies, and residential care centers. While articles included a variety of stakeholders, such as service  providers, caregivers/family, government agencies/employees, age‐friendly leaders or committee members, communities, students, and hospitals, the majority focused on older adults and clinicians/healthcare providers. The structure, process, outcomes map revealed that most evidence to date has focused on structural factors of age‐friendly health systems, followed by processes for delivering age‐friendly practices. Least common was data on outcomes of age‐friendly systems in the rural ecosystem, especially at the interpersonal and organizational level. While the SWOT analysis revealed many strengths, including its impact on persons' outcomes,4The purpose of this review was to map the state of the science on rural age‐friendly systems and make person, practice, and policy‐level recommendations to support active aging and development of age‐friendly communities. Overall, much work remains to be done in this important area. The unifying element in this scoping review was the focus on the rural age‐friendly ecosystem. Further, it was discovered that while many terms and places were identified in this review, age‐friendliness was framed in similar ways. The most common framework for age‐friendliness in articles in this review was the World Health Organization age‐friendly cities framework, which has 8 domains: community and healthcare, transportation, housing, social participation, outdoor spaces and buildings, respect and social inclusion, civic participation and employment, communication, and information.4.1This review reveals some limitations of existing empiric literature on the rural age‐friendly ecosystem. While articles included a variety of stakeholders, the significant majority focused on older adults and clinicians/healthcare providers. This focus fits the purpose of improving care for older persons and clinicians/healthcare providers being positioned to impact this movement. However, we found limited data on caregiver and family involvement in age‐friendly systems and its impact on their outcomes/experience. This gap is critical as supporting family caregivers is a key aspect of age‐friendly acute and community care.4.2Because most of the articles in this review involved quality improvement projects, we completed a SWOT analysis of age‐friendly system implementation and sustainability in rural areas. While the SWOT analysis revealed many strengths, it also revealed several weaknesses, threats, and gaps. Age‐friendly systems have weaknesses due to reliance on specially‐trained volunteers4.34.3.1Development of age‐friendly systems and the associated evidence base present an opportunity to align healthcare and communities with needs and priorities of the older adult population in rural ecosystems. Evidence suggests that age‐friendly systems have a positive impact on patient and community outcomes, including quality of life,4.3.2Challenges for implementation and sustainability of age‐friendly systems in practice include often limited funding and differences in impact observed in urban versus rural areas.Implementation of age‐friendly systems is an important public health issue impacting the health and well‐being of our older adult populations.4.3.3Policy recommendations of this work begin with the need to promote opportunities to leverage improvements in age‐friendliness at a community level. Age‐friendly systems are commonly established and maintained at the community level, so that interventions fit with local needs. One challenge with both increasing older adults' access to existing community services and increasing collaboration or shared services between rural communities is transportation and availability of transportation services.4.4Our literature search was limited to articles published in English only. The decision to preclude a “gray” literature or a comprehensive bibliography review was made given the involvement of an expert health sciences librarian, multiple test searches, use of multiple databases, and detailed inclusion/exclusion criteria. In addition, we targeted main sources and types of evidence to identify key concepts in the body of literature.5This international scoping review maps the science on rural age‐friendly systems and makes person, practice, and policy‐level recommendations to support active aging and development of age‐friendly communities. While age‐friendly systems in this review were heterogeneous, there is an opportunity to focus on unifying elements including the World Health Organization age‐friendly cities framework or 4Ms framework for age‐friendly care. This article summarizes many potential barriers  and facilitators (integration of age‐friendly systems into existing structure and practices) to consider when implementing and sustaining an age‐friendly system in rural areas. Despite the many potential benefits of age‐friendly systems in healthcare systems and communities, we must acknowledge limitations of the evidence base, pursue opportunities to examine organizational metrics to support implementation and sustainability of age‐friendly systems, and leverage improvements in age‐friendliness at a community level.Daniel Liebzeit: Conceptualization; data curation; formal analysis; investigation; methodology; visualization; writing—original draft; writing—review and editing. Anna Krupp: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Jacinda Bunch: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Shalome Tonelli: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Emily Griffin: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Sarah McVeigh: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Nai‐Ching Chi: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Saida Jaboob: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Lynn Nakad: Data curation; formal analysis; investigation; visualization; writing—original draft; writing—review and editing. Alicia I. Arbaje: Formal analysis; investigation; writing—original draft; writing—review and editing. Harleah Buck: Conceptualization; data curation; formal analysis; investigation; methodology; supervision; visualization; writing—original draft; writing—review and editing.The authors declare no conflict of interest.The lead author Daniel Liebzeit affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.This project did not involve human participants. This research was carried out in accordance with recognized standards for scoping reviews.Supporting information.Click here for additional data file.


Amyloidosis due to the transthyretin Ser77Tyr mutation (ATTRS77Y) is a rare autosomal‐dominant disorder, characterized by carpal‐tunnel syndrome, poly‐ and autonomic‐neuropathy, and cardiomyopathy. However, related symptoms and signs are often nonspecific and confirmatory tests are required. We describe the age and frequency of early symptoms and diagnostic features among individuals of Jewish Yemenite descent in Israel.Records of mutation carriers were retrospectively reviewed. ATTRS77Y diagnosis was defined by the presence of amyloid in tissue and/or amyloid‐related cardiomyopathy.We identified the Ser77Tyr mutation at the heterozygous state in 19 amyloidosis patients  and 30 amyloid‐negative carriers. The probability for disease diagnosis increased from 4.4% at age 49 to 100% at 70 and occurred earlier in males. Initial symptoms preceded diagnosis by 5 ± 3.8 years (range 0–12) and were commonly sensory changes in the extremities. Erectile dysfunction predated these in 8/13 (62%) males. In two patients cardiac preceded neurological symptoms. Two patients declined symptoms. Electrophysiological studies near the time of diagnosis indicated a median neuropathy at the wrist in 18/19 (95%) and polyneuropathy in 13/19 (68%). Skin biopsy revealed epidermal denervation in 15/16 (94%) patients. Cardiomyopathy was identified in 16/19 (84%). Sensory complaints or epidermal denervations were present in 17/30 (57%) of amyloid‐negative carriers and co‐occurred in 10/30 (33%).ATTRS77Y symptoms commonly occur after age 50, but may begin earlier. Median neuropathy, skin denervation and cardiomyopathy are frequently identified. Symptoms may be absent in patients and common in amyloid‐negative carriers. TTR) gene cause variant TTR amyloidosis (ATTRv), inherited in an autosomal dominant manner. ATTRv is a severe but treatable multisystem disorder, characterized by a combination of entrapment‐, autonomic‐ and poly‐neuropathy, cardiomyopathy, gastrointestinal and renal disease as well as ocular, leptomeningeal and brain involvement.TTR. Some show a genotype–phenotype correlation, with predominant or initial neurological or cardiological manifestations, consistent with amyloid accumulation mainly in peripheral nerves or heart.Pathogenic mutations in the transthyretin , 99mTc‐3,3‐diphosphono‐1,2‐propano‐ dicarboxylic acid (DPD) or 99mTc‐hydroxymethylene diphosphonate if a monoclonal gammopathy is excluded.Echocardiography is commonly used to screen for increased left ventricular hypertrophy (LVH), which is typical for cardiac amyloidosis. When present, amyloidosis may be confirmed and specified by identifying amyloid deposits within an endomyocardial or an extra‐cardiac biopsy.In this study, we employed tissue biopsies, most commonly of skin, and/or cardiac criteria for diagnosis of ATTR in Ser77Tyr mutation carriers. The age and frequency of early symptoms, and of clinical, electrophysiological, and cardiac findings at the time of diagnosis in 19 ATTRS77Y patients were determined. We also describe the characteristics of 30 amyloid‐negative carriers and estimated the age of disease onset according to the age at diagnosis.TTR Ser77Tyr mutation carriers evaluated at the Sheba Medical Center (SMC) neuromuscular clinic, a tertiary referral center in Israel, between January 2016 and December 2022. The study was approved by the institutional review board (7476‐20‐SMC and 7238‐20‐SMC).We retrospectively reviewed medical records of all All participants underwent a detailed neurological examination. Sensory, motor, autonomic, and cardiac‐related symptoms and signs were collected.TTR gene coding region by commercial laboratories: Centogene (Germany) or Genetic Medico‐diagnostic laboratory Genica and Genome Center (Bulgaria). For the rest, targeted Sanger sequencing of the Ser77Tyr mutation in the TTR gene was performed at SMC or Hadassah Medical Center in Israel.All patients gave informed consent for genetic testing. Some of the patients underwent sequencing of the entire Nerve conduction studies (NCS) and electromyography (EMG) were performed at SMC as previously described.99mTc‐PYP or 99mTc‐DPD) or typical CMRCardiac evaluation included echocardiography when complaints were suspected as cardiac‐related and for all carriers above age 45. Heart failure symptoms were classified according to the New York Heart Association (NYHA).th percentile for age was determined abnormal, indicating small‐fiber polyneuropathy.Skin biopsies (3.0‐mm punch) were performed at the distal leg 10 cm proximal to the lateral malleolus (ankle) using standard procedures.Congo red staining was performed on at least 5 additional 16 μm glass‐mounted sections obtained from the same biopsy used for IENFD analysis. Sections were lightly counterstained with hematoxylin. Amyloid deposition was identified by autofluorescenceTTR Ser77Tyr mutation carriers were suspected to be affected by amyloidosis if a related objective sign, or symptom accompanied by an abnormal test was present.Carriers were determined as amyloid‐negative if (1) two skin biopsies were Congo‐red negative and (2) echocardiography ruled‐out LVH in individuals above age 45 or when cardiac‐related symptoms were suspected at any age. If LVH was present, CMR or scintigraphy was indicated and had to be negative as well.t‐test. Fisher Exact test was conducted to compare the risk of disease between age groups. The probability of becoming diagnosed with increasing age was calculated by Kaplan–Meier survival analysis. Statistical analysis was performed using GraphPad Prism 9.Patient characteristics are reported as mean ± standard deviation and range. Differences between independent groups were calculated by independent TTR Ser77Tyr mutation in 56 subjects, all self‐reportedly descendants of 5 Jewish families that emigrated to Israel from Yemen. Evaluation sufficient to define subjects as amyloidosis patients or amyloid‐negative was available for 49 mutation carriers.We identified the heterozygous Nineteen ATTRS77Y patients which fulfilled the diagnostic criteria for amyloidosis were identified , commonly with sensory complaints of tingling, numbness, pain or sensory loss in the hands in 13/19 (6%) patients (Table Erectile dysfunction predated these symptoms in the limbs in 8/13 (62%) males. Early gastrointestinal symptoms were reported by only one patient, but later in the course constipation was reported by 5/19 (26%), diarrhea by 4/19 (21%), and weight loss by 10/19 (53%) patients. Importantly, 2 patients  had no complaints and declined any neuropathic, autonomic, or dysfunctional symptoms.Neurological examination near the time of diagnosis showed a distal sensory loss in only about a third of patients: pain in 7/19 (37%), vibration in 8/19 (42%) and proprioception in 6/19 (32%). Distal weakness and absent Achilles deep tendon reflex were more common, present in 10/19 (53%) and 13/19 (68%) of patients, respectively.Additional disorders which are associated with polyneuropathy were present in 16/19 (84%) patients. In all of them, these delayed evaluation and identification of amyloidosis. These included T2D or hyperglycemia in 12 subjects, and monoclonal gammopathy of undetermined significance (MGUS) in 2, which both also had T2D. One patient exhibited a smoldering myeloma on bone marrow biopsy. Three patients had renal failure, requiring hemodialysis in two followed by renal transplantation, related in one case to polycystic kidney disease.Electrodiagnostic studies performed in proximity to diagnosis indicated a median neuropathy at the wrist in 18/19 (95%) patients, which was bilateral in 16/19  patients, and EMG showed active denervation in 11/17 (65%), as indicated by lower extremity fibrillations. The 6 patients which showed no evidence of a large‐fiber polyneuropathy had normal SSR, providing no evidence for the presence of small‐fiber sympathetic neuropathy.Skin biopsy was performed by 16/19 patients at age 61 ± 5.5 (range 49–70); two samples were obtained in 11/16 cases. The IENFD was abnormally low, indicating epidermal denervation in 15/16 (94%) patients  that had no clinical complaints but did show evidence for a median neuropathy at the wrist, polyneuropathy, and in one a cardiomyopathy  patients. In one, this was related to ischemic heart disease. Palpitation and syncope were not reported as early symptoms. Cardiomyopathy consistent with cardiac amyloidosis was present in 16/19 (84%) patients  in only 2/9 (22%), which both employed 99mTc‐PYP (Table Scintigraphy with The diagnosis of ATTRS77Y was established 5 ± 4.2 years (range 0–12) following initial neurological or cardiac symptoms.Seventeen patients were treated with Tafamidis and/or Patisiran. Eleven patients received Tafamidis 20 mg, and two Tafamidis 61 mg (due to cardiomyopathy). After Patisiran became available in Israel, 10 were treated with this agent (7 of them were switched from Tafamidis to Patisiran).One patient who was treated with liver transplantation at age 62 died due to amyloid‐related cardiac complications at the age of 70, 13 years following symptom onset. Four patients which were not treated by liver transplantation died due to amyloidosis‐related cardiac complications. The mean age at death was 70 ± 3.7 years (range 64–72), 2–13 years after sensory symptoms onset (average 9 ± 4.7). Two of these patients received no disease‐modifying treatment, since at that time treatment was unavailable, or not indicated due to a high disease score. One patient started Tafamidis treatment 8 years after symptom onset and died 2 years later, prior to the availability of Patisiran. Another patient started Tafamidis treatment 10 years after symptoms' onset, switched to treatment with Patisiran 1 year later due to disease progression, and died 16 months later due to amyloidosis‐related cardiac complications.Additional 37 individuals with the Ser77Tyr mutation at a heterozygous state were identified, of whom 30  had evaluation for the presence of amyloid disease Tables . Skin bip < 0.0001; Fig. p = 0.165).Most amyloid‐negative carriers were younger than patients  amyloid‐negative carriers, 5 in the group with and 2 in those without neuropathy symptoms  of amyloid‐negative carriers; in 10/17 (59%) with and in 7/13 (54%) without neuropathy‐related symptoms, but this difference was not significant.Seven carriers did not complete the required evaluation, and therefore were excluded from the analysis. Their mean age at clinical evaluation was 40 ± 11.9 years (range 25–56). Upper and/or lower limb sensory symptoms were reported by 3 of them, and clinical exam was normal in all. Electrodiagnostic studies were performed by 4 and echocardiography by 3, and were all normal. One 48‐year‐old female had a skin biopsy which showed epidermal denervation but no amyloid, and was reluctant to performing cardiac imaging. These findings, despite the incomplete data, are similar to that of the reminder of unaffected carriers.p < 0.001). At the age of 49, 4.4% of carriers become affected by amyloidosis, and 100% at the age of 70, with a median at 63 years. Male carriers were diagnosed with amyloidosis at an earlier age in comparison to females  amyloidosis increased with age, as most of the patients were older than 50 years , while most of the amyloid‐negative carriers were younger than that . We aggregated the data of patients and amyloid‐negative carriers that were older versus younger than 50 and found a significant risk for becoming affected above this age  over time, and this will be hopefully addressed in the future. In addition, the possible involvement of other body systems or organs  was not consistently investigated. Last, seven mutation carriers did not complete the required evaluation and were excluded from the analysis.TTR Ser77Tyr mutation causes a rare disease with typical but common presenting symptoms, usually due to a median and polyneuropathy, and cardiomyopathy. Identification of these in a mutation carrier, with or without clinical complaints and findings, suggests amyloidosis, particularly after the age of 50 years. This may be confirmed by Congo red staining of tissue, readily from skin, or detection of heart involvement by echocardiography.To conclude, the Amir Dori, Michael Arad and Lior Greenbaum contributed to the conception and design of the study; Amir Dori, Michael Arad, Yishay Wasserstrum, Arthur Pollak, Vera Nikitin, Merav Ben‐David, Jana Shamash, Ayelet Hashachar Nahum, Efrat Shavit‐Stein, Liran Domachevsky, Rafael Kuperstein, Dan Dominissini, Natalia Shelestovich, Menachem Sadeh, Elon Pras and Lior Greenbaum, contributed to the acquisition and analysis of data; Amir Dori, Michael Arad, and Lior Greenbaum contributed to drafting a significant portion of the manuscript or figures.Detection of TTR mutations in Israel was supported by a research grant from Pfizer [#59354385].This is to acknowledge that A. Dori received consultancy fees, honoraria for lectures and travel meeting attendance support from Pfizer, and investigator‐initiated research grant support from Pfizer and Alnylam Pharmaceuticals industry (companies that manufacture the drugs described is in this study). Additionally, A. Dori is the local site's primary investigator in Pfizer's Transthyretin Amyloidosis Outcome Survey (THAOS) and Alnylam's Global Observational Study of Patients with Transthyretin‐Mediated Amyloidosis (ConTTRibute) study. M. Arad received lectures honoraria and fees for participation in an advisory board from Pfizer.Table S1Click here for additional data file.Table S2Click here for additional data file.


Implantable cardioverter‐defibrillators (ICDs) are frequently used to prevent sudden cardiac death in patients with high‐risk arrhythmias. However, the use of ICD therapy in elderly patients beyond the predicted age of life expectancy is still controversial. We aimed to evaluate the predictors of mortality and clinical outcomes following ICD implantation in elderly patients.We conducted a retrospective analysis of 145 elderly patients aged 72 years and older who received ICD implantation between January 2010 and August 2015. We collected and analyzed baseline data, including clinical, demographic, and medical history, the reason for ICD therapy, procedural data, and echocardiography results. Follow‐up data included the development of complications and mortality. The predictors of mortality were identified using the univariate and multivariable Cox regression models.p = 0.008).During the median follow‐up duration of 30.5 [18.0–48.0] months, 141 cases completed follow‐up (mean age = 76.0 ± 3.7 years). Forty‐four patients experienced at least one episode of ICD therapy. Inappropriate shock, recurrent shock, and device‐related infection were the most frequent complications observed in our study. Of the 145 patients, 42 died during the follow‐up period, with an average survival time of 22.4 months after ICD implantation. Among these patients, 11 received ICD for primary prevention, and 31 received it for secondary prevention. Cardiovascular problems were the leading cause of death. We found that a low baseline ejection fraction (EF) was an independent predictor of mortality (hazard ratio = 0.93, 95% confidence interval: 0.90–0.98; Our study suggests that ICD therapy is a valuable treatment option for elderly patients beyond their predicted age of life expectancy. The study highlights the importance of baseline EF as a significant predictor of mortality in these patients. For this study, we included older adults who underwent ICD implantation for primary or secondary prevention and followed up for 5 years above the predicted life expectancy in Iran . This study conforms to the ethical principles outlined in the Declaration of Helsinki and its updates.2.2We comprehensively evaluated patient data, including baseline information such as demographic and clinical characteristics, medical history, procedural data, and echocardiography results. A number of clinical and demographic data were considered as baseline characteristics, including age, gender, hypertension, diabetes mellitus, history of coronary artery disease and cardiovascular disease, history of the coronary artery bypass graft, cardiomyopathy, chronic obstructive pulmonary disease, chronic kidney disease, and other comorbidities such as hepatic failure, Alzheimer and dialysis history, lead fracture, and malignancy as well as the type of ICDs device and baseline ejection fraction (EF). It should be clarified that baseline data was obtained before ICD implantation.After implantation, patients visited our pacemaker clinic every 6 months, and we retrieved their follow‐up data from hospital records to ensure standardized device programming and ongoing monitoring. In cases where we were unable to obtain postoperative clinical records, we contacted patients via telephone to assess their vital status. All‐cause mortality was the primary endpoint of our study. In addition, we will also investigate the survival rates, cost, and benefit of ICD therapy as secondary endpoints.2.3t‐test after ensuring the normal distribution of the variable. Due to non‐normal distribution, the follow‐up period was reported as median [Interquartile range]. Univariate Cox regression analysis was performed to determine the predictors of mortality. The independent predictors of survival were identified by multivariate Cox regression analysis by including variables with a p‐value < 0.200 in the univariate analysis. Data analyses were performed using SPSS version 23.0 (IBM) software. A p‐value less than 0.05 was considered statistically significant for all tests.Categorical variables were expressed as frequencies and percentages, and the Chi‐square test was used to compare the dead and alive patients. Continuous variables were presented as mean ± standard deviation and were compared between the study groups using the Student's 33.1From January 2010 to August 2015, 1198 patients underwent ICD implantation in our center. Based on our inclusion criteria, 145 patients were older than 72 years, and four were excluded due to incomplete follow‐up data. Finally, 141 patients were included in this analysis. The mean age of the participants at the time of implantation was 76.0 ± 3.7 years; 29 (20.5%) individuals were ≥80 years old, and 109 (77.3%) were men. Moreover, the mean implant time for all cases was 89.8 ± 2.1. In addition, 107 (75.8%) patients received an ICD, and 34 (24.1%) cardiac resynchronization therapy device (CRT‐D). Thirty‐five patients received the device for primary prevention, with the main indication in most patients being ventricular tachycardia. The baseline characteristics of the participants are entirely depicted in Table 3.2The median follow‐up duration was 30.5 [18.0–48.0] months. During this period, and based on the device analysis report, 44 patients received at least one shock episode. Of these patients, 31 (77.5%) had an appropriate shock. In terms of ICD complications, nine patients (6.4%) had an inappropriate shock, one patient (0.7%) had a fractured lead, four patients (2.8%) suffered device‐related infections, and four (2.8%) patients had a recurrent shock.3.3n = 5), sepsis or infection in 2.8% (n = 4), pulmonary edema in 7% (n = 3), myocardial infarction in 7.1% (n = 3), cerebrovascular accident in 9.5% (n = 4), gastrointestinal bleeding/disorder in 4% (n = 2), malignancy in 4.8% (n = 2), renal failure in 4.8% (n = 2), hepatic failure in 4.8% (n = 2), congestive heart failure in 2.4% (n = 1), accident in 2.4% (n = 1), and bowel obstruction in 2.4% (n = 1). In the remaining patients, the leading cause of death was unclear.During the follow–up period, the overall survival rate was 67.3%, and 42 (29.7%) patients died. The leading causes of death were cardiac arrest in 11%  were in the primary prevention group, while 31 (29.2%) were in the secondary prevention group, and the difference was not statistically significant. The median survival benefit after ICD implantation was approximately 36.0 [18.0–48.0] months for the primary prevention group and 30.0 [18.0–48.0] months for the secondary prevention group, which was not statistically significant. The mean survival after ICD implantation was 18.0 [6.0–38.5] months for the deceased group and 36.0 [24.0–48.0] months for the group still alive at their last follow‐up.3.4p = 0.040) (Table p = 0.008)  Table . Notably8) Table .4The main findings of the current study are that in elderly patients ( ≥ 72 years) undergoing ICD therapy, a low baseline EF is an independent predictor of mortality. During a median follow‐up of 30.5 months, 29.7% of the patients died. The survival benefit after ICD implantation was not statistically significant between primary and secondary prevention groups, and the mean survival was shorter in deceased patients than in those still alive at the last follow‐up. These findings highlight the importance of patient selection and monitoring in the elderly population undergoing ICD therapy.Despite the rising number of elderly individuals in society and the growing need for ICD therapy, there is a dearth of reliable data regarding the effectiveness and consequences of this treatment. The current guideline acknowledges ICD therapy as a viable treatment option for the elderly.Several studies have been conducted to evaluate the benefits of ICD therapy in elderly patients, but the definition of the elderly age group has not been clarified. Regarding Rees et al.'sBased on our study results, we found that a low baseline EF is an independent predictor of mortality. This result is closely related to other literature in which low EF, and advanced age, are the most significant predictors of mortality following ICD therapy.Nonetheless, the primary endpoint of our study was any cause of death, whether related or unrelated to ICD therapy. A multivariable and univariable regression model was used to account for the influence of unrelated causes. In utilizing these models, we sought to neutralize unrelated causes and focus on the specific effects of ICD therapy. However, establishing a definitive relationship between death causes and ICD therapy may require further investigation.In summary, clinicians should balance the cost and benefit of ICD therapy in elderly candidates and consider the probable complications and potential injury to the myocardium.4.1Our study has some shortcomings. First, the therapeutic policies of our referral center are based more on secondary prevention, which may cause a selection bias and reduce the generalizability of the study. Although this study was able to demonstrate the safety of ICD in the elderly, but can not demonstrate efficiency because of the lack of a control group. Therefore, a longitudinal study comparing patients with and without ICD therapy after the predicted age of life expectancy can clarify the definite clinical benefit of this treatment in elderly patients and its pure impact on survival. Without a control group in a randomized trial, the utility of ICD therapy cannot be judged. Lastly, the limited number of our study population and the used age life expectancy specific to Iran limit our findings’ generalizability. Thus, drawing decisive conclusions about the benefits and identifying the risks of ICD therapy beyond the age of life expectancy demands further studies.5Aging is accompanied by life‐threatening cardiac conditions, including ventricular tachycardia and heart failure, and thereby ICD therapy could be an effective treatment for elderly patients. However, decision‐making about ICD implantation in elderly patients is not as straightforward as it may seem. The present study shows an acceptable survival benefit in ICD recipients after the predicted age of life expectancy. It underlines the role of baseline EF as the main predictor of mortality. Based on the current knowledge and until obtaining evidence from a clinical trial designed specifically for elderly patients, we should consider predictors of mortality plus the individual characteristic while deciding on ICD therapy in an elderly patient. This information may help physicians better select patients who would most benefit from ICD therapy and improve clinical outcomes in elderly patients.Alireza Malekrah: Conceptualization; data curation; writing—original draft. Akbar Shafiee: Formal analysis; investigation; project administration; writing—review & editing. Amirhossein Heidari: Data curation; writing—review & editing. Ali Vasheghani‐Farahani: Supervision; writing—review & editing. Ali Bozorgi: Project administration; supervision. Saeed Sadeghian: Project administration; supervision. Ahmad Yaminisharif: Conceptualization; project administration; supervision; visualization; writing—review & editing.The authors declare no conflict of interest.In accordance with our hospital's routine, all patients provided written informed consent upon admission, granting permission for their clinical data to be used anonymously for research purposes. Tehran University of Medical Sciences and the Cardiology Department Research Committee approved the study protocol. . This study conforms to the ethical principles outlined in the Declaration of Helsinki and its updates.The lead author Ahmad Yaminisharif affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


EGFR mutation detection in advanced non‐small cell lung cancer (NSCLC). In clinical practice, whether EGFR variants in baseline plasma ctDNA of advanced NSCLC can predict prognosis in addition to guiding targeted therapy remains to be further explored.The plasma sample has emerged as a promising surrogate sample for EGFR mutation data from tissue detected by ARMS‐PCR and paired plasma samples within 1 month of admission detected by SuperARMS or ARMS‐PCR were collected. The correlation between baseline plasma ctDNA EGFR mutation status and survival was compared.In total, 315 NSCLC patients were retrospectively enrolled. EGFR mutation detection rates in tumor samples and plasma samples were 65.1% (205/315) and 43.8% (138/315). Referred to tissue results, the consistent rate of test ctDNA EGFR alteration by SuperARMS was higher than that detected by ARMS , either in stage I–IIIA patients  or stage IIIB–IV patients . Patients' treatment status and pathological subtype were the two factors that affected plasma ctDNA EGFR alteration detection accuracy. The concordance in non‐adenocarcinoma patients was obviously higher than that in adenocarcinoma (p = 0.02), and the concordance in treatment naïve patients was significantly higher than that in relapse patients (p = 0.047). In treatment naïve patients, the median PFS (mPFS) in plasma ctDNA EGFR‐positive patients was shorter than that in plasma ctDNA EGFR negative patients . In relapsed patients, the mPFS in plasma ctDNA EGFR‐positive patients was 9.0 months versus 11.0 months in plasma ctDNA EGFR negative patients (p = 0.1).EGFR mutation positivity not only guides targeted therapy but also predicts a worse prognosis.A plasma sample could be an alternative for a molecular test when tissue samples was unavailable. The SuperARMS‐PCR detection method has high sensitivity in real‐world clinical practice. Furthermore, in patients with stage IIIB‐IV, baseline plasma ctDNA  EGFR alteration by Super‐PCR was higher than detected by ARMS‐PCR. And in ctDNA EGFR mutation positivity not only guide targeted therapy, but also predict a worse prognosis. Confiremed the concordance of test ctDNA  Currently, among the various blood EGFR variants. And further explored the prognostic value of ctDNA monitoring in advanced NSCLC therapies. In the present study, neither the molecular detection method including ARMS‐PCR or SuperARMS‐PCR nor the follow‐up treatment regimens have been intervened. The assay performance of the reagents thus evaluated may be more suggestive for clinical practice. The factors that could affect the detection rate of ctDNA EGFR mutation were deeply analyzed. The data from this study showed that the SuperARMS can be used to detect plasma samples from NSCLC patients in early stages, whereas previous data reported in the literature almost all focus on samples from patients in advanced stages.In our study, we collected paired tissue and plasma samples to explore the consistency of plasma cfDNA 22.1EGFR mutation detection records were retrospectively screened in the Department of Pathology, Beijing Chest Hospital. Among them, the exclusion criteria were set as follows: (1) Patients who cannot be pathologically diagnosed with non‐small cell lung cancer, (2) The interval detection time between tissue and plasma exceeded 1 month. Therefore, 315 pathologically diagnosed NSCLC patients were finally enrolled in this study. The patients' clinicopathological information, including age, gender, tumor‐node‐metastasis (TNM) stage, smoking history, EGFR molecular detection results of tissue and plasma samples, and treatment history were investigated and collected in the clinicopathological system and pathological information system of our hospital  according to the manufacturer's instructions. EGFR mutation status of the tumor tissues was detected using the ARMS EGFR Mutations Detection Kit (Amoy Diagnostics) according to the manufacturer's instructions.The 2.3EGFR mutation in plasma before treatment, regardless of subsequent treatment regimens.Among the 315 enrolled patients, a total of 268 patients received chemotherapy or targeted therapy after plasma testing, and only 227 of 268 patients had relatively complete treatment follow‐up information, among which 139 treatment naïve patients and 82 relapsed patients were in stage IIIB‐IV. Patients' treatment efficacy data were collected and progression‐free survival (PFS) was calculated. PFS differences were compared between patients with positive and negative ctDNA 2.4p < 0.05 was considered statistically significant. The Kaplan–Meier was used for PFS analysis, and survival curves were compared using the log‐rank test.The data were analyzed using SPSS software version 20 (IBM). The relationship between the two groups was evaluated using a standard chi‐squared test, and 33.1Among the 315 enrolled NSCLC patients, 149 (47.3%) were males, 166 (52.7%) were females, 193 (61.3%) patients were over 60 years old, 184 (58.4%) had never smoked; the number of treatment naïve patients were 232 (73.7%), 299 (94.9%) were in stage IIIB‐IV, and 279 (88.6%) had adenocarcinoma. The clinicopathological characteristics of the patients are shown in Table EGFR mutation was detected in 205 (65.1%) tissue samples and 138 (43.8%) plasma samples. In tissue samples, a total of 237 EGFR mutation sites were detected, including L858R  and 19‐Del , T790M , G719X , 20‐Ins , and L861Q . In plasma samples, a total of 156 EGFR mutation sites were detected, including L858R , 19‐Del , T790M , G719X , 20‐Ins , and L861Q . The EGFR mutation profile in detail is shown in Figure In the present study, 3.2EGFR mutation detection in tumor tissue DNA, the total concordance rate of ctDNA detected by SuperARMS was higher than that of ARMS , especially in patients in stage I‐IIIA  compared with patients in stage IIIB‐IV . Besides, in treatment naïve patients the concordance of ctDNA detected by SuperARMS were higher than those ctDNA detected by ARMS . Reverse results were observed in relapsed patients with no significant difference  , and positive predictive value (PPV) of ctDNA detected by ARMS was 59.7% , 92.9% , 95.6% , and 47.3% . The total sensitivity, specificity, NPV, and PPV of ctDNA detected by SuperARMS were 68.4% , 97.6% , 97.8% , and 65.6% . Compared with EGFR detection and plasma ctDNA EGFR detection by SuperARMS were analyzed. The results showed that there were statistically significant differences in treatment status and pathological types. Eight of the 28 non‐adenocarcinoma patients detected by SuperARMS had EGFR mutation, seven of which were positive both in tissue and plasma, one of which was only positive in tissue. The concordance in non‐adenocarcinoma patients was obviously higher than that in adenocarcinoma (p = 0.02) (Table p = 0.047)  Table . The con7) Table , Table 33.3EGFR‐negative had a significantly longer PFS than those of EGFR‐positive  regardless of treatment regimen  of the 37 patients with plasma ctDNA EGFR‐negative was significantly longer than that of the 55 patients with plasma ctDNA EGFR‐positive    Figure . Forty‐s) Figure .EGFR‐negative and EGFR‐positive patients regardless of treatment regimen  , and that was 6.5 months vs. 6.5 months (p = 0.5) in the chemotherapy group  Figure . Fifty‐ep Figure .4EGFR mutation has been determined. Genetic testing based on tumor tissue is considered the gold standard method, However, it was reported that only 18% of advanced NSCLC patients have sufficient tumor specimens for full tissue genotyping of all eight guideline‐recommended genomic biomarkers, thus new alternative tissue samples are needed for genetic testing in NSCLC in clinical practice.EGFR mutation detection in paired tissue samples and blood samples. Based on the highly sensitive SuperARMS‐PCR method to detect plasma ctDNA EGFR mutation, the concordance rate with tissue detection was as high as 79.5%.EGFR mutation status and prognosis in advanced patients. Stratified analysis of the treatment status of patients, compared to the intention‐to‐treat population, treatment naïve patients with baseline plasma EGFR‐negative would have better PFS which was slightly higher than previously reported.EGFR‐negative patients was also found in relapsed patients. So precise stratification of patients' treatment status can better benefit clinical patients. And since these data were obtained from real clinical practice, it has greater significance for clinical diagnosis and treatment guidance.EGFR‐TKI has been widely used in the treatment of advanced NSCLC, but the precondition of EGFR‐TKI application is whether EGFR detection methods were included in this study, ARMS‐PCR and SuperARMS‐PCR, because the SuperARMS‐PCR method was available after the year 2018.EGFR mutation detection was only 59.7%, which was lower than those detection results of SuperARMS‐PCR (68.4%) as well as the published data.EGFR mutation was more prominent in plasma samples of I‐IIIA patients . This further illustrated the advantages of SuperARMS in the detection of plasma samples with low abundance.EGFR in relapsed patients was lower than that of ARMS‐PCR, but there was no statistical difference, which may be caused by the low number of samples between the two groups. To further analyze the factors affecting the detection rate of EGFR mutation in SuperARMS‐PCR ctDNA, the patient's pathological subtype was an influencing factor. In this study, the consistent rate of ctDNA EGFR detected by SuperARMS‐PCR in patients with non‐adenocarcinoma was higher than that in patients with adenocarcinoma. This phenomenon has also been mentioned in other studies.Two plasma ctDNA EGFR mutations at baseline had better EGFR‐TKI efficacy than patients with detectable plasma EGFR mutations at baseline.EGFR mutation was detected.In patients with advanced lung cancer, what is the prognostic value of baseline blood ctDNA mutation status in addition to guiding targeted drugs? Patients from the FLAURA study with undetectable plasma p = 0.004) or chemotherapy , patients with baseline ctDNA EGFR mutation had worse PFS. No difference (p = 0.1) in the PFS was observed in the chemotherapy group because of the few number of ctDNA EGFR‐positive patients (N = 4). Among the relapsed patients who received targeted therapy, patients with plasma ctDNA EGFR‐positive before treatment also had worse PFS compared with patients with plasma ctDNA EGFR‐negative . The patients with detectable plasma ctDNA mutation at baseline maybe have a high tumor load, caused by spatial heterogeneity of the tumor, leading to a poor clinical outcome.In our study, the median duration of follow‐up was 15.0 months (IQR 0.33–140.0). Since the patients in this study were treated with chemotherapy or targeted monotherapy, and the first‐generation or second‐generation EGFR TKIs was used in targeted therapy, the median follow‐up time of 15 months could track more than 50% of patients' relapse.EGFR mutation sites has no statistical value, so a stratified analysis of EGFR classic mutation sites and rare mutation sites was not performed. Second, when analyzing the prognostic value of baseline ctDNA mutation status, PFS was used as the study endpoint rather than OS due to the limited follow‐up time. The survival status of these patients will be followed to obtain OS data. Finally, more patients receiving chemotherapy and immunotherapy will be included in follow‐up studies to observe the correlation between baseline ctDNA EGFR mutation status and patient prognosis.There were several limitations in this study. First, due to the limited sample size, a more detailed analysis of the detection rate of 5EGFR mutation in plasma. And in patients with stage IIIB‐IV, baseline plasma ctDNA EGFR mutation positivity not only guides targeted therapy but also predict a worse prognosis.This study confirmed that plasma samples can be used as a surrogate for molecular testing when tissue samples were not available in real‐world studies, especially in patients with advanced NSCLC. Compared with the ARMS‐PCR detection method, the SuperARMS‐PCR detection method had higher sensitivity for the detection of ctDNA Chaolian Long: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; supervision ; visualization ; writing – original draft ; writing – review and editing . Kun Li: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; project administration ; supervision ; visualization ; writing – original draft ; writing – review and editing . Zichen Liu: Formal analysis ; investigation ; visualization . Nana Zhang: Formal analysis ; investigation ; visualization . Xuya Xing: Formal analysis ; investigation ; visualization . Liming Xu: Data curation ; formal analysis ; methodology . Fei Gai: Data curation ; formal analysis ; methodology . Nanying Che: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition ; investigation (lead); methodology (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); visualization (lead); writing – review and editing (lead).This work was supported by grants from the National Natural Science Foundation of China (Grant Numbers: 82072381), Beijing Municipal Science and Technology Project .Liming Xu and Fei Gai are full‐time employees of Amoy Diagnostics Co, Ltd.The study was approved by the Ethical and Institutional Review Boards for Human Investigation of the Beijing Chest Hospital (number: 2020‐keyan‐linshen14).Figure S1.Click here for additional data file.Table S1.Click here for additional data file.


When an audience member becomes immersed, their attention shifts towards the media and story, and they allocate cognitive resources to represent events and characters. Here, we investigate whether it is possible to measure immersion using continuous behavioural and physiological measures. Using television and film clips, we validated dual-task reaction times, heart rate, and skin conductance against self-reported narrative engagement. We find that reaction times to a secondary task were strongly positively correlated with self-reported immersion: slower reaction times were indicative of greater immersion, particularly emotional engagement. Synchrony in heart rate across participants was associated with self-reported attentional and emotional engagement with the story, although we found no such relationship with skin conductance. These results establish both dual-task reaction times and heart rate as candidate measures for the real-time, continuous, assessment of audience immersion.The online version contains supplementary material available at 10.1186/s41235-023-00475-0. The average UK adult spends almost a third of their waking hours (5 hours and 40 minutes per day) watching television, film, or other online video content Ofcom, . One ofta state of deep mental involvement in which their cognitive processes (with or without sensory stimulation) cause a shift in their attentional state such that one may experience dissociation from the awareness of the physical world’  and in the context of individual audience members viewing alone. In this paper, we are referring to the latter: synchrony arising from audience member’s cognitive processing of the content.In this experiment, we also investigate whether synchrony in reaction times (e.g. correlations across participants arising from similar cognitive processing of the media) may be driven by immersion. We note that audience synchrony has been used both in the context of viewer In Experiment 1, we explore if the dual-task reaction times task performance is related to immersion as measured by a standard immersion questionnaire. Our aim is to validate dual-task reaction times against questionnaire-based, self-reported immersion. We selected the Narrative Engagement Scale . Participants were excluded who: did not watch all clips (n = 2), did not meet the eligibility criteria (n = 1), or had an error rate on the dual-task paradigm above or equal to chance (n = 3); leaving a final sample size of n = 164 . Note that excluding participants performing below chance deviates from our preregistered exclusion criteria of < 75% correct responses  and low (600 Hz) tones at 15-s intervals and were required to make a button press response  as soon as they heard the tone. Tones were 1 s length sine waves, and so distinctive from the audio characteristics of the content. Tones were presented at approximately 10% louder (measured using root mean square energy) than the average volume of all clips. Participants were instructed to set their volume to a comfortable listening level and completed 10 practice trials before the main task to ensure they could discern the tones. As tones themselves may necessarily disrupt the experience of immersion, we elected to compromise varying the interval between tones (and reducing predictability), by creating maximal distance between each tone to avoid potential interference.Participants completed the Narrative Engagement Scale , version January 2022. All data were analysed using R 4.1.1 , it was not possible to fully counterbalance the design and so the clip order was random. The breakdown of clips in each order is provided in Additional file M = 999 ms, SD = 427 ms with M = 92.7%, SD = 26.9% correct responses , emotional engagement , narrative presence , and narrative understanding . In subsequent references to ‘narrative engagement’, we describe the mean of all 12 items. When referring to a dimension of narrative engagement, we describe the mean of the subscale items which assess that dimension. The mean single-item immersion rating was M = 4.41, SD = 1.83.Mean dual-task reaction times were b = 29.24, 95% CI ). Our single-item question of immersion (hereafter referred to as ‘immersion’) also positively influences reaction time . Individual p values are not provided for linear mixed model estimates, due to the problems associated with interpreting p values from linear mixed models  = 6.82, p = 1.69 × 10–10. Similarly, participants’ individual correlation between reaction times and self-reported immersion was significantly greater than zero: mean r = 0.185, t(163) = 5.85, p = 2.58 × 10–8.To assess whether this relationship was robust within participants, we computed the correlation between reaction time and narrative engagement for each participant and compared this distribution against zero. This approach accounts for individual differences in preference, as the test makes no assumptions about which content participants may rate as most engaging. Using a one-sample, two-tailed t test, we found that this overall distribution was significantly greater than zero: mean r(5) = 0.580, p = 0.172), but was significantly associated with error rate (r(5) = 0.823, p = 0.023). As such, we have evidence to conclude that louder clips may be masking the detection of the tones. We therefore included RMSE as a fixed effect in subsequent linear mixed-effects models, to account for this effect of clip volume.One interpretation of these data is that the more engaging clips may simply be louder, and as such it may be more difficult to discern reaction time probes, leading to slower responses or increased errors from participants. To address this possibility, we calculated the root mean square energy (RMSE) for each clip’s audio track, as a measure of loudness. Given we only have 7 RMSE values, we are constrained to make comparisons based on those values and have averaged reaction times and tone discrimination error rate per clip for this analysis. RMSE was not significantly correlated with reaction times . We also included fixed effects for single-item immersion, clip order , and for familiarity (whether participants had seen the clip before). Participant was set as a random slope to account for participant-level differences in average reaction time.To assess which dimensions of the Narrative Engagement Scale were influencing reaction time, we fitted a linear mixed model to our data using the ‘lme4’ package in R , narrative understanding , familiarity , and clip volume  did not significantly affect reaction time. Interestingly, despite the dual-task paradigm being a measure of attention, attentional focus also did not affect reaction times . We can conclude therefore that increases in reaction time are predominantly driven by emotional engagement in the story.As shown in Fig. RT) was not significantly related to narrative engagement (r(5) = 0.554, p = 0.197) or immersion (r(5) = 0.490, p = 0.264). As with mean reaction times, we then looked to assess whether this correlation was robust across participants, by calculating the relationship between individual participant’s ISCRT and narrative engagement. Individual participant’s ISCRT was significantly related to narrative engagement  = 6.73, p = 2.84 × 10–10) and immersion  = 5.75, p = 4.26 × 10–08).We then looked to assess whether synchrony in reaction time was related to narrative engagement. We rely on the most widely used method to measure synchrony: inter-subject correlation  = − 11.06, p < 0.001) but did not show differences in reaction times  =  − 0.22, p = 0.82). Similarly, participants who had seen the specific clip before were more engaged  =  − 11.62, p < 0.001) but did not show differences in reaction times  = − 1.45, p = 0.15). To summarise, familiarity was associated with higher narrative engagement, but did not affect reaction time.Additional file S1: Figure S4 (left) presents a breakdown of participant’s familiarity scores in Experiment 1. To assess if familiarity affected our measures, we used Welch’s two-sample, two-tailed r(1146) = 0.797, p = 2.2 × 10–16. To assess which dimensions immersion was related to, we fit a linear regression predicting immersion from each dimension of narrative engagement. Immersion was predicted by attentional focus , emotional engagement , and narrative presence , but not narrative understanding . This offers a promising indication that most dimensions of engagement (excluding understanding) could be indexed by a single-item questionnaire.Finally, we looked to assess whether our single-item question of immersion was related to overall narrative engagement. We found a significant correlation between single-item immersion and the Narrative Engagement Scale: Experiment 1 provides strong evidence that greater levels of immersion are associated with slower dual-task reaction times. This provides strong support for the view that immersion arises from changes in attention Murray, . This isA simple way to interpret these results is in terms of an enveloping of perceptual apparatus , their allocation of attentional resources towards the content remains unchanged. We do note that this experiment did not ask participants how long ago they previously saw the content; participants who recently viewed the content may show differences in attentional orientation.While dual-task reaction times are able to index the focussed attention arising during immersion, they are not without their own pitfalls. The regular probe intervals may themselves act as a distraction from becoming fully immersed within the media. As an example of this, Hammond et al. (unpublished) find evidence that reaction time probes cause subsequent physiological responses which may be associated with a startle reflex. Further, while providing moment-to-moment estimates, reaction times are not truly continuous , and as such could not be used to ascertain faster-moving changes in attention and immersion.Experiment 2 looked to further explore the relationship between immersion and two physiological measures: heart rate and skin conductance. These measures have both been widely used within media psychology . The sample size was preregistered at https://osf.io/ckx8q.Experiment 2 recruited 50 participants and used the same eligibility criteria as Experiment 1. Participants were recruited from the University of Bristol student and staff population and were reimbursed with course credit, or £10 financial reimbursement if they were not Psychology students. Two participants were excluded as the equipment failed to capture synchronisation information between the content and physiological responses, leaving a final sample of Experiment 2 used 10 short clips between 132 and 252 s . Artefacts were identified as regions with no clear R waves and were corrected by linear interpolation from the preceding and following signals.Skin conductance was recorded using the EDA100C module, and electrodes were placed on the distal phalanges of the index and middle fingers, on the participant’s non-dominant hand. Skin conductance was downsampled to 125 Hz and filtered using a 1 Hz low-pass filter.The narrative engagement was obtained through the same procedures as Experiment 1.As in Experiment 1, we had a within-subjects design , and separated into constituent dimensions this was: attentional focus , emotional engagement , narrative presence , narrative understanding . For single-item immersion, mean scores were M = 4.92, SD = 1.64.For each participant, physiological measures were first averaged over 1-s intervals and difference scores were calculated by subtracting the grand mean for each participant from their scores in each clip. This standardises responses across participants. Mean heart rate was b = − 0.057, 95% CI ), and heart rate and immersion . Individual participant’s heart rate and engagement correlations were significantly lower than zero for engagement  =  − 2.61, p = 0.012) but not immersion  = -1.95, p = 0.057), offering some indication that higher self-reported engagement is associated with lower heart rate. For a discussion of heart rate variability, see Additional file First, we investigated the relationship between heart rate and narrative engagement. Using linear mixed-effects models again, we found a negative relationship between heart rate and narrative engagement (HR) and narrative engagement: narrative engagement increases heart rate synchrony . A similar relationship was found between ISCHR and immersion: b = 0.012, 95% CI . ISCHR is defined in the same way as ISCRT in Experiment 1.We find strong evidence that synchronicity in heart rate is a predictor of engagement. Figure HR and engagement for each participant  = 7.38, p = 2.79 × 10–9) as well as between ISCHR and immersion  = 6.54, p = 4.95 × 10–8).This relationship between heart rate synchrony and engagement was also robust across participants. Again, we computed correlations between ISCant Fig. : Bottom.As with reaction times above, we then built a linear mixed model, including fixed effects for each dimension of narrative engagement, single-item immersion, clip order, familiarity, and a random intercept for participant. Figure b = 0.009, 95% CI ), emotional engagement , clip order , and familiarity  significantly increase HRISC, while narrative presence , narrative understanding  and immersion  do not significantly affect ISCHR. Higher narrative engagement drives synchrony between participants' heart rate then, and this is predominantly related to attentional and emotional engagement with the narrative.As shown, attentional focus , or immersion . Individual participant’s correlations between skin conductance and narrative engagement  =  − 0.58, p = 0.57) or immersion  = 0.11, p = 0.912) were also nonsignificant. Additionally, we did not find evidence of a relationship between inter-subject correlation of skin conductance (ISCSC) and narrative engagement  or immersion . This relationship was also not significant at an individual participant level for engagement  = − 0.50, p = 0.617) or immersion  = − 0.69, p = 0.492).Mean skin conductance level for Experiment 2 was M unfamiliar = 4.52, M familiar = 5.12, t(101) = -5.49, p = 2.99 × 10–7) and when they had seen the specific clip before  = -6.02, p = 6.23 × 10–8). Heart rate was not affected by familiarity with the series before  = -0.27, p = 0.791) or the specific clip  = -0.44, p = 0.664). Similarly, skin conductance was not affected by participants seeing any of the series before  = 0.15, p = 0.880) or the specific clip before  = 0.65, p = 0.516). From this, we can conclude that while participants found familiar content more engaging, this did not affect physiological responses. Additional file In Experiment 2, familiarity was related to higher engagement both when participants had seen any of the series before (r(478) = 0.755, p = 2.2 × 10–16. Again, immersion was predicted by attentional focus , emotional engagement , narrative presence , but not narrative understanding .The same pattern of results was present as in Experiment 1. Our single-item question of immersion was significantly correlated with narrative engagement: In Experiment 2, we find evidence that self-reported narrative engagement is related to synchronisation across audience’s heart rate. These results add to the growing body of literature which suggests media stimuli can evoke synchronised responses across viewers—in this case at a physiological level. This evidence began with research by Hasson  demonstrWhile we argue that heart rate synchrony in our study is driven by immersion, a critical reader may suggest that the result is due to an unintended confound, such as laughter. Through this view, our more engaging stimuli are also more comedic, which may evoke laughter and associated increases in heart rate and blood pressure (Sugawara et al., In our study, skin conductance was not sensitive to changes in immersion. This is in contrast to other findings such as Sukalla et al.  that skiWhen immersed in narratives, we allocate attentional resources to construct detailed representations of events, characters, and their intentions and emotions; to understand the content and anticipate outcomes Zwaan, . AlongsiIn both experiments, our measures were related to narrative engagement through the attentional and emotional dimensions of the scale. This complements existing research that film stimuli narrow attention (Bezdek & Gerrig, Film has previously been considered an emotional machine Tan,  and therHowever, we did not find relationships between the narrative understanding or presence dimensions of the Narrative Engagement Scale. The former is consistent with previous research indicating an absence of relationship between comprehension and gaze synchrony. In a study by Hutson et al. , comprehThe absence of a relationship between immersion and narrative presence is perhaps more surprising. Presence is generally conceptualised as an individual’s perception being oriented from the real world towards the world of the media (Waterworth et al., In these experiments, we employed a design which was indifferent to each participant’s ratings of immersion in each clip, and individual’s propensity to become immersed. Several recent attempts have been made to classify individual’s tendency to become immersed: for example, the narrative engageability scale (Bilandzic et al., As immersion itself is in part characterised by a lack of meta-awareness (Agrawal et al., HR may be applied to index narrative moments.These experiments demonstrate that reliable changes across audiences’ attention and physiology can be seen during the everyday context of watching television and film. These mediums have evolved to hold attention across extended periods, through the use of cinematic conventions and editing techniques (Cutting et al., In a traditional psychology experiment, participants are often bored and disengaged and are only studied across discrete single-trial intervals. In contrast, the real world is continuous and rich in engaging stimuli. The above experiments present an opportunity to study how attention is sustained and disrupted in a naturalistic context.The purpose of the current study was to validate behavioural and physiological measures of audience immersion across several types of film and television content. Further, these experiments demonstrate the narrowing of attention and robust emotional engagement which occurs as we watch engaging media. This toolkit can now be applied to a wider range of contexts, including different media formats, or real-world situations. Our measures of immersion should additionally be sensitive to fluctuations in immersion within a single stimulus, such as a feature-length film. The dual-task paradigm and heart rate ISCs both offer the opportunity for continuous measures of audience immersion, while heart rate ISC offers this in a truly non-invasive way.Additional file1. Supplementary materials.