This systematic review and meta-analysis investigates the association of professional-led and layperson-led weight loss interventions with short- and long-term weight reduction among children and adolescents with overweight and obesity. What are the short- and long-term associations of professional- and layperson-led behavioral interventions with weight loss for children and adolescents with overweight and obesity?In this network meta-analysis of 78 unique clinical trials including 5780 participants, professional-led, but not layperson-led, interventions were associated with short-term reductions in absolute and relative weight compared with standard care. These reductions were not sustained long term.Professional-led behavioral weight loss interventions were associated with weight reduction among children and adolescents with overweight and obesity; there was a lack of direct evidence for the association of layperson-led approaches. The appropriate approach for weight loss among children and adolescents with overweight and obesity remains unclear.To evaluate the difference in the treatment outcomes associated with behavioral weight loss interventions led by laypersons and professionals in comparison with unsupervised control arms among children and adolescents with overweight and obesity.For this systematic review and meta-analysis, the Medical Literature Analysis and Retrieval System Online (MEDLINE), Embase, the Cochrane Library, and Cumulative Index of Nursing and Allied Health Literature (CINAHL) databases were searched from January 1, 1996, to June 1, 2019.Included in this study were randomized clinical trials (RCTs) of behavioral interventions lasting at least 12 weeks for children and adolescents (aged 5-18 years) with overweight and obesity. Exclusion criteria included non-RCT studies, interventions lasting less than 12 weeks, adult enrollment, participants with other medical diagnoses, pharmacological treatment use, and articles not written in English. Two of 6 reviewers independently screened all citations. Of 25 586 citations, after duplicate removal, 78 RCTs (5780 participants) met eligibility criteria.A bayesian framework and Markov chain Monte Carlo simulation methods were used to combine direct and indirect associations. Random-effects and fixed-effect network meta-analysis models were used with the preferred model chosen by comparing the deviance information criteria. This study was registered with the International Prospective Register of Systematic Reviews (PROSPERO) and followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline.The immediate and sustained changes in weight and body mass index (BMI) standardized mean difference (SMD) were primary outcomes planned before data collection began, whereas waist circumference and percent body fat were secondary outcomes. The hypothesis being tested was formulated before the data collection.P < .001) and BMI  that were not sustained long term . There was no association between layperson-led interventions and weight loss in the short-term  or long-term  compared with standard care. No difference was found in head-to-head trials .Of 25 586 citations retrieved, we included 78 RCTs (5780 participants), with a follow-up of 12 to 104 weeks. Compared with the control condition, random-effects models revealed that professional-led weight loss interventions were associated with reductions in weight (mean difference [MD], −1.60 kg ; 68 trials; This systematic review and meta-analysis found that professional-led weight loss interventions were associated with short-term but not sustained weight reduction among children and adolescents with overweight or obesity, and the evidence for layperson-led approaches was insufficient to draw firm conclusions. While efficacious, these approaches are costly and often impractical in real-world settings. Less intensive interventions delivered in community settings are less costly but often yield less significant weight loss.8 Few studies have directly compared the association between short-term and sustained interventions on weight management in children and adolescents with overweight and obesity.11Child and adolescent obesity are a global public health concern.13 In some settings, community health workers or peer leaders yield meaningful improvements in lifestyle behaviors and health outcomes among persons living with chronic disease.15 A series of recent trials suggested that behavioral interventions led by nonprofessionals yield similar results to those led by trained professionals.8 In the context of pediatric obesity, a limited number of trials suggest that peer- or layperson-led approaches may be associated with the achievement of successful weight loss.8 To the best of our knowledge, this has yet to be investigated by a systematic literature review with meta-analysis.Engaging laypersons or community-based health workers to deliver health interventions is an attractive public health model for disease management, as it is cost-effective and can be tailored to local needs.16 Network meta-analyses can assess treatment outcomes or safety when few direct head-to-head trials exist.18 With the abundance of therapeutic trials for weight loss among children and adolescents with overweight or obesity,2 a network meta-analysis is an attractive model for comparing the associations of layperson- and professional-led approaches with weight loss. Accordingly, we conducted a systematic review and network meta-analysis to assess the association of behavioral interventions led by lay individuals vs those led by professionals, compared with the standard of care, with short- and long-term weight loss among children and adolescents younger than 18 years with overweight or obesity.Network meta-analysis allows for the comparison of multiple treatments in 1 statistical model.PRISMA) reporting guideline. We searched the Medical Literature Analysis and Retrieval System Online (MEDLINE), Ovid , Embase Ovid , Cumulative Index of Nursing and Allied Health Literature (CINAHL), EBSCOhost (EBSCO Information Services), and the Cochrane Library (Wiley) databases. A combination of controlled vocabulary  and keywords , in addition to free-text terms, were used. A search for randomized clinical trials (RCTs) from January 1, 1996, to June 1, 2019, was conducted without any restriction on the language of publication  and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (overweight (between 1 and 1.99 SD) and obesity (>2 SD for age and sex) were defined according to age- and sex-specific body mass index  criteria for children and adolescents.20 We excluded trials evaluating pharmacotherapy for weight loss as well as cluster RCTs or quasi-experimental studies and those published in languages other than English. Cluster randomized trials were excluded as they would potentially increase trial heterogeneity, introduce difficulties in estimating intervention-type associations, and potentially be more common among layperson-led interventions, introducing a design bias into our analysis. RCTs that met inclusion criteria were classified into 3 comparisons: (1) professional-led vs standard, (2) layperson-led vs standard, and (3) professional-led vs layperson-led weight loss interventions.We included RCTs of parallel group design evaluating weight loss interventions administered for a minimum of 12 weeks in children and adolescents with overweight or obesity and younger than 18 years. The terms Professional-led interventions were defined as led by health care professionals, such as dieticians, nurses, kinesiologists, physicians, and other relevant certified health care professionals, at least twice during the conduct of the RCT. Direct involvement of laypeople  in participants’ schools, communities, neighborhoods, and families was considered a layperson-led weight loss intervention. Standard weight loss interventions  were defined as receiving recommendations for behavioral change without additional support provided to the participants at or before the baseline.Abstracts and titles of relevant citations were independently screened by 2 of 6 reviewers  to determine eligibility. Two reviewers independently assessed the eligibility of full-text articles of citations using a standardized prepiloted form outlining the inclusion and exclusion criteria. Disagreements were resolved by consensus or with the involvement of a third reviewer.Data were extracted independently by 2 of the 6 reviewers, with disagreements resolved by consensus or with the involvement of a third reviewer. For continuous data, we extracted change over time and the final reported measures of weight, as well as measures of variances, for each intervention type. We extracted outcome data from 2 time points—(1) immediately following the intervention and (2) at the end of the follow-up period—to assess long-term associations of the intervention. We used DistillerSR, version 2 (Evidence Partners Inc) to manage study selection, data extraction, and trial-level risk of bias assessments.z score and BMI percentile) at the end of the intervention period. As outcomes for BMI were not consistently reported, we used the J correction factor for an unbiased estimate of the standardized mean difference (SMD).21 We also assessed change in percent body fat, waist circumference, and overall study withdrawals as secondary outcomes. For RCTs that reported long-term follow-up data after the end of the intervention, we also examined changes in these outcomes to assess the sustainability of the interventions. The change in the outcome variables was calculated as the difference between baseline and immediate postintervention measurements to calculate the short-term weight loss. The difference from baseline to the last follow-up after the intervention was completed was used to calculate the sustainability of the intervention.The primary outcomes were the change from baseline in weight and any measure of BMI  and a categorization of the overall risk of bias. Each separate domain was judged as low, unsure, or high risk of bias. The overall assessment was based on the responses to individual domains. If 1 or more individual domains were assessed as having a high risk of bias, the trial was judged as having a high risk of bias. The overall risk of bias was considered low only if no domain was rated as having an either high or unclear risk of bias. The source of funding was also extracted.We evaluated the internal validity of included RCTs using the Cochrane risk of bias tool.23 We fit both random-effects and fixed-effect network meta-analysis models24 . We used a bayesian framework and Markov chain Monte Carlo simulation methods to combine direct and indirect evidence implemented in WinBUGS software, version 1.4.3 (University of Cambridge).Results are summarized by point estimates presented as medians with 95% CIs established using the 2.5 and 97.5 percentiles obtained via Markov chain Monte Carlo simulations. The 95% CI represents the interval in which the pooled effect is expected to lie with 95% probability. We also generated treatment rankings from best to worst and their corresponding probability estimates.115 (5780 participants) met the eligibility criteria , 2.3 , and 37.5% , respectively. Only 25% of the RCTs were judged to have a low risk of bias  of participants that completed the trials was similar in professional- and layperson-led interventions. On average, each intervention type consisted of 1 to 1.5 hours of contact time, delivered 1 to 3 times per week for approximately 24 weeks.Summary information for professional- and layperson-led interventions is provided in P < .001) and BMI  compared with standard care. Layperson-led weight loss interventions did not show an association with a difference in weight  or BMI  compared with standard care. No differences were observed in RCTs that directly compared professional-led to layperson-interventions .Data from each randomized trial on primary outcomes are presented in eFigure 2 in the P = .06) or layperson-led interventions  were associated with reduction in weight following discontinuation of the intervention, compared with standard care  and waist circumference  compared with standard care. No differences in percent body fat  or waist circumference  following layperson-led interventions were seen compared with standard care  regardless of intervention type.4 This review tested for differences in treatment outcomes across studies that had different contact time with participants, but did not directly compare different intervention models. Among the 42 behavioral therapeutic trials included in the analysis, several were not randomized, cluster trials were included, and no long-term follow-up data were provided. Despite these differences between the network meta-analysis presented here and the analysis from the US Preventive Services Task Force, the effect size for professional-led interventions was similar and comparable to previous systematic reviews of weight loss interventions among children and adolescents with overweight or obesity.117 The data presented here extend previous systematic reviews by demonstrating that the short-term benefits of professional-led weight loss interventions are not sustained following the end of the intervention and that similar effect sizes may be achieved with layperson-led interventions. Adherence to lifestyle change is a critical determinant of intervention effectiveness. Unfortunately, very few trials reported adherence to prescribed lifestyle change, and therefore it is unclear whether the lack of maintenance and the relatively modest weight loss following these interventions are associated with low adherence to intervention attributes. Collectively, these data provide robust evidence that professional-led behavioral interventions are associated with achieving modest weight loss among children and adolescents with overweight or obesity; however, this association was not sustained in the long term.In 2017, the US Preventive Services Task Force released the results from an extensive systematic review of trials examining weight loss interventions for children and adolescents with overweight or obesity.119 school-,118 and community-based120 behavioral interventions for obesity prevention in children and adolescents suggest that these nonprofessional-led interventions yield minimal or no weight change. In contrast to previous systematic reviews, we excluded cluster randomized trials, quasi-experimental trials, trials lasting less than 12 weeks, and trials that included children and adolescents of a healthy weight. For the current review, layperson-led interventions were delivered either by parents or older peers without formal training in a health profession. In contrast to results from quasiexperimental121 or cluster randomized trials,8 the RCTs of layperson-led interventions examined here did not show an association in the short or long term. The effect sizes for absolute (–1.60 vs –1.40 kg) and relative  weight loss were similar between layperson- and professional-led interventions. The few trials of layperson-led interventions, however, lacked precision. Larger trials of layperson-led interventions, particularly trials directly comparing layperson- and professional-led interventions, are needed to understand the association of this approach for weight management among children and adolescents with overweight or obesity.Systematic reviews of home-,125 or obesity-related comorbidities.126 Layperson- or community-led interventions have proved to be associated with low-resource areas or settings in which culturally tailored approaches are preferred by community members.13 The meta-analysis conducted here found that layperson-led behavioral trials were not associated with statistically significant reductions in body weight among children and adolescents living with obesity. Weight status is only 1 of multiple measures of health that can be influenced by behavioral change in children and adolescents with overweight or obesity, particularly cardiometabolic risk factors. Children and adolescents with overweight or obesity also are more likely to live in families with low income,128 to have been exposed to adverse childhood experiences, and to suffer from mental health comorbidities. We did not include these outcomes in our analysis; however, it is possible that these outcomes could be responsive to layperson-led interventions. The promising association of layperson-led approaches in other settings and populations130 reinforces the need for large-scale, well-designed, multiarm RCTs to determine the effectiveness of interventions led by lay individuals for supporting weight change among children and adolescents with overweight or obesity.There is some evidence that layperson- or peer-led approaches support positive behavioral change and improved health outcomes among adults living with obesityThe advantage of conducting a network meta-analysis, relative to a conventional meta-analysis, lies in the capacity to estimate the relative efficacy of 2 given interventions when few or no direct head-to-head trials exist. We were only able to identify 3 to 5 trials that directly compared layperson- to professional-led interventions for weight loss in children and adolescents with overweight or obesity. Performing a meta-analysis on the results of these RCTs did not reveal an association with either intervention. The few RCTs directly comparing layperson- and professional-led interventions were relatively low-powered and were considered to have a high risk of bias. Based on the limited available evidence, professional-led approaches were ranked as being associated with short-term weight loss; however, over the long-term, layperson- and professional-led interventions appeared to perform equally. Adequately powered, head-to-head trials of layperson- and professional-led approaches with long-term follow-up are needed to confirm these observations.z scores. Additionally, we only searched for trials appearing in the last 20 years in an effort to limit the number of low-quality RCTs. We did not include non-English publications or RCTs that were unpublished in order to increase feasibility and the homogeneity between weight loss interventions; this may have introduced selective reporting bias . Furthermore, only 25% of the included trials were judged as having a low risk of bias. As these were behavioral trials, blinding was not possible; however, as the outcomes are semiobjective, blinding may not be as effective as in pharmaceutical trials with subjective outcomes.131 As mentioned previously, this review was restricted to weight-related outcomes and did not include other outcomes that could be responsive to behavioral lifestyle change. Finally, there were very few trials that directly compared the effectiveness of layperson- and professional-led interventions. This limits our ability to provide an accurate estimate of the effects and also resulted in very low precision. Similarly, with so few layperson-led interventions with long-term follow-up, the precision was very low for the indirect comparisons generated by the network meta-analysis.The study is strengthened by limiting the analyses to trials focused exclusively on children and adolescents with overweight or obesity, an a priori published protocol, and the relatively large number of RCTs available for the network meta-analysis. Despite these strengths, there are limitations to consider. The strict criteria we imposed on the search limited the inclusion of designs, including cluster RCTs and quasi experiments, which limits the generalizability of our findings. Additionally, differences in intervention designs could have influenced the point estimates between professional- and layperson-led trials; however, with only 5 trials led by laypersons, we were underpowered to adjust for these differences. We also recognize that age- and sex-standardized measures of adiposity are the best practice for reporting weight-related outcomes in children. As we relied on published outcome data and not individual-level data, we were largely unable to use BMI or waist circumference In this systematic review and meta-analysis, professional-led behavioral interventions were associated with modest but statistically significant weight loss among children and adolescents with overweight or obesity, compared with standard weight loss interventions. Layperson-led behavioral interventions were no associated with weight loss. Weight loss achieved by both professional and layperson-led interventions were not sustained following the intervention among children and adolescents with overweight or obesity. These findings suggest a need for trials assessing the immediate and sustained effectiveness of layperson-led behavioral weight loss interventions among children and adolescents with overweight or obesity.


We studied the effect of visible absorber dyes on the photo-actuation performances of liquid crystalline elastomer (LCE) materials under quasi-daylight irradiation. The dye-doped LCE materials were prepared through infiltrating visible absorber dyes into a polysiloxane-based LCE matrix based on its solvent-swollen characteristic. They demonstrated well absorption properties in visible spectrum range and performed strong actuation upon the irradiation from quasi-daylight source, thus indicating that the presence of visible absorber dyes effectively sensitized the LCE materials to light irradiation since the light energy was absorbed by the dyes and then converted into heat to trigger the phase change of LCE matrix. The photo-actuation properties of dye-doped LCE materials with different visible absorber dyes, varied dye contents, and irradiation intensities were investigated. It was shown that the visible absorber dyes with different absorption bands created different photo-actuation performances of LCE materials, the one whose absorption band is near the intensity peak position of quasi-daylight spectrum created the optimum photo-actuation performance. The result disclosed a valuable light utilization way for photo-controlled LCE materials since it revealed that a light-absorbing dye, whose absorption band is in the high intensity region of light spectrum, is capable of effectively utilizing light energy to drive the actuation of LCE materials. Liquid crystal elastomer (LCE) materials, as a type of fascinating smart polymer deformable materials, have been intensively studied since Finkelmann and co-workers reported in the 1990s ,3,4,5. TThermal stimuli and light stimuli play major roles in various applications of LCE materials ,11. HoweQuasi-daylight sources, with the emission spectra being similar to nature sun-light, generally possess the advantages of economy, convenience, and health, thus it would be more valuable to effectively utilize the energy of quasi-daylight, even the nature sunlight, to drive the actuation of LCE materials. In this work, three visible absorber dyes, whose absorption bands are in the range of visible spectrum, were used to develop the dye-doped LCE materials which can be actuated by quasi-daylight, and a classical polysiloxane side-chain LCE matrix was used as the host material.The dye-doped LCE materials are conventionally prepared via in situ synthesis by adding the dye molecules into synthetic reaction mixtures ,54,55,57bis(undec-10-enyloxy)benzene (11UB), and the catalyst solution was dichloro  platinum (II) (Pt (COD) Cl2) dissolved in toluene. They were synthesized or prepared by following our previous work [The mesogenic monomer was 4-methoxyphenyl 4-(3-butenyloxy)benzoate (MBB), the cross-linker was 1,4-ous work . The pol2 catalyst solution dissolved in 0.8 mL of toluene was filled into a Teflon mold, and then underwent the first step crosslinking in an oven at 65 °C for 40 min to generate a partially crosslinked toluene-swollen gel elastomer. After the contained toluene was fully evaporated through drying in air, the de-swollen elastomer was immersed in 0.2, 0.4, or 1.2 mL of dye-dissolved toluene solution with a visible absorber dye concentration of 7.5 mg/mL for about 10 min, 20 min, or 40 min. When the dye-dissolved toluene solution was fully absorbed by the elastomer, the re-swollen elastomer was dried again for 40 min to let the contained toluene fully evaporate while the dye molecules retained. The dye containing elastomer was uniaxially stretched under a load to attain a stable length and form a monodomain structure, and then heated in the oven at 70 °C overnight to complete the second step of crosslinking and stabilize the monodomain structure. Through the above procedures, dye-doped nematic LCE materials with dimensions of 40 mm × 10 mm × 0.6 mm, and mass content of visible absorber dye of 0.1 wt %, 0.3 wt %, or 0.6 wt % according to composition ratios, were respectively prepared. A blank nematic LCE material without dye doping was prepared by the same way but without the procedure of infiltrating dye molecules into the LCE matrix. Herein, the first crosslinking step was critical and the reaction condition for it was optimal in our work. The preparation of LCEs would fail if the reaction time of the first crosslinking step is too short or too long, as expatiated in the The synthesis of the side-chain LCE network with polysiloxane backbone was via a sol–gel process of hydrosilation reaction which achieved an effective 15% cross-linking density, as shown in The mesomorphic and phase transformation properties of LCE materials were investigated by polarizing optical microscopy  observations and differential scanning calorimetry  measurements, respectively. The UV-vis-NIR absorption spectra of the dyes and dye-doped LCE materials were measured by using a USB4000 spectrometer. The photo-responsive actuations of LCE materials were performed by using a quasi-daylight source . As shown in Suitable optical wavelength absorptions of dyes are needed in order to achieve the photo-actuation of dye-doped LCE materials. Though our dye-doped LCE materials might be fabricated via in situ synthesis since the used visible absorber dyes are toluene soluble and thus can be dissolved in the reaction solution. It should be noted that the dye molecules may influence the synthesis reaction, leading to poor material quality, if directly mixing them into the reaction mixture solution, while the phase separation may be induced by the synthesis reactions in solutions, resulting in the tiny aggregations of dye molecules . In our Tg) were in the range of about 3–8 °C, and their nematic-isotropic transition temperatures (Tni) were in the range of about 70–73 °C. The LCE materials all had an enantiotropic nematic phase. Because of the very low doping ratios (0.6 wt %) of the dyes, the Tni of the dye-doped LCE materials were close to that of the blank LCE with a small deviation of below 3 °C.The DSC measurement data curves of the prepared blank LCE and three kinds of dye-doped LCE material are shown in Tni were about one third of their original lengths. The mechanism lied in the temperature dependence of mesogenic order. When monodomain LCE materials are heated above the Tni, the mesogenic orders decrease and the LCE networks become isotropic state, hence the LCE materials show contraction along the alignment directions. When the temperature decrease below the Tni, the mesogenic orders increase and the LCE networks revert to nematic structure, hence the LCE materials experience an expending process and restore to the initial dimensions [The prepared blank LCE material and dye-doped LCE materials demonstrated inherent thermo-actuation performance. As shown in mensions .5 lux in intensity. When being exposed to the incident light irradiation, the dye-doped LCE materials behaved axial contraction and the finally reached maximum contraction amplitudes were about one-third of their original lengths, which were consistent with those under heating. After the light source was switched off, the dye-doped LCE materials restored to their initial lengths, indicating completely reversible photo-actuation. The blank LCE demonstrated almost no discernible response under the same light irradiation. The visible absorber dyes dispersed in LCE matrix could absorb the energy of quasi-daylight irradiation since their main absorption bands are in the region of quasi-daylight spectrum. The absorbed light energy was converted into thermal energy through molecular vibrations, and then the generated thermal energy heated the LCE matrix, resulted in the axial contraction behavior of the dye-doped LCE materials. When the LCE matrix was heated up to the nematic-isotropic phase transition, the axial contraction reached the maximum amplitude. The blank LCE had almost no light absorption ability, and thus was weakly responsive to light irradiation. The photothermal conversion effect of the visible absorber dyes was confirmed by comparing the temperature changes of the dye-dissolved toluene solutions and the pure toluene solvent upon light irradiation, as expatiated in the The dye-doped LCE materials also demonstrated attractive photo-driven deformation behaviors. Tni, as shown in The axial strains of the blank LCE and three kinds of dye-doped LCE material with different dye contents, under different irradiation intensities, versus irradiation time, measured by the universal material mechanical analyzer shown in 5 lux intensity drove very small contraction strains, the final contraction strains of the dye-doped LCE materials were below 6.5% and had no clear difference each other. The achieved highest temperatures in dye-doped LCE materials were below 45 °C and also had no clear difference each other, as shown in 5 lux, the contraction strains of the three kinds of dye-doped LCE material with different dye contents increased but obviously did not reach the maximum contraction ratio since their final contraction strains were below 20%. Tni. But among the three kinds of dye-doped LCE material, the strain rate, final contraction strain, and temperature rise under irradiation of the 594/LCE were all higher than those of the 407/LCE and 694/LCE with the same dye content, as shown in 5 lux of irradiation intensity, the dye-doped LCE materials still could not reach the maximum contraction ratio, and the highest achieved temperatures under irradiation were still below their Tni, although their final contraction strains and temperature rises under irradiation were larger than the states under 4.0 × 105 lux of irradiation intensity because of the increase of light energy absorption, as shown in 5 lux, the final contraction strains and temperature rises of the three kinds of dye-doped LCE materials under irradiation began to gradually approach each other as the dye contents increased from 0.1 wt % to 0.6 wt %, but the 594/LCE still demonstrated an obvious higher strain rate compared to 407/LCE and 694/LCE at every dye content because of the increased absorption of quasi-daylight energy.At 4.5 × 105 lux, the final contraction strains of the three kinds of dye-doped LCE materials began approaching each other at every same dye content, and were all above 30%, as shown in Tni, as shown in When irradiation intensity was increased to 5.0 × 10In addition, our experiments exhibited that the strain rates of the dye-doped LCE materials in response to light irradiation slightly decreased as the material thickness increased. The reason is that a larger material thickness could cause a higher resistance of thermal diffusion, resulted in a slower photo-actuation rate of LCE material, as expatiated in the 5 lux of intensity are plotted in The axial strains of 407/LCE, 594/LCE and 694/LCE with 0.6 wt % of dye content versus irradiation time under repeated on-off switch of quasi-daylight irradiation with 5.0 × 10The utility of photo responsive dyes in LCE matrices involves a novel route of light energy absorption and energy conversion molecular units which provide effective photo-actuation performance to the LCE materials, while incorporating the visible absorber dyes into LCE matrices is an effective way to realize the actuation of LCE materials by using quasi-daylight. By utilizing the toluene-swollen characteristic of polysiloxane-based LCE materials, we prepared three kinds of dye-doped LCE materials through infiltrating the toluene solution of visible absorber dyes into the polysiloxane LCE matrix. The prepared dye-doped LCE materials exhibited basically coincident photo absorption properties as the corresponding pure visible absorber dyes, and demonstrated prominent photo-thermo-mechanical actuation performances. The maximum axial contractions of these dye-doped LCE materials under the irradiation of quasi-daylight were consistent with the situation of thermo-actuation, and the reversible axial contraction/extension deformations could be steadily performed upon the repeated on-off switch of light irradiation. The photo-actuation properties of every kind of dye-doped LCE material depended on the light intensity and dye content. However, the 594/LCE demonstrated better photo-actuation properties compared to the 407/LCE and 694/LCE at every light intensity and dye content. The reason is that the absorption band of the dye molecules contained in 594/LCE is in the high intensity region of quasi-daylight spectrum, thus they can more effectively utilize the energy of quasi-daylight to drive the actuation of LCE material. The research results revealed that the addition of specially selected dyes to LCE materials could offer a useful alternative route for the programming of photo-controlled smart actuators based on LCE materials, and would be of great guiding significance for the effective energy utilization of wide spectrum lights, including the nature sunlight, in the actuation of photo-controlled LCE materials. Many of the cyanine dyes and coordination complex dyes have good light absorption properties and are suitable for the development of dye-doped LCE materials; their properties can be improved or tuned through by constituting coordinative bonds, incorporating functional chromophores or substitute groups, changing the coordinative metal, or constituting different conjugation effect ,62,63, e


The objective of this randomized controlled trial was to test whether a commercially available, mindfulness meditation mobile app, , was effective in reducing fatigue (primary outcome), pre-sleep arousal, and daytime sleepiness (secondary outcomes) in adults with sleep disturbance (Insomnia Severity Index Score >10) as compared to a wait-list control group. Associations between the use of the Calm app  and changes in sleep quality was also explored in the intervention group only. Adults with sleep disturbance were recruited (N = 640). Eligible and consenting participants (N = 263) were randomly assigned to the intervention (n = 124) or a wait-list control (n = 139) group. Intervention participants were asked to meditate using the Calm app ≥10 minutes/day for eight weeks. Fatigue, daytime sleepiness, and pre-sleep arousal were assessed at baseline, mid- (4-weeks) and post-intervention (8-weeks) in both groups, whereas sleep quality was evaluated only in the intervention group. Findings from intent-to-treat analyses suggest the use of the Calm app for eight weeks significantly decreased daytime fatigue (p = .018) as well as daytime sleepiness (p = .003) and cognitive (p = .005) and somatic (p < .001) pre-sleep arousal as compared to the wait-list control group. Within the intervention group, use of the Calm app was associated with improvements in sleep quality (p < .001). This randomized controlled trial demonstrates that the Calm app can be used to treat fatigue, daytime sleepiness, and pre-sleep arousal in adults with sleep disturbance. Given that the Calm app is affordable and widely accessible, these data have implications for community level dissemination of a mobile app to improve sleep-related symptoms associated with sleep disturbance.Trial registration: ClinicalTrials.gov NCT04045275. More than 50–70 million adults suffer from a sleep disorder , 2 and 6Effective treatment for sleep disturbance (depending on type and severity) include both pharmacological and non-pharmacological strategies, although cognitive behavioral therapy for insomnia (CBT-I) is recommended as the first line of treatment for insomnia by the American Academy of Sleep Medicine , 12. Ind,Mindfulness meditation, a less intensive intervention, has been found to be effective in the treatment of sleep disturbance, with evidence of non-inferiority to the gold standard in CBT-I , 15. SucAs the field of medicine moves towards digital health, especially in the midst of the COVID-19 pandemic, research on the delivery and efficacy of health-supportive interventions via mobile technologies such as smartphones  is needed. Approximately 81% of US adults own a smartphone and the high utilization of this technology has opened up many opportunities for delivering mindfulness meditation via mobile apps , yet theN = 11,870) typically downloaded the app to improve sleep (62.9%) and adults with sleep difficulties were more likely to notice changes in their sleep after using the app compared to those without sleep difficulties , or a wait-list control. Those randomized to the wait-list control group received the intervention after eight weeks.d = 0.50). Results indicated that analyses with 211 participants across three time points would yield .80 power. To account for attrition, we aimed to enroll up to 250 participants.Based on previous studies assessing the effects of in-person meditation interventions on fatigue , 31, powParticipants were recruited nationally between June and September of 2019 via Internet-based strategies including social media , social networking sites, email listservs and ResearchMatch.org. The study was advertised as a “mobile app intervention for physical and mental health”. Interested participants completed a brief (5–10 minutes) eligibility screener via Qualtrics . Inclusion criteria were: 1) sleep disturbance defined by a moderate level of elevated insomnia symptoms  . Becauserandom.org). Group randomization was unstratified. The research coordinator and participants were unblinded to group allocation.To conceal allocation, randomization occurred after enrollment, sequentially, on a one-by-one basis. After a participant completed baseline assessments, the research coordinator allocated that participant to a group using an online randomization tool  grounded in mindfulness-based stress reduction (MBSR) and Vipassana meditation and Sleep Stories, grounded in sensory immersion and present moment awareness. Participants were asked to download the Calm app on their phone from the Apple App Store or Google Play. Participants then received a study email address and password allowing them to access their Calm account . Participants were asked to meditate for at least 10 minutes each day and encouraged to use Calm as much as they would like during the intervention. We chose 10 minutes/day for the intervention because previous research using the Calm app suggests 10 min/day may lead to improvements in mental health . The resParticipants randomized to the wait-list control group were asked to wait eight weeks from the time they signed consent and asked not to change their normal routine or download or use any apps for relaxation, meditation or sleep. After eight weeks, participants received a Qualtrics link to the post-intervention surveys (same surveys that the intervention group received) and received free access to Calm (in the same way as the intervention group) for eight weeks upon completion of the surveys.Information about demographic characteristics, self-reported chronic health and sleep diagnoses was collected at baseline. During the study period, both the intervention and wait-list control groups were administered three self-reported surveys at baseline, mid- (four weeks) and post-intervention (eight weeks) that assessed fatigue, daytime sleepiness, and pre-sleep arousal. Among intervention participants only, sleep quality was assessed via daily sleep diaries throughout the study period. All data was collected using Qualtrics (online survey software).The Fatigue Severity Scale (FSS) was used to measure fatigue , 36. TheThe Epworth Sleepiness Scale (ESS) was used to measure daytime sleepiness . The ESSThe Pre-Sleep Arousal Scale (PSAS) was used to measure pre-sleep arousal . The PSASleep quality was measured using the gold-standard Core Consensus Daily Sleep Diary questionnaire in the intervention group only . The CorAdherence to the intervention was tracked using data provided by Calm and confidentially shared with Arizona State University. Reports included the corresponding date and time, title, and duration of engagement for each participant.Due to limited funding, monetary compensation was provided for the first 100 enrolled participants. Incentives were provided based on the percentage of sleep diaries completed: $25 for 70%, $15 for 50%, and $10 for 25%. Participants enrolled after the first 100 who completed at least 70% of their sleep diaries were entered into a drawing to win one of two $99 Amazon gift cards. All participants were provided with an additional free eight-week membership to Calm at the end of the intervention.SDs) were analyzed in IBM SPSS 26.0 to and used to characterize the sample.Descriptive statistics  analyses . Effect ectively . A p-vald was calculated by dividing the unstandardized regression coefficient by the standard deviation of the outcome variable ), followed by in the afternoon/evening  and morning . General meditations  were the most commonly used component of the app , followed by Sleep Stories , and sleep-specific meditations .The most common time of day to use Calm was at night , ≥10 mins/day for eight weeks on fatigue in those with elevated insomnia symptoms as compared to a wait-list control group. We also assessed the effects of using Calm on daytime sleepiness and pre-sleep arousal and explored associations between using Calm and changes in sleep quality. Results suggest that using Calm led to decreases in fatigue, daytime sleepiness, and cognitive and somatic pre-sleep arousal. Using Calm was also associated with improvements in sleep quality.Our findings suggest that those in the intervention group had significantly larger reductions in fatigue as compared to the wait-list control group. Specifically, average decreases in intervention participants’ FSS scores decreased by an average of 0.3 points (0.2 points larger than the decreases observed in wait-list control participants). Previous studies reported minimally important differences (MIDs) for improvement in fatigue to be between 0.08 and 0.4 points , which sThe findings in this study also show promise in reducing daytime sleepiness, and cognitive and somatic pre-sleep arousal in the intervention group at eight weeks when compared to the wait-list control group. Similar to the literature in fatigue, there is limited data related to mindfulness meditation mobile app use and its effects on daytime sleepiness or pre-sleep arousal in adults with elevated insomnia symptoms. A review by Garland and colleagues (2016) suggests that mindfulness-based interventions may be particularly useful for cognitive deactivation and physiological de-arousal by allowing the individual to disengage from daily concerns and strivings and ultimately encouraging a shifting of ones relationship to sleep-related thoughts  , 55. OurIntervention group participants reported significant improvements in perceived sleep quality over the course of the intervention period and more time spent using the mindfulness meditation app was associated with greater improvements in sleep quality. These findings are similar to mobile apps delivering CBT-I (no meditation component) suggesting that mindfulness meditation apps could be a less intensive and more affordable alternative, or adjunctive treatment, to gold standard treatment for those with sleep problems , 57. ForParticipants reported robust usage of the mindfulness meditation mobile app with an average of approximately 14.98 minutes/day (6.36 sessions/week). Interestingly, participants spent substantially more time using the general meditations  than other sleep-specific components of the app . However, the most common time for to meditate was at night (made up 52% of sessions). Despite participants using general meditations rather than sleep-specific meditations, changes in sleep outcomes were observed. It would be interesting to test if the magnitude of changes would be greater (or the same) if participants were instructed to use sleep-specific meditations prior to bed. Perhaps the effects of meditation are driven via other processes  rather than directly impacting sleep. Future studies testing different types of meditation  on sleep outcomes as well as mechanisms of change is warranted.SD 80) for an average of 24 days (SD 36) with a 26% attrition rate. Their attrition was higher than our study and because objective adherence was reported as number of logins and days of use, it is unclear how many minutes of meditation were actually completed. A review by Donker and colleagues (2013) suggests that app-based health interventions may have high adherence rates because of the portability and flexible usage of mobile apps [The attrition rate in our study was 44/263 (17%), which is similar to or lower than what has been reported in other studies of app-based meditation interventions . Howeverile apps . Even soAlthough the findings in this study are promising, there are important limitations noted. The sample recruited was primarily female and highly educated, and therefore limits the generalizability of our findings. Although there were fairly high rates of sleep-related diagnoses in our sample (40%), it is also unclear whether the effects of the intervention would be similar in clinical settings or in populations with very severe sleep disturbance. In addition, we collected data online which may result in unrepresentative samples. Due to the remote nature of the study, we cannot determine whether or not participations were actually meditating or actively engaging with the Calm app . This is common in digitally delivered interventions . FatigueThis is one of the first randomized controlled trials to examine the effects of a mindfulness meditation mobile app  on sleep outcomes in adults reporting elevated insomnia symptoms. Calm is an easily accessible and cost-effective resource that may be used to help sleep disturbed adults improve their sleep. Mobile apps provide a convenient way to improve sleep disturbance as users have access 24/7 in the comfort and privacy of their homes or even on the go as they please. As Calm is widely accessible, disseminating this resource as a tool for sleep can be done easily and effectively and has the potential for large reach as it is available globally.S1 FileCONSORT 2010 checklist of information for reporting randomized trials.(DOC)Click here for additional data file.S2 FileArizona State University Institutional Review Board approved study protocol.(DOCX)Click here for additional data file.


Exosomes are a group of nano-sized membrane vesicles and are important mediators of intercellular communication, particularly in tumor microenvironment. Recently, researchers have found that circular RNAs (circRNAs), with the great research significance, are enriched and stable in exosomes. In this review, we summarize the research significance of exosomal circRNAs, sorting mechanisms and their functioning mechanisms in tumor progression. Their clinical applications as clinical tumor biomarkers and as therapeutic targets in inhibiting tumor metastasis, anti-cancer immunity response and drug resistance have been widely discussed. Exosomes were first identified in sheep reticulocytes by Following microRNA (miRNA) and long non-coding RNA (LncRNA), circRNA is an emerging endogenous non-coding RNA (ncRNA) with excellent research potential, which is a new member of the non-coding RNA family. In 1976, Sanger  first foIntriguingly, recent research found that the expression of circRNAs in exosomes is abundant and stable. Exosomal circRNAs can play a significant role after being absorbed by recipient cells . In thisCircRNAs are generated from specific alternative splicing of pre-mRNA  and can Compared to other ncRNAs, circRNAs have some unique characteristics. Firstly, there are thousands of tissue-specific circRNAs in eukaryotes . For exaExosomes are a class of extracellular vesicles (EVs), which are membrane vesicles secreted by various types of cells. Compared with other EVs, exosomes have unique ways of formation and release: the cytoplasmic membrane is enmeshed, and some extracellular components and cell membrane proteins are wrapped together to form early-sorting endosome (ESEs), which can exchange materials with other organelles, or fuse with different ESEs to form late-sorting endosomes (LSEs), and then further form into intracellular multivesicular bodies (MVBs), which contain many intraluminal vesicles (ILVs) . During Exosomes contain many RNAs, including mRNA and ncRNAs . SurprisAlthough RNA loading is a random event, different levels of RNA enrichment in EVs suggest that RNA sorting is regulated by specific mechanisms . The mecAlthough there is no clear evidence to show how lncRNAs target EV-producing sites, they may share the similar sorting mechanisms with mRNAs . MRNAs hConsidering that the types of circRNAs in exosomes are different from those in multiple cell lines, the mechanism of circRNAs entering exosomes deserves further investigation. Research showed that the abundance of exo-circRNAs was only moderately correlated with the level of cellular circRNAs, indicating that the sorting of specific circRNA species to exosomes may be actively regulated . Since cBesides, other possible mechanisms include RNA-associated proteins binding to circRNAs . NonetheMiRNAs are a class of endogenous, regulatory ncRNAs with a length of about 20–25 nucleotides in eukaryotes . MiRNAs Studies have shown that exosome-derived circRNAs can still function as miRNA sponges after targeting recipient cells. The transfer of exosomes makes the circRNAs in exosomes more flexible and generally regulate the downstream target genes. There may be two situations: (1) CircRNAs bind with miRNAs and enter the exosomes together. Upon arrival at the recipient cells, miRNAs detach from circRNAs and then bind to the mRNAs in the recipient cell, thereby silencing mRNAs . (2) AnoThe latter has found the following main types so far:1.Target at adipocytes:  found th2.Target at normal liver cells (L-02):  found th3.Target at human microvascular vein endothelial cells (HUVECs): HUVECs is a barrier that controls the exchange of substances between surrounding tissues and blood, it is an important factor to prevent the invasion of pancreatic carcinoma (PADC) cells. Studies have shown that exosomes from PADC cells transferred circ-IARS to HUVECs, which could absorb miR-122 and remove its inhibition on target gene RhoA and activate the RhoA signaling pathway, thus increasing endothelial monolayer permeability and promoting invasion and metastasis of PADC .4.Target at tumor cells: in addition to affecting normal cells in the body, exosomal circRNAs secreted by tumor cells also can perform effects on the same type of cancer cells with low metastatic ability. For example, Recent researches have shown that normal cells can also transmit circRNAs to tumor cells through exosomes and function as sponges, thus promoting or inhibiting tumor progression in recipient cells. For example, Exosomes can gather circRNAs within vesicles with an abundance of 2–4 times greater than that in the cytoplasm and transport them to target cells . With thRNA binding proteins are a group of proteins that bind to RNA and regulate the metabolism. More than 800 RBPs have been identified in the human genome . They caExosomal circRNAs can also interact with binding proteins in recipient cells to perform biological functions. + cells isolated from colorectal cancer (CRC) cells could notably enhance cell stemness, sphere formation and metastasis. Next, they further cocultured CD133– cells with exosomes from CD133+ cells, the results revealed that cell stemness, sphere formation and metastasis were remarkably enhanced. To further explore the mechanism of exosomes from CD133+ cells in CRC, researchers screened out the upregulated circ-ABCC1 which is a CRC-related circRNAs in exosomes by means of circRNADisease database. Naturally, researchers speculated that exosomes from CD133+ cells carrying circ-ABCC1 mediate cell stemness and metastasis in CRC. Subsequently, researchers inferred and preliminarily confirmed that circ-ABCC1 could bind with β−catenin into the cell nucleus and activate the Wnt pathway to regulate CRC progression via RNA Immunoprecipitation (RIP) and RNA pull-down experiments , and the EVs of CCA cells carried circ-CCAC1 into vascular endothelial cells selectively. CCLP1 cell-derived EVs could significantly down-regulate the expression of ZO-1/Occludin in HUVEC, while the EVs overexpressed circ-CCAC1 in CCLP1 could significantly down-regulate the expression of Zo-1/Occludin. The RPISeq database predicted proteins that might interact with circ-CCAC1, and the prediction results indicated that EZH2, DNMT1, and STAU1 might interact with circ-CCAC1. The RIP and RNA pull-down experiments showed that there was an obvious interaction between EZH2 and circ-CCAC1. The interaction was also verified by fluorescence in situ hybridization (FISH) co-location analysis. Increased circ-CCAC1 in EVs significantly increased the cytoplasmic localization of EZH2, while decreased the expression of ZO-1 and Occludin. SH3GL2 is a negative regulator of ZO-1/Occludin, and circ-CCAC1 in the extracellular vesicle may increase the expression of SH3GL2. Chromatin Immunoprecipitation (ChIP) experiments showed that EZH2 could directly bind to the promoter of SH3GL2 and promote its H3K27me3 methylation level, and thus inhibit the expression of SH3GL2. Circ-CCAC1 derived from EVs could reduce the efficiency of EZH2 binding to the SH3GL2 promoter, thereby promoting the expression of SH3GL2, which further inhibited the expression of ZO-1/Occludin .Therefore, we concluded that exosomal circRNAs also interact with binding proteins in recipient cells to exert biological functions. As is mentioned above, with the assistance of binding proteins, circRNAs enter the exosomes and are transported by exosomes to the recipient cells. Another situation is that circRNAs are transported by exosomes and released into the recipient cells, thus binding to the RBPs of the recipient cell to perform functions. More experiments are needed to verify these hypotheses. The potential interactions between exosomal circRNAs and RBPs are shown in Since circRNAs do not contain 5′caps, the translation is hat-independent. Some circRNAs have an internal ribosomal entry site (IRES)  or transIn glioblastoma, circ-SHPRH can be translated into SHPRH-146aa, which is involved in the occurrence and development of tumors of the central nervous system by regulating the protein ubiquitination pathway. Overexpression of SHPRH-146aa in U251 and U373 glioblastoma cells can reduce the degree of malignancy and tumorigenicity both in vivo and vitro . SHPRH-1Although it has not been reported that exosomal circRNAs could encode protein or peptides to regulate the development of tumors, theoretically, circRNAs may take advantage of exosomal targeting merit to transfer encoded protein to cells in need. They may also transmit circRNAs to recipient cells to encode the corresponding proteins to regulate the activity of life. The model will be more “humane” to satisfy the need of our body, which need more experimental verification. The potential of exosomal circRNAs encoding proteins or peptides is shown in Since the early symptoms of most tumors are not obvious and lack of specific early diagnosis biomarkers, patients tend to miss the optimal opportunity for treatment before being diagnosed ultimately. Therefore, it is an urgent clinical challenge to find accurate therapeutic targets. At present, some ncRNAs (miRNA and lncRNA) have shown to have the potential to be tumor biomarkers . CircRNAThe area under the curve (AUC) of exosomal circRNA-002178 is 0.9956. These results confirmed that serum exosomal circRNA-002178 could be a novel diagnostic biomarker for LUAD. with the AUC of 0.88, which suggested that circulating has_circ_0004771 may serve as an early diagnostic biomarker of CRC . More importantly, the exo-FECR1 level in patients with extensive stage SCLC was significantly higher than that in limited stage patients with SCLC, indicating that exo-FECR1 is closely associated with tumor stage . In addiIn Bladder urethral epithelial carcinoma (UCB), circPRMT5 is overexpressed and its high expression is positively correlated with low survival in patients. Mechanism studies have found that circPRMT5 can promote Epithelial-Mesenchymal Transition (EMT) progression by binding to miR-30c. CircPRMT5 was found to be up-regulated in the serum and urine exosomes of patients and was closely related to tumor progression. It is expected to be a prognostic marker for UCB patients to evaluate its postoperative efficacy . AdditioCompared with the normal control group, the expression of CircNFIX in the exosomes of the patients’ serum samples was significantly increased, and the expression of CircNFIX in serum exosomes of patients with tumor recurrence was higher than that of the serum of patients with primary tumor. According to ROC curve analysis, it could be concluded that the exosomal CircNFIX has the potential to be used as a biomarker to detect the postoperative recovery for glioma patients .The exosomal circRNAs can be released into the body fluid, making sampling more convenient and having the potential to become tumor biomarkers. If the detection of exosomal circRNAs based on blood or urine can be effectively applied to the clinic, the tumor patients can be diagnosed as early as possible and avoid the pain of invasive exploration.Exosomes play an important role in the cellular microenvironment. Cells can convey information to nearby or distant cells through packing molecules into exosomes and other EVs. Studies showed that tumor-derived exosomes could enhance cell metastasis by conveying some circRNAs with specific functions.There is growing evidence that communication between cancer cells and the surrounding stroma contributes to metastasis. CircSHKBP1 was highly expressed in tumor and serum exosomes of GC patients, and overexpression of circSHKBP1 promoted the growth and metastasis of GC cells, while knockdown circSHKBP1 inhibited the development of GC cells. More importantly, with the ectopic expression of circSHKBP1 in GC cells, more circSHKBP1 was loaded into exosomes, thus interfering with the biological functions of nearby or distant GC cells .Further research shows that circ-PDE8A promotes tumor cell growth by upregulation of MET, a tyrosine kinase receptor that is one of the key oncogenes in a subset of epithelial tumors including PDAC. In addition, circ-PDE8A excreted by the tumor can be released into the blood circulation through exosome transport, acting as ceRNA of miR-338, regulating MACC1 and promoting invasive metastasis through MACC/MET/ERK or AKT pathways . UltimatA novel circRNA, circPACRGL, is derived from CRC exosomes. CircPACRGL was significantly upregulated after the addition of tumor-derived exosomes to CRC cells. In addition, circPACRGL acts as a sponge for miR-142-3p/miR-506-3p to promote the expression of transforming growth factor-1 (TGF-1). TGF-β is a multifunctional cytokine implicated in tumor initiation, progression, and metastasis. CircPACRGL promoted the proliferation, migration and invasion of CRC cells and the differentiation of Neutrophils from N1 to N2 via miR-142-3p/miR-506-3P-TGF-1 axis. This study is the first to reveal the role of cancer-derived exosomal circPACRGL in CRC proliferation and metastasis .Another study screened a set of differentially expressed circRNAs from plasma exosomes from CRC patients and normal subjects. Circ-133 is associated with hypoxia and regulates the distribution of E-cadherin protein on the cell membrane. Then, it was verified in vivo and vitro that exosomal circ-133 from hypoxic cells were transported to normal oxygenated cells through miR-133a/GEF-H1/RhoA axis, which regulated the distribution of E-cadherin membrane and promoted tumor metastasis .In addition to be biomarkers, exosomal circRNAs have potential to be therapeutic targets. For example, using the targeting properties of exosomes, we can encapsulate drugs or siRNA in exosomes, which are absorbed by specific cells and bind to the corresponding circRNAs in the cells, thus alleviating the metastasis and proliferation of tumors .Extracellular vesicles-RNAs mediate cross-talk between cancer cells and immune cells and within immune cells to regulate malignant behavior of cancer cells. Tumor-derived EV-RNAs can contribute to the immunosuppression, reduce the anti-tumor activity of various immune cells .For example, T cell immunoglobulin and mucin domain 3 (TIM-3) is a kind of immunomodulatory receptor which can bind with ligands on tumor cells in the microenvironment to inhibit antitumor immunity in a variety of cancers, including HCC. TIM-3 is one of the main inhibitory receptors on the natural killer (NK) cells. The expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. CircUHRF1 in the plasma of HCC patients was mainly delivered in the form of HCC cell exosomes. It upregulated the expression of its target gene TIM-3 by degrading miR-449c-5p, thereby inhibiting NK cell function, promoting immune escape of HCC, and driving resistance to PD1 immunotherapy. Overexpressing circUHRF1 can improve the resistance of HCC to PD1 therapy. Therefore, targeting circUHRF1 may be an effective method to restore the sensitivity of HCC to PD1 therapy .Chemotherapy and radiotherapy are still the most commonly used methods in the clinical treatment of many tumors, but these methods are prone to drug resistance and the therapeutic effect is not satisfactory. Currently, exosomes have been proved to be able to transfer nucleic acids and multi-drug resistance (MDR)-related proteins from resistant cells to target cells in order to expand the drug-resistance of cancer cells. For example, exosomes secreted by cancer-related fibroblasts can transport lncRNA H19 to CRC cells and enhance Oxaliplatin (OXA) resistance of cells . It has There is growing evidence that some epithelial cancers, such as CRC, are preferentially resistant to many advanced drugs. KRAS is an intracellular signaling molecule, and about 40% of colorectal tumors have KRAS mutations, so identifying the key signaling pathways affected by KRAS mutations help us understand how to conduct drug-targeted therapy . InitialIn vitro and vivo studies have shown that exosomes from drug-resistant cells can deliver ciRS-122 to drug-sensitive cells and enhance cell glycolysis and drug resistance by reducing miR-122 and upregulating Human pyruvate kinase M2 (PKM2). In vitro experiments, exo-si-ciRS-122 could reverse oxaliplatin resistance by inhibiting the ciRS-122/miR-122/PKM2 pathway, and injection of exo-si-ciRS-122 in nude mice could also reverse oxaliplatin resistance. It can be seen that exosomal circRNAs are expected to be therapeutic targets for inhibiting tumor drug resistance.Recently, new studies have found that some circRNAs with abnormal expression in drug-resistant cell lines can be transferred to drug-sensitive cell lines through exosomes, which promotes the development of drug resistance in tumors to some extent. Through exosome uptake and co-culture experiments, Previous studies have reported that tumor-derived exosomes regulate progression and drug resistance of glioma by transmitting lncRNAs or miRNAs to recipient cells . Ding etAn overview of roles of exosomal circRNAs in tumors is shown in Exosomes are a kind of EVs which contain numerous different varieties of bioactive cargoes secreted from cells. Notably, circRNAs are abundant and stable in exosomes, particularly in tumor-derived exosomes, compared with parent cells. One hypothesis suggested that the mechanism of circRNAs entering exosomes is partially related to miRNAs levels in parent cells. Another speculated that RBPs may function in circRNAs incorporating into exosomes. It has been reported that RBPs can promote miRNAs into exosomes, so it is reasonable to speculate that the related RBPs can also promote circRNAs into exosomes, or even that RBPs with different functions can load different types of circRNAs into exosomes.Upon entering the recipient cells, most exosomal circRNAs function as miRNA sponges to regulate tumor progression. However, the regulatory mechanisms of exosomal circRNAs in tumors are still in the preliminary stage of exploration. Besides, a small number of exosomal circRNAs have been discovered to interact with RBPs in recipient cells to exert biological functions. Binding proteins may be encapsulated with circRNAs into exosomes and function in recipient cells. It should be a relatively common situation for the released circRNAs to bind to the RBPs in the recipient cells after exosomes are fused with plasma membrane. In addition, circRNAs have recently been found to encode proteins or peptides, so we hypothesize that exosomal circRNAs may also encode proteins. Exosomes may transport circRNA-encoded proteins to cells in need, or circRNAs transported to target cells may encode functional proteins which regulate the activity of recipient cells. However, more experiments are needed to confirm these speculations. The ultimate goal of scientific researches is to improve the clinical treatment effect of patients. Due to their unique characteristics and high specificity, the combination of exosomes and circRNAs is beneficial to its potential clinical application as a biomarker for cancer diagnosis and prognosis. Besides, exosomal circRNAs can be used as therapeutic targets in inhibiting tumor metastasis, regulating tumor anti-cancer immunity response and mediating tumor drug resistance.Despite these studies, there still remain some fundamental questions that need further clarification and exploration. (1) The sorting mechanisms of exosomal circRNAs are unclear. (2) In terms of tumor drug resistance, we considered whether interference can be designed to reverse exosomal circRNAs-mediated tumor drug resistance. For example, we can design corresponding drugs or small interfering fragments targeted at recipient cells for intervention, or during the entry of drug-resistant circRNAs into exosomes or transporting. (3) It is a controversial topic that whether the functions of exosomes mediated only by their circRNAs. There are a lot of contents inside exosomes, and we can’t rule out whether other substances also play a similar role in inhibiting or promoting cancer. (4) There are no intuitive and effective methods to confirm that the process of circRNAs passing from cell to cell via exosomes. The commonly used method is to detect the difference in the expression level of circRNAs between donor cells and recipient cells. (5) It is not clear whether exosomal circRNAs function in vivo. The changes in the behavior of recipient cells may be partly due to the human regulation of exosomal circRNA expression. In addition, the lack of a suitable model is a major obstacle to the study of the roles of exosomal circRNAs in cancer. The cell model system in vitro could not adequately simulate intercellular communication mediated by exosomal circRNAs in vivo. The incorporation of a large number of exosomes into cultured cells may amplify the true effect of their circRNA vectors on recipient cells. After fully elucidating the function and molecular mechanism of exosomal circRNAs related to human cancer, new ways of understanding will be opened up to provide new methods for the treatment of malignant tumors.YX and SK collected the related manuscript and drafted the manuscript. SQ revised the manuscript. SJ and XS participated in the design of the review and helped to draft and modify the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The nanoscale organization of the F-actin cytoskeleton in neurons comprises membrane-associated periodical rings, bundles, and longitudinal fibers. The F-actin rings have been observed predominantly in axons but only sporadically in dendrites, where fluorescence nanoscopy reveals various patterns of F-actin arranged in mixed patches. These complex dendritic F-actin patterns pose a challenge for investigating quantitatively their regulatory mechanisms. We developed here a weakly supervised deep learning segmentation approach of fluorescence nanoscopy images of F-actin in cultured hippocampal neurons. This approach enabled the quantitative assessment of F-actin remodeling, revealing the disappearance of the rings during neuronal activity in dendrites, but not in axons. The dendritic F-actin cytoskeleton of activated neurons remodeled into longitudinal fibers. We show that this activity-dependent remodeling involves  The lattice was later observed in dendrites of multiple types of neurons, albeit to a lesser extent compared to axons4. Several isoforms of spectrin have been observed in the lattice, with variable prevalence in axons compared to dendrites and during development4. In addition to forming periodical rings, other F-actin structures have been described at the nanoscale in axons and dendritic shaft, including longitudinal fibers7.One of the hallmark discoveries made possible by fluorescence nanoscopy methods is the existence of a periodical lattice of F-actin, spectrin, and associated proteins under the surface membrane of neuronal processes. This lattice, containing F-actin rings periodically spaced 180–190 nm apart, was initially discovered in axons8. It was demonstrated that the submembrane lattice destabilization triggers axonal degeneration10. A recent study provided evidence that it serves as a signaling platform for receptor tyrosine kinase transactivation in neurons11.The role and regulatory mechanisms of the periodical submembrane skeletal structure remain unclear. It has been shown to be regulated during development in axons, dendrites, and dendritic spines12. A clear discrimination between spatially overlapping axons and dendrites, using a combination of specific markers, has been lacking in studies comparing the properties of the lattice, which may have impacted the analyses of its prevalence in dendrites. Furthermore, the greater diversity of F-actin nanostructures in dendrites compared to axons poses an additional challenge for quantitative analysis of the submembrane lattice. Several features of neuronal culture conditions, age, or fixation methods may have also contributed to discrepancies across studies of the dendritic lattice. One feature that has not been controlled or assessed is the level of electrical or synaptic activity. Ample evidence has shown, using conventional microscopy methods, that neuronal activity regulates the F-actin cytoskeleton14.The more variable appearance and sporadic presence of the F-actin/spectrin lattice in dendrites compared to axons suggests that the structure is differently regulated in these distinct processes8 prompted a high throughput analysis framework for the detection of those patterns on nanoscopy images. Recently, deep learning methods have been developed for automated feature detection in microscopy images from cells19. We show here that, using a weakly supervised deep learning approach22, we could train a modified U-Net architecture15 to segment regions containing fluorescent F-actin rings and/or longitudinal fibers in axons and dendrites. We demonstrate that this can be done without a significant decrease in segmentation quality, even in the presence of incomplete or coarse labeling. Our quantitative analysis highlights the profound diversity of F-actin patterns in dendrites, revealing an activity- and Ca2+-dependent remodeling, from a lattice pattern to longitudinal fibers, which does not occur in axons. The activity-dependent remodeling of F-actin structures in dendrites may explain the previous sporadic observations of the submembrane F-actin lattice in dendrites and may be necessary to regulate membrane dynamics and protein transport required for dendritic signaling and plasticity.We thus set out to test whether neuronal activity regulates the F-actin-based lattice in dendrites and axons of cultured hippocampal neurons, using STimulated Emission Depletion (STED) nanoscopy. The complexity of the nanoscale F-actin patterns in dendrites and axons required the use of image segmentation to quantify their dynamics. The high diversity and variability of the reported F-actin patterns23)  F-actin lattice in somatic regions, as previously described4 , imaged with STED nanoscopy. We observed complex and diverse patterns of fluorescence inside the neuronal processes that were only distinguishable with super-resolution microscopy , revealed both F-actin rings and longitudinal fibers, as previously shown , Lifeact, and Actin-Chromobody. However, these labels were shown not to reveal the F-actin periodical ring structure31. Using STED nanoscopy, we observed activity-dependent remodelling of F-actin nanostructures on dendrites that could not be resolved with diffraction-limited confocal microscopy  affect the spatial relation between fluorescent structures.To achieve reliable and high throughput quantification of the F-actin patterns at the nanoscale, we implemented a deep learning approach for the precise segmentation of F-actin rings and longitudinal fibers on STED images Fig. . We chos20. The expert had to label the F-actin patterns using polygonal bounding boxes instead of carefully drawing contours of each pattern  score of manual bounding box labeling -amino-5-phosphonovaleric acid (APV) on the 0To test whether the impact of the 0Neuronal depolarization and NMDAR activities drive Finally, we assessed whether the F-actin remodelling was reversible by washing the neurons with high 12. In our study, we specifically controlled both the neuronal activity level and the developmental stage of the preparation and found that the extent of dendritic remodeling is more pronounced in more mature and synaptically connected neurons. Another source of variability may have arisen from inconsistent use of specific axonal and dendritic markers to discriminate these processes. We found it critical to have double labeling of both axonal and dendritic markers in our cultures, as these distinct processes exhibit considerable spatial overlap, while their F-actin-based membrane lattice reacts differently to neuronal stimulation.Our results demonstrate that the somatodendritic F-actin-based lattice is highly dynamic compared to that of the axon. While it was known that the dendritic lattice is distributed in a more patchy fashion, we show here that this difference is likely due, at least partially, to a distinct response to neuronal activity, which destabilizes the lattice only in dendrites and somata Fig. . The spo34. Meanwhile the lattice remodeling might also be modulated by spectrin cleavage through 35. The reasons why the axonal lattice is resistant to enhanced neuronal activity remain to be determined. The elevation in axonal 2, may have a stabilizing role for the submembrane lattice.We demonstrated that the activity-dependent disappearance of the somatodendritic submembrane lattice coincided with the formation of F-actin longitudinal fibers. The simplest interpretation of this observation is that the F-actin underwent restructuring from a ring to fiber pattern. This putative remodeling would be demonstrated more clearly using live imaging, however, as we described and discussed above, currently available F-actin probes either do not reveal, or interfere with, the F-actin lattice dynamics. Nevertheless, the 11. The activity-dependent remodeling of the F-actin based lattice into F-actin longitudinal fibers might, on the other hand, be necessary for some dendritic signaling mechanisms, such as endo/exocytosis, membrane receptor lateral movement, intracellular trafficking or spine formation. In contrast, the stable lattice in axons might limit transmembrane receptor movement36, which could support a tight location of voltage-dependent channels critical for action potential conduction. It might also serve to restrict exo/endocytosis to pre-synaptic terminals, where the lattice is less organized12. In contrast, the removal of the lattice in dendritic segments may release a break on membrane receptor diffusion and exo/endocytosis, or spine formation. Meanwhile, the activity-dependent formation of F-actin longitudinal fibers in dendrites may serve to promote active cargo transport37 and targeting of material to synapses7. As such, the activity-dependent conversion of membrane-associated rings to intracellular fibers of the dendritic F-actin cytoskeleton may be essential for synaptic plasticity. In this context, we found that NMDA receptor function, which is crucial for synaptic plasticity, was essential in supporting the remodeling of the dendritic F-actin rings to longitudinal fibers. Hence, of the numerous signaling processes downstream of NMDA receptor function and The stability of the membrane-associated cytoskeleton was shown to be important for RTK signalingThe quantification of these fluorescent patterns is highly difficult because of the heterogeneity and complexity of the signals, and because it is prone to biases. Our study emphasizes the potential of machine learning approaches, as an expert surrogate, for high throughput analysis of complex and highly variable neuronal structures observed with optical nanoscopy. It became essential in order to pick up subtle differences associated with developmental stages, activity levels or drug action. The segmentation task shown here presented a number of challenges related to the context of experimental biology: (1) limited number of biological samples and labeled data , (2) high variability across samples, (3) expert-dependent determination of the ground truth, (4) no direct link to other problems that could be used as a pre-training step. We demonstrated how a deep learning approach can be applied, using weak supervision and harnessing the deep network extrapolation power. We also characterized how two types of labeling problems (e.g. incomplete and decreased precision) influence the performance of the FCN in this weakly supervised learning framework. We showed that our approach was sufficiently robust to the use of coarse labeling and label omission. This should broaden the possibilities of exploiting deep learning-based analysis for high throughput biomedical image analysis, especially when the generation of a large precisely labeled training dataset is impracticable. The weakly supervised deep learning approach used in our study provided the necessary tool to investigate more effectively and accurately the activity-dependent remodeling of the dendritic F-actin cytoskeleton, and should be useful for similar types of optical imaging-based investigations of cellular signaling.Neuronal cultures were prepared from neonatal Sprague Dawley rats. We followed the guidelines of the animal care committee of Université Laval for the care and use of the rats. Before dissection of hippocampi, animals were sacrificed by decapitation, in accordance to the procedures approved by the animal care committee of Université Laval. Dissociated cells were plated on poly-d-lysine coated glass coverslips . Dendrites were identified using a staining against the microtubule-associated-protein-2 with Rabbit-anti-MAP2 PAB  and GAR-STAR488 SAB . Axons were identified using a staining against the phosphorylated neurofilaments (SMI31) stained with the PAB mouse-anti-SMI31  and the SAB GAM-STAR580 .Cultured hippocampal neurons were fixed in 4% PFA solution, permeabilized with 0.1% Triton X-100, blocked with 2% goat serum and immunostained as described previously39.Super-resolution imaging was executed on a 4 color Abberior Expert-Line STED system . Imaging of F-actin stained with Phalloidin-STAR635 was performed using a 640 nm pulsed diode (40 MHz), a 775 nm depletion laser (40 MHz) and a ET685/70  fluorescence filter. MAP2-STAR488 and SMI31-STAR580 were imaged in confocal mode using excitation diodes at 485 nm and 561 nm (40 MHz), 525/50 and 615/20  fluorescence filters, respectively. Scanning was conducted in a line scan mode with a pixel dwell time of For the training of the FCNs presented in this work, two labeled datasets  were built using a custom labeling application developed in Python.40 applied after a gaussian blur with For the segmentation of dendritic F-actin ring and fiber regions, we collected a set of large STED images , and we used early stopping on the validation set to ultimately keep a model with good generalization properties. The Adam optimizer was used with a learning rate of 0.001, with the other parameters kept to their default values. Batch size was set at 72.To obtain a binary segmentation map, a hard threshold had to be applied on the network predictions. To compute the optimal thresholds for the two independent channels, we generated receiver operating curves (ROC) using the validation set. We computed the euclidean distance between the false positive and true positive rate  coordinate of the thresholds to the optimal operation point  and took the threshold having the smallest distance . In other words, the network is able to infer precise segmentation rules using only coarse examples. To assess the improvement in segmentation performance, we asked the expert to take the time to precisely label (P) 70 regions (38 for rings and 32 for fibers) randomly sampled from the testing dataset. We then compared the precision-recall curve (PR), an analysis method that is insensitive to class imbalance (e.g. number of background pixels in our images being more important than the number of foreground pixels) Fig. b. The coWe also set out to analyse how coarse labels can impact on the precision of the predictions. To do so, we trained 5 different instances of networks for different stepwise dilation of the labels  randomization test (shuffling) for statistical analysis43. The bootstrapping experiments consisted in sampling the data points for each condition with replacement to generate an estimate of the mean values distribution. For each condition (N data points per condition), a bootstrap sample consisted of randomly selecting N data points with replacement from the raw data and calculating the mean value of the distribution. The number of repetitions was set to 10,000 for all conditions. It resulted in a distribution of the bootstrapped mean values that could be used for data visualization  bootstrappingon Figs. e, 7a, d.44. The first metric is the F1-score and measures the overlap between the segmentation map and the ground truth. The F1-score is defined asWe define common pixel-wise metrics that were used to evaluate the performance of the architecturesFor the morphological analysis of the dendritic and axonal processes we used two measurements that were performed on each image: (i) total length of analysed branches and (ii) maximal length of a branch. Briefly, we generated a skeleton image from the binary masks used to identify dendrites and axons. We extracted all nodes, i.e. a joint or an end point, from the skeleton image. The skeleton was subdivided in multiple segments, where each segment has a start and an end node. The total length of branches per image was calculated using the sum of each segment length in the skeleton image. To calculate the maximal branch length per image, we used the NetworkX python library to efficiently create and manipulate a complex network. The complex network (or graph) was created from an adjacency matrix. The weights of the adjacency matrix were set according to the length of the segment between two connected nodes. The weight is set to 0 if two nodes are not connected by a segment.Supplementary Information.


The ultrasound measurement of inferior vena cava (IVC) diameter change during respiratory phase to guide fluid resuscitation in shock patients is widely performed, but the benefit on reducing the mortality of sepsis patients is questionable. The study objective was to evaluate the 30-day mortality rate of patients with sepsis-induced tissue hypoperfusion (SITH) and septic shock (SS) treated with ultrasound-guided fluid management (UGFM) using ultrasonographic change of the IVC diameter during respiration compared with those treated with the usual-care strategy.This was a randomized controlled trial conducted in an urban, university-affiliated tertiary-care hospital. Adult patients with SITH/SS were randomized to receive treatment with UGFM using respiratory change of the IVC (UGFM strategy) or with the usual-care strategy during the first six hours after emergency department (ED) arrival. We compared the 30-day mortality rate and other clinical outcomes between the two groups.p > 0.05 by log rank test). Neither was there a difference in six-hour lactate clearance, a change in the sequential organ failure assessment score, or length of hospital stay. However, the cumulative fluid amount given in 24 hours was significantly lower in the UGFM arm.A total of 202 patients were enrolled, 101 in each group  for intention-to-treat analysis. There was no significant difference in 30-day overall mortality between the two groups (18.8% and 19.8% in the usual-care and UGFM strategy, respectively; In our ED setting, the use of respiratory change of IVC diameter determined by point-of-care ultrasound to guide initial fluid resuscitation in SITH/SS ED patients did not improve the 30-day survival probability or other clinical parameters compared to the usual-care strategy. However, the IVC ultrasound-guided resuscitation was associated with less amount of fluid used. Sepsis is a significant burden for emergency departments (ED) worldwide.16Inferior vena cava (IVC) diameter measurement during respirophasic change is widely used to help physicians predict the fluid responsiveness in shock patients and is reasonable to tailor fluid therapy during the resuscitation.ClinicalTrials.gov (identifier NCT03020407).This randomized controlled trial was conducted in a 1,500-bed urban, university-affiliated tertiary-care hospital. This ED has more than 80,000 new patient visits per year. Our institutional review board approved this study, and written informed consent was required for trial participation. Patient recruitment started in January 2017 and concluded at the end of January 2020. This study was registered at Adult (≥18 years old) nontraumatic SITH/SS patients (see Study definitions) who presented to the ED during the study period were enrolled in the study. Patient eligibility was assessed by emergency physicians during all work shifts. Patients were excluded if they met any of the following criteria: 1) congestive pulmonary edema or known poor systolic cardiac function (left ventricular ejection fraction ≤ 40%); 2) known right heart pathology; 3) had or were suspected of having marked ascites, significant bowel dilatation, or conditions that could cause abdominal hypertension; 4) body mass index ≥ 30 kilograms/meters squared; 5) concomitant attack of a severe airway disease  IVC could not be identified, or its diameter could not be measured correctly; 7) end-stage renal disease with or without dialysis; 8) noninfectious disease as a final diagnosis; 9) pregnancy; 10) were referred from or treated at other healthcare facilities; 11) active hemorrhage; 12) duplicated or multiple case visits; 13) had a living will stating “do not resuscitate”; and 14) declined to consent.What do we already know about this issue?Ultrasonographic inferior vena cava (IVC) diameter measurement is used to tailor fluid therapy in shock patients. Its benefit in reducing sepsis mortality is unclear.What was the research question?To compare the 30-day mortality of sepsis patients treated with IVC ultrasound-guided resuscitation and the usual-care strategy.What was the major finding of the study?IVC ultrasound-guided resuscitation did not improve survival of sepsis patients but was associated with less fluid volume.How does this improve population health?Assessment of ultrasonographic IVC diameter change did not affect overall survival of patients with sepsis.In this study we specifically defined patients with SITH as those with infections and systolic blood pressure equal to or less than 90 millimeters of mercury (mm Hg) or initial lactate equal to or greater than 2 millimoles/liter (mmol/L) at ED presentation. However, we used the most recent definitions of septic shock and other related terms recommended in the literature.23In our protocol we prepared the preplanned, permuted block-of-four randomization list that was blinded to the investigators before patient allocation. Randomization was set at a 1:1 ratio of the ultrasound-guided and usual-care arms. When an eligible patient was identified and informed consent was obtained, demographic data, preexisting condition, bloodwork, diagnostic investigations, microbiologic workups, and blood lactate were collected at ED arrival (hour 0). Prompt empirical antibiotics were given to every patient within one hour after ED arrival. Then, the patient was rapidly assigned in accordance with the randomization and treated with one of the two treatment strategies as follows:In this treatment arm, the treating emergency physician promptly assessed the IVC diameter to obtain the IVC collapsibility index (IVCCI)  of each patient while venous access was performed and initial laboratory specimens were collected. A previous study showed that IVCCI > 40% was strongly associated with fluid responsiveness.In our protocol, IVC was identified in longitudinal section in the subcostal area using the curvilinear or phased array transducers (cardiac) of a standard ultrasound machine. The selected area of IVC diameter measurement was set at 2 centimeters distal to the confluence of the hepatic vein by M-mode coupled with two-dimensional mode on frozen screen images using the Sonosite X-porte . All treating physicians including attending staff and residents regularly participated in hands-on training twice a year  by a qualified international instructor in critical care ultrasonography (the third author). The residents who were allowed to perform the study protocol required at least six months exposure in real clinical experience and had passed formal performance evaluation on ultrasonographic IVC measurement. If the patient was breathing spontaneously, the IVCCI, which reflects the decrease in IVC diameter on spontaneous inspiration, was used. IVCCI is calculated as follows:If the patient required MV for respiratory support, the IVCDI, which reflects the increase in IVC diameter on mechanical inspiration, was used. IVCDI is calculated as follows:Sample images of ultrasonographic landmark and respirophasic diameter changes of an IVC during volume expansion are shown in Patients were promptly treated with 30 mL/kg loading of NSS in this treatment arm. After theNSS bolus, treatment with either the additional IV fluid or a vasopressor was given at the physicians’ discretion during the six-hour study period. The threshold for the need of a vasopressor was set at mean arterial pressure below 65 mm Hg if a patient did not respond to fluid therapy during each treatment protocol, and the time of vasopressor administration was noted. However, ancillary fluid administration was allowed at treating physicians’ judgment in both treatment arms. Other adjunctive therapies, such as colloid administration, central venous catheterization, or surgical removal of the infectious source, were not prohibited in our protocol and were used at the discretion of the treating physicians. The study patients were closely monitored while we recorded their clinical parameters every two hours for study purposes. Our resuscitative study protocol stopped at six hours after initiation of the treatment. After this period, patients were treated according to the physicians’ judgment.At six hours after treatment, the cumulative fluid volume was recorded, and blood lactate was obtained for lactate clearance calculation. At 72 hours after ED presentation, the cumulative fluid volume from the initial presentation was again recorded, and the patients were followed for clinical condition evaluation and blood chemistry tests to calculate the SOFA score and assess its change from the hour-zero baseline. The in-hospital requirement and time to start renal replacement therapy or MV were followed and recorded by searching the electronic data summary of a patient. The indication to initiate these life-saving procedures was at the discretion of the treating physicians. To identify the deceased patients for mortality analysis, we retrieved the electronic database of in- and outpatient clinical records or made a telephone call to the patients or their personal contact in every case at 30 days after the day of hospital presentation. The clinical data retrieval was performed and recorded by the trained non-investigators.According to the results of large trials of septic shock treatment, the 90-day mortality was 30% in the usual-care group.252 test with odds ratio (OR) was performed to compare the proportions between the groups. No data were imputed for any missing information. We used the Kaplan-Meier curve and the log-rank test to compare the 30-day mortality between the treatment arms. All tests were two-sided for superiority testing and considered statistically significant at a p < 0.05.We used Stata version 14.0  for all statistical tests and production of graphics. The normally and non-normally distributed data were analyzed using the two independent-samples t test and Mann-Whitney U test, respectively. A χIn accordance with the data safety monitoring board, the investigators decided to stop the trial before the target number of participants was recruited due to the possible ineffectiveness of the intervention and safety concerns. In total, 514 patients were screened for eligibility; 106 and 105 eligible patients were randomized to the usual-care and UGFM treatment arms, respectively. In summary, there were 101 patients in each treatment group available for intention-to-treat analysis. The patient flow chart and exclusion details are shown in p > 0.05 by log rank test). For secondary outcome analyses, we did not find a significant difference between the treatment groups in six-hour lactate clearance, SOFA score at 72 hours or the length of hospital stay. However, the rate of vasopressor use and the cumulative fluid administration in 24 hours was lower in the UGFM arm. The comparisons of the study variables are shown in There was no significant difference in 30-day mortality between the two groups , the analysis also revealed a potential negative effect of the intervention on those with initial lactate ≥4 mmol/L. However, neither of the subgroups reached statistical significance. A forest plot of the subgroup analysis and related information is shown in The use of point-of-care ultrasound (POCUS) is helpful in rapidly identifying causes of shock in the ED.32The results from an impactful research on a large database of the ED patients who had been admitted to the ICUs with SS showed that the use of large fluid volume (over five liters) during the first day of SS resuscitation was associated with increased risk of hospital mortality.Our in-depth study analysis showed that, regardless of the treatment strategies, the median amount of fluid used during the initial phase of resuscitation in non-survivors was strikingly higher than that of survivors. . These sInterestingly, we found a significantly lower rate of vasopressor use in patients treated with UGFM, while a recent study revealed a higher incidence of vasopressor need in septic patients resuscitated with a restrictive fluid strategy.The equivocal outcomes in the specific subgroups of SITH/SS patients also require additional scientific investigation in a larger population. According to our results in this ED, the initial use of respirophasic change in IVC diameter with POCUS in resuscitating SITH/SS did not improve the overall survival probability of patients compared to those treated with the usual-care strategy. It also did not improve lactate clearance or SOFA score. However, it was associated with a reduced amount of IV fluid given during the initial resuscitation.Our study had noteworthy limitations. First, this study was conducted with the specific study protocol at a single, tertiary-care hospital. The results may not be generalizable to other settings with various resuscitative techniques or protocols or if different target parameters are set. Second, since our recruited patients had median initial SOFA scores of 4, and one-third of them required a vasopressor or MV during initial phase of their treatment, physicians should use cautious clinical judgment or different approaches when treating more severe septic shock patients in their practice. Third, bias may have occurred during patient allocation, data collection or outcome measurement due to the unconcealed nature of the study interventions, although this was minimized by the appropriate randomization. Moreover, the primary study outcome was the mortality rate of the patients, which is generally unaffected by blinding of the assessors.Fourth, the higher rate of achieving the IVC collapsibility targets in patients treated in the ultrasound-guided arm may have affected the outcome. Finally, although the standard protocol and location of IVC diameter measurement was determined before the study, the interpersonal variation and sampling position may have affected the consistency of IVC diameter measurement.In our ED setting, where a relatively restricted amount of IV fluid administration is generally practiced, we did not demonstrate the benefit of the use of respiratory change of IVC diameter determined by point-of-care ultrasound to guide initial fluid resuscitation in SITH/SS patients in the ED in improving the 30-day survival probability or other clinical parameters compared to the usual-care strategy. However, it was associated with less amount of fluid used. Further studies are required to identify the optimal physiologic targets and fluid resuscitation approach in the initial treatment of sepsis-induced hypoperfusion and septic shock patients in the ED.