Young Australians (16-25 years) have the highest rates of past-month cannabis use in the world. Cannabis use increases the risk of alcohol and other drug disorders and depressive disorders, and has a robust dose-response association with psychotic experiences (PEs) and disorders. PEs are subthreshold positive psychotic symptoms, including delusions and hallucinations, which increase the risk of substance use, depressive or anxiety disorders, and psychotic disorders. Access to effective web-based early interventions targeting both cannabis use and PEs could reduce such risk in young people.Keep it Real web-based program compared to an information-only control website among young cannabis users (16-25 years) with PEs.The objective of this study is to determine the efficacy and cost-effectiveness of the Keep it Real web-based program (n=249) or an information-only control website (n=249). Both websites are self-guided (fully automated). The baseline and follow-up assessments at 3, 6, 9, and 12 months are self-completed online. Primary outcome measures are weekly cannabis use, PEs, and the relative cost-effectiveness for quality-adjusted life years. Secondary outcomes include other substance use and related problems, PE-related distress, cannabis intoxication experiences, severity of cannabis dependence, depression/anxiety symptoms, suicidality, and mental well-being and functioning.Participants are recruited online, and consenting individuals meeting inclusion criteria  are automatically randomized to either the Recruitment commenced in February 2019, and the results are expected to be submitted for publication in mid-2021.Keep it Real could help reduce growing public health concerns about the significant social, economic, and health impacts of cannabis use.This study protocol describes a large randomized controlled trial of a new web-based program for young cannabis users experiencing PEs. If effective, the accessibility and scalability of Australian New Zealand Clinical Trials Registry ACTRN12618001107213; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=374800DERR1-10.2196/15803 Young adults aged 18-24 years 23%) have the highest rates of past-year cannabis use in Australia % have th,6. Canna2.6 times the risk of developing a psychotic disorder, with the level of risk increasing in a dose-response manner  score of >18). The pilot study recruited 1089 past-month cannabis users over a period of 6 months using university student emails and social media and website posts. Almost all young people (95%) accessed the CAPE-15 PE feedback as well as a mean of 2.94 (SD 1.82) of the six Keep it Real modules. On average, they gave the program a rating of 4 out of 5 for overall objective quality on the eHealth Rating Scale , with good engagement  and high levels of functionality , aesthetics , and information  quality. High retention rates were achieved in the 3-month  and 6-month  follow-ups. Participants achieved significant reductions  in the frequency of cannabis use  and related problems  as well as the frequency of PEs  and their associated distress  at 3- and 6-months follow-up, respectively. Moderate effect sizes were found for reductions in cannabis use and related problems and PE-related distress, and large reductions in the frequency of PEs were found. Neither the frequency of cannabis use nor PEs at baseline had an impact on website engagement, retention rates, or treatment outcomes. Together, these results provide preliminary evidence that the Keep it Real web-based program could be effective and acceptable among young cannabis users with PEs.To address this gap, we developed the stand-alone Keep it Real (Version 2) compared to an information control website (ICW) among young people: (i) aged 16-25 years, who have (ii) used cannabis in the past month, and (iii) experienced PEs in the past 3 months. It is hypothesized that Keep it Real will result in significantly greater improvements in primary outcome variables of cannabis use and PEs, and will be more cost-effective than the ICW control condition. Secondary outcomes such as cannabis, alcohol, tobacco, and other substance use and related problems, including PE-related distress, cannabis intoxication experiences, the severity of cannabis dependence, depression/anxiety symptoms, suicidality, mental well-being, and functioning; and baseline moderators of intervention effects  will also be examined.This protocol paper describes an RCT aimed at determining the efficacy and cost-effectiveness of Keep it Real web-based program, compared to an ICW. Assessments are completed at baseline, and at 3, 6, 9, and 12 months follow-up. The trial has ethics committee approval, is registered (ACTRN12618001107213), and follows Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) research protocol guidelines  as well as Consolidated Standards of Reporting Trials (CONSORT) eHealth guidelines  superiority RCT that will determine the efficacy and cost-effectiveness of the ines see  42,43].,43.Keep Cannabis and Psychotic Experiences, and repeat the survey 6 and 12 months later. They are recruited online via university student emails and posts and paid advertising to social media  and substance use-related websites  containing a link to an online consent form and survey. Snowballing techniques will be used and participants will be offered a reimbursement incentive of Aus $10 for referring a friend to the study. Consenting participants (via a checkbox) self-complete all baseline and follow-up assessments online. Survey logic automatically screens participants for eligibility. Participant contact details  and the IP address of respondents will be manually checked to identify repeat or duplicate surveys. Participants who give contact details that appear false, for example, multiple similar names or email addresses, will be excluded. All baseline, 6-, and 12-month survey completers will be entered in a draw to win 1 out of 10 Aus $100 gift vouchers.This project will utilize a secure, automated web-based project and data management system for RCTs. A two-step recruitment and informed consent process will be used. Young people aged 16-25 years who have used cannabis in the past month will be first asked to complete an online survey on Keep it Real website (via a checkbox) on the online survey consent form, and again at the end of the baseline survey. Those who express interest in trialing the website and score a minimum of 18 on the CAPE-15, which is indicative of at least three PEs “sometimes” or one PE “nearly always” in the past 3 months, are eligible to participate in the RCT. They are also required to have internet access and to provide a mobile number and email address. People who use alcohol or other illicit drugs are included as long as cannabis is the most frequently used drug (other than tobacco). Exclusion criteria will be (i) history of traumatic brain injury or organic brain disease, (ii) current acute suicide risk, and (iii) insufficient English fluency. Eligible participants are automatically sent the consent form for the RCT, and those who provide consent (via a checkbox) are randomized and sent an email and SMS link to either the Keep it Real web-based program or information-only control website. They are not informed about which website program they are allocated to. The study information sheet states that both programs provide information on PEs, psychosis, and cannabis use. They differ by their level of interactivity.Young people are asked if they are interested in trialing the Keep it Real website. Those in the RCT are automatically sent follow-up surveys at 3, 6, 9, and 12 months postbaseline via SMS and email. Noncompleters will be contacted via telephone by outcome assessors blind to treatment allocation to remind them to complete the survey online or over the phone. Participants are reimbursed Aus $20 for completing each follow-up survey (maximum of Aus $80). If a participant chooses to withdraw from the study, they will still be able to continue using Keep it Real or the ICW. The trial statistician will be blind to treatment group allocation.Young people who are ineligible for the RCT remain participants in the original survey study and are provided with immediate access to the All measures are self-reported and the follow-up frequency is provided in The primary outcome measures for cannabis use and psychotic like experiences are described below:Cannabis use: The AOD section of the Opiate Treatment Index (OTI) will be used to assess the frequency  and the mean amount of cannabis (in 1/4 gram standard units) per day in the past month . As cannPsychotic-like experiences: PEs are assessed with the 15-item version of the 20-item positive scale of the Community Assessment of Psychic Experiences (CAPE) measure. The CAPE-15 measures the frequency  and level of distress (1=not distressed to 4=very distressed) related to PLEs in the past 3 months . This veA number of secondary outcomes will also be measured, as outlined below:AOD-related problems: The 8-item Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST) provides a measure of the frequency of substance use and related problems in the past 3 months for 10 substance classes . It has Depression/anxiety symptoms: The 9-item Patient Health Questionnaire (PHQ-9) and the 7-item Generalized Anxiety Disorder (GAD-7) scales will be used to assess depression and general anxiety symptoms in the previous 2 weeks. PHQ-9 and GAD-7 have been validated across a number of populations including adolescents, young adults, and substance users -53.Suicidality: Suicidal ideation, plans, and attempts will be assessed using the 6-item Suicidality Scale of the Mini-International Neuropsychiatric Interview (MINI) .Well-being: The 14-item version of the Mental Health Continuum-Short Form (MHC-SF)  ask partCannabis experiences: The Cannabis Experiences Questionnaire-Intoxication (CEQ-I) scale is a 13-item measure of the paranoid-dysphoric (PEs) and euphoric effects of cannabis intoxication validated in young adults .Dependence: The Severity of Dependence Scale (SDS), a 5-item measure, will be used to assess the severity of psychological dependence on cannabis in the past 3 months. The SDS is validated for use in cannabis users and people with early psychosis ,59.Functioning: The Multidimensional Adolescent Functioning Scale (MAFS) is a 23-item self-reported measure of general, family, and peer-related functioning with good psychometric properties .Health-related quality of life will be assessed using the 5-dimension version of the European Quality of Life 5-level (EQ-5D-5L) scale . InformaA number of other measures will also be included in the survey:Lifetime psychotic disorders: The psychosis screen (patient form), which contains 6 items and up to 4 supplementary items for positive responses, will be used to identify lifetime psychotic disorders . A cutofKnowledge: In all, 19 questions will assess the young person’s level of knowledge about cannabis and its effects, with response options of “true” or “false.” The total score reflects the number of correct answers.Impulsivity: The 20-item Short UPPS-P Impulsive Behavior Scale (SUPPS-P) provides a measure of the 5-factor model of impulsivity consisting of: (i) negative urgency (tendency to act rashly under extreme negative emotions), (ii) lack of premeditation (tendency to act without thinking), (iii) lack of perseverance (inability to remain focused on a task), (iv) sensation seeking (tendency to seek out novel and thrilling experiences), and (v) positive urgency (tendency to give into impulses when experiencing high positive affect) . Higher Childhood trauma: The Childhood Trauma Questionnaire (CTQ) is a valid and reliable 28-item measure of childhood abuse and neglect . ChildhoWebsite evaluation: The 26-item eHealth Rating Scale, which is based on the Mobile App Rating Scale user version, will be utilized to measure the usability, engagement, aesthetics, information quality, and perceived impact of web-based programs at the 3-month follow-up . ParticiKeep it Real or (ii) ICW. Stratification will be by cannabis usage (daily or less), sex, age (16-20 years and 21-25 years), and psychosis screen result (present vs not present). The randomization sequence is concealed within the secure website and the allocation is automatically released when full eligibility criteria are met.A computerized random number sequence generator incorporating random permuted blocks will be used to allocate participants to: (i) Keep it Real or ICW program, and are sent an email and SMS link to the relevant program. Both programs are accessible via login on any computer, tablet, or mobile device with an internet connection. They are password protected and are sited on parallel, secure servers. Both programs are self-guided (fully automated), and all website modules are unlocked and can be accessed by the user at any time. SMS reminders to access both programs will be sent on day 7, 14, and 21 after baseline. Participants’ “backend” usage data will be automatically captured by the website, including logins, modules accessed/completed, inserted text, and dates of data entry. All participants have continuous access to the Get Help section of the website, which provides the contact details of mental health and AOD services (including crisis care).Young cannabis users who meet RCT inclusion criteria and consent to participate are automatically randomized to either the Keep it Real program (Version 2) consists of seven modules that can be completed in 30-90 minutes over 1-3 sessions (via login). Version 1 of Keep it Real, which was tested in the initial pilot study, only targeted cannabis use. Version 2 targets cannabis, alcohol, methamphetamine, and heroin use. Each module ends with a printable summary screen. PEs are targeted first, given young people are more likely to be concerned about PEs than their cannabis use. The first three modules aim to increase the users’ ability to identify, understand, and reduce distress associated with PEs. The program content was informed by the Think You’re Crazy? Think Again [Get Real web-based program for young people with PEs. A small pilot study in 12 young people found that the program had high levels of acceptability and perceived utility, and resulted in significant reductions in the frequency and level of distress associated with PEs at the 3-month follow-up [The nk Again  self-helnk Again -72. Moduollow-up .Modules 4 and 5 use motivational interviewing techniques to target cannabis, alcohol, methamphetamine, and heroin use. The program content was informed by evidence-based brief motivational interviewing interventions for cannabis use, including web-based programs ,38,74. AModule 6 targets users’ coping skills by providing training in cognitive behavioral techniques including stress management, problem solving, behavioral activation, and attention control retraining (mindfulness). Module 7 encourages appropriate help seeking for PEs and/or cannabis use and addresses any barriers to doing so.Participants can track their PEs (CAPE-15), cannabis intoxication effects (CEQ-I), substance use and related problems over time, and receive feedback on age-related and gender-specific norms each time they complete the measures. An interactive graphical summary of their PE, CEQ-I, cannabis, alcohol, methamphetamine, and heroin use and related problems over various time frames (self-selected) is also available to increase participants’ awareness of the relationship between their substance use and PEs.Keep it Real program in a 4-module web-based format including: (i) What are weird experiences? (ii) How does cannabis affect me? (iii) How does other substance use affect me? (iv) Should I seek help? Further reading sheets are also available from a “Fact Sheets” menu containing: (i) What is psychosis? (ii) Am I at risk of developing psychosis? (iii) What is schizophrenia? (iv) What is bipolar disorder? (v) Facts about cannabis/alcohol/amphetamines/heroin. This control condition was designed to provide participants with access to the type of web-based information they may have found naturally online. It does not provide personal feedback from assessments or normative feedback on substance use or PEs at baseline or follow-up. No information is given on how to manage PEs nor do the resources focus on building motivation to change substance use or increasing users’ understanding of the relationship between their substance use and PEs.This program delivers the information sheets from the Weekly meetings monitor project implementation, clinical , and research integrity . Email or telephone consultations will be used to determine urgent issues, and a record of decision precedents will be kept to ensure consistency. Suicide and psychosis safety protocols identify and manage risk at all survey time points. Participants with a positive psychosis screen or who report a low level of suicide risk  receive a message in the online survey and an email providing details of appropriate support services and helplines. They are also asked to contact the research team if they have additional questions or would like assistance finding support. Those who report a moderate  or high (10+) level of suicide risk are asked if they are receiving adequate support and if their feelings have recently improved (due to the 1-month time frame of the measure). If the participant answers yes to both questions, no further action is taken. If they answer yes to one of these questions or request further support, they are called within 48 hours by a research assistant to assess how further support can be provided . Those who report a suicide plan or a suicide attempt within the past month are contacted by a trained clinical psychologist, who assesses the level of risk and helps the participant connect with appropriate supports.Keep it Real pilot study, which found a moderate effect size for reductions in the frequency of cannabis use (d=0.46). Due to the uncontrolled nature of the pilot study, a conservative approach using small to moderate effects was used. To measure a moderate effect size of f=.20, alpha (α) set at 0.025, and power set at 0.95, we would require 191 participants per group . If baseline covariates accounting for about 40% of the variability in the outcome model were included in the calculations, then we would have 0.99 power to detect a small to moderate treatment effect. We predict a 20%-30% attrition rate at 12 months based on our pilot data and previous work and will therefore need to randomize a total of 498 participants.Power calculations to determine sample size were based on the effects found in the Keep it Real pilot trial  and 213  were randomized. We will, therefore, need to recruit 1992 cannabis users in order to randomize 498 participants (see The pilot study recruited 1089 past-month cannabis users over a period of 6 months (using the same recruitment strategies) to identify 860 monthly cannabis users meeting our PE inclusion criterion (CAPE-15≥18). Of these, 295 (34%) expressed interest in participating in the ants see .Keep it Real and (ii) the ICW. The primary outcome variable is the frequency and average amount of cannabis/day (in 1/4 gram standard units) in the past month (on the OTI) and the frequency of PEs  in the past 3 months. Secondary outcome variables include the typical quantity and frequency of alcohol, tobacco, cannabis, and other drug use and related problems in the past 3 months (ASSIST), cannabis knowledge, PE-related distress (CAPE-15), cannabis intoxication experiences (CEQ-I), severity of cannabis dependence (SDS), depression (PHQ-9)/anxiety (GAD-7) symptoms, suicidality (MINI), mental well-being (MHC-SF), and functioning (MAFS). To determine whether there are group differences in the primary and secondary outcome measures at 3, 6, 9, and 12 months, a series of mixed effects models for repeated measures (MMRM) will be employed. The within-groups factor will be time  and group  will be the between-subjects factor to examine the timexgroup interaction. This technique can also control for potential confounds  and examine potential baseline moderators of intervention effects . Missing data will be handled using full information maximum likelihood estimation and an intention-to-treat analysis is performed.The independent variable is a treatment condition with two levels: (i) Keep it Real and ICW will be conducted, taking a societal perspective, including costs of treatment provision and intervention (including website development and maintenance) as well as productivity costs. The analysis will display the cost-effectiveness in both natural units and as QALYs (based on the EQ-5D-5L). Models will assess Keep it Real’s offset on reduced health care service use and productivity losses are avoided. In a second modeled cost-effectiveness analysis (CEA), the estimated change in cannabis consumption from the trial will be extrapolated to the general Australian population of 16-25-year olds. Uncertainty will be assessed using a combination of nonparametric bootstrapping for the effect size (cannabis frequency) and Monte Carlo simulation for all other variables (with sampling uncertainty).A two-pronged economic analysis comparing Recruitment commenced in February 2019, and is now closed for recruitment. The results are expected to be submitted for publication in mid-2021.Keep it Real program resulted in substantial reductions in both PEs and cannabis use, and young people found the program engaging and easy to use. This study protocol describes a large RCT that will determine if Keep it Real is more efficacious and cost-effective than the minimal web-based information they may otherwise receive.Large numbers of young cannabis users report PEs, which increase their risk of developing psychotic, substance use, and depressive/anxiety disorders. Current services are failing to engage these young people—and would struggle to meet the need if they did seek help. An effective, safe, low-cost, nonstigmatizing accessible intervention is needed. Our pilot data showed that the Keep it Real, as young people gave the program an average rating of 4 out of 5 for overall objective quality, and the functionality, aesthetics, and information subscales and a 3 out of 5 rating for engagement. Moreover, the pilot study provided preliminary evidence for the efficacy of Keep it Real as well as valuable data on the participation/retention rates and effect sizes to inform sample size which was used to ensure that this RCT is well powered.Engaging large numbers of young nonhelp-seeking cannabis users into web-based treatment is challenging, as they are unlikely to view their cannabis use as problematic or responsible for their PEs. However, the pilot study clearly demonstrated the feasibility of recruiting young cannabis users with PEs (79% of survey completers), with 72% of the 34% who expressed interest agreeing to be randomized to the trial. The pilot study also highlighted the acceptability of Young people will be asked if they were interested in participating in the RCT prior to receiving feedback on their PEs. While this recruitment strategy will ensure that all young people are given the opportunity to participate, it may increase the risk of selection bias by potentially excluding those with little insight into their PEs. Nevertheless, the inclusion of people with a lifetime history of a psychotic disorder and the stratification of the randomization sequence by psychosis screen result may help reduce such risk of bias.Keep it Real participants are able to monitor their PEs and substance use within the program. The extent of monitoring observed in this study will provide an indication of young cannabis users' willingness to engage in regular assessments for future studies exploring potential mediators of change.The inclusion of a range of potential control variables and moderators of treatment outcomes is a further strength of the study. However, mediators were not included due to concerns about potential assessment reactivity, the existing length of the assessment schedule, and participant burden. Keep it Real and ICW programs are not matched, but the ICW program contains the same number of information sheets as the Keep it Real program. While it could be argued that the smaller amount of content in the ICW may be insufficient to produce an effect, this control condition was designed to control access to a web-based program and the minimal amount and type of information users may otherwise receive online. Nevertheless, it is possible that the overlap in content may wash out any differential treatment effects between programs.The number of modules in the Keep it Real is the first web-based program to target PEs in cannabis users. The program content is based on evidence-based CBT treatments for people at ultrahigh risk for psychosis (which includes a subgroup with PEs) [Keep it Real will be freely available and users will have unlimited 24/7 access to the program, including the PE and substance use monitoring and feedback, enabling the user to engage in continuous self-management. The program also has broad applicability as it can be used as a stand-alone treatment or as an adjunct to usual treatment for help-seeking cannabis users with PEs accessing clinical services. Different components of the program can also be used to address cannabis, alcohol, methamphetamine, or heroin use, PEs, cannabis intoxication effects, and to provide coping skills training. If Keep it Real demonstrates a substantial and cost-effective impact on cannabis use and PEs, its low unit cost and scalability would mean that it has substantial potential to make a population-wide impact on both immediate and longer term consequences of cannabis use and their significant social, economic, and health costs.Web-based programs are ideal platforms for delivering self-guided treatments to nonhelp-seeking young people with PEs. ith PEs)  and motiith PEs) ,38. Keep


To evaluate the effect of a dialogue-based intervention targetingpsychosocial well-being at 12 months post-stroke.Multicenter, prospective, randomized, assessor-blinded, controlled trial withtwo parallel groups.Community.n = 166) or control group(n = 156).Three-hundred and twenty-two adults (⩾18 years) with stroke within the lastfour weeks were randomly allocated into intervention group.Secondary outcome measures included the Stroke and Aphasia Quality of LifeScale-39g, the Sense of Coherence scale, and the Yale Brown single-itemquestionnaire.The mean (SD) age of the participants was 66.8 (12.1) years in theintervention group and 65.7 (13.3) years in the control group. At 12 monthspost-stroke, the mean (SE) GHQ-28 score was 20.6 (0.84) in the interventiongroup and 19.9 (0.85) in the control group. There were no between-groupdifferences in psychosocial well-being at 12 months post-stroke : −3.08, 1.60). The secondaryoutcomes showed no statistically significant between-group difference inhealth-related quality of life, sense of coherence, or depression at12 months.The results of this trial did not demonstrate lower levels of emotionaldistress and anxiety or higher levels of health-related quality of life inthe intervention group  as compared to thecontrol group  at 12 months post-stroke. Depressive symptoms, anxiety, general psychologicaldistress, and social isolation are prevalent.3 About one-third of strokesurvivors report depressive symptoms, and 20% report anxiety post-stroke.5 Psychosocial problems persistover time, and the prevalence of post-stroke depression remains high at 25% in theperiod from 1 to 5 years post-stroke.4 Psychosocial difficulties may significantly impact long-term functioning andquality of life,7reduce the effects of rehabilitation services, and lead to higher mortality.8Stroke is one of the leading causes of death and disability in the adult population worldwide.10 targeted treatments to promote psychosocial adjustment mayimprove psychosocial well-being.11 In our work, psychosocialwell-being was defined as consisting of a basic mood of contentment, a self-conceptcharacterized by self-acceptance, usefulness, and a belief in one’s abilities.Having social relationships and support, a feeling of loving and being loved inrelationships are included in the definition, as well as participation andengagement in meaningful activities beyond oneself.13 The feasibility work precedingthis randomized controlled trial (RCT) suggested that it is possible to promotepsychosocial well-being and coping through a dialogue-based intervention.1315Despite inconclusive evidence,We hypothesized that a dialogue-based intervention would lead to higher levels ofpsychosocial well-being expressed through lower levels of emotional distress andanxiety at 12 months post-stroke. Secondary hypotheses were that stroke survivorswho received the intervention would experience significantly higher levels ofhealth-related quality of life and sense of coherence at 12 months post-stroke.15In this study, patient enrollment started in November 2014 and concluded datacollection in November 2017. The Regional Committee for Medical and Health ResearchEthics, South-East (REC no. 2013/2047) and the Data Protection Officer serving allparticipating hospitals (Case number: 2014/1026) approved the study. Writteninformed consent was obtained from all participants. Due to strict regulations andconditions for informed consent enforced by Norwegian law, the data set thatsupports the findings of this study is not publicly available. A subset of the datamay be made available from the corresponding author upon reasonable request. Thestudy was registered with ClinicalTrials.gov (NCT02338869), and the study protocoloutlining the full details of the study was published in BMC Psychology in 2018.The study was a multicenter, prospective, randomized, assessor-blinded, controlledtrial with two parallel groups (intervention and standard stroke treatment) and anequal size allocation ratio of 1:1. Eleven hospitals with stroke units orrehabilitation centers in South-Eastern Norway enrolled patients. Eligibleparticipants were adults aged ⩾18 years, had suffered an acute stroke within thelast four weeks, were medically stable, had sufficient cognitive functioning toparticipate, were able to understand and speak Norwegian before stroke onset, andwere able to give informed consent. Exclusion criteria were moderate to severedementia or other serious physical or psychiatric diseases and severe receptive orexpressive aphasia.15 Based on the results of comparable studies,16 we deemed an odds ratio of 1.6or higher between groups (intervention/control) with normal mood after six and12 months to be clinically relevant. With 80% power across both time points and asignificance level α at 0.05, the sample size was estimated to 300 patients (150 pergroup), which was inflated to a total of 330 to allow for a potential 10% drop-out.15The sample size was determined based on the primary outcome measure General HealthQuestionnaire-28 (GHQ-28). The calculations were based on a repeated measureslogistic regression model of the binary output variable “normal mood”(GHQ-28 < 5) with two measurements for each patient (i.e. one at six months andone at 12 months).A computer-generated block randomization procedure with blocks of 10 stratified byhospital and with an allocation ratio of 5:5 was used in this study. An assistantindependent of the research team prepared opaque randomization envelopes. Tworegional trial coordinators carried out the allocation following the baselineassessment. Participants were informed about group allocation immediately. To ensuremasking of group allocation at the follow-up assessments, a message was issued fromthe trial coordinators to participants with a reminder not to reveal their groupallocations to the assessors.18 In light of the extensiveliterature of the high prevalence of emotional distress following stroke, we assumedthat well-being in this population would premise the absence of emotional distress.Consequently, in this study, we operationalized psychosocial well-being as lowerlevels of emotional distress and used the GHQ-28 to measure it. For additionaldetails on the scoring of the GHQ-28 in this study, please refer to the online supplementary material . Clinical characteristics such as strokeclassification, side localization of the stroke symptoms, stroke severity, cognitivefunction, and language difficulties were assessed at the hospital and were collectedfrom the patients’ medical records.The primary and secondary outcomes and measures are presented in Data were collected in-person via structured interviews conducted by trainedhealthcare professionals  atbaseline, four to six weeks post-stroke (T1), and at six months (T2) and 12 (T3)months post-stroke. The data collectors were blinded to group allocation. Theparticipants’ ages, sexes, living situations, caring responsibilities, previousillnesses and comorbidities, and current rehabilitation services were recorded inaddition to the structured outcome measures.31 In Norway, patients with minor stroke are typically discharged home withaccess to interdisciplinary rehabilitation services in the municipality according tothe need and availability of the service. Services typically include physicaltherapy and/or occupational therapy and/or speech and language therapy and/or homenursing care. Systematic psychosocial follow-up is rarely part of the servicesprovided. Patients with severe stroke are typically discharged to a specialized,in-patient rehabilitation unit for specialized rehabilitation services.All participants randomized into the study received standard stroke treatment in theacute phase according to the Norwegian stroke treatment guideline.15 The intervention was delivered in the community, primarily in theparticipants’ homes. The same intervention provider worked with each participant inall sessions.Participants randomized to the intervention group were offered a dialogue-basedintervention to promote psychosocial well-being. The intervention consisted of eightindividual 1–1½-hour sessions between the participants and a specially trained nurseor occupational therapist (intervention providers). The intervention providerscompleted a three-day training program to learn how to guide the sessions and how towork with the participants based on the principles outlined in the protocol.15 the intervention started shortly after randomization; four to eight weeksafter stroke onset. It lasted 17 weeks, and the last session was completed withinsix months post-stroke.32 A guide of stroke-related topics and work-sheets for eachsession were supplied as part of the intervention.15 The intervention provider and the participant were encouraged to individuallyadapt the order of topics and the time in-between sessions to suit the needs of theparticipants. Additional details on theoretical perspectives underpinning theintervention, themes, and content of the intervention are outlined in the protocol.15In line with the protocol,32 The assessment of implementation fidelity included a separate analysis ofintervention adherence. The composite adherence score showed that 117 (80.1%) of theintervention trajectories satisfied the criteria for high-fidelity intervention adherence.32Implementation fidelity was assessed and previously published as part of the processevaluation of this RCT.34 All reported results of thestatistical analyses were pooled across five imputations based on Rubin’s rule.35 The statistical software R v3.6.136 with package mitools v2.4 was used to pool the results across all fiveimputed data sets. For additional details of the imputation model, please see theonline supplementary material .The data were analyzed using an intention-to-treat approach. Missing data wereimputed using multiple imputations by chained equations in the Statistical Packagefor the Social Sciences (SPSS).ttests for continuous outcomes. A linear mixed model was used to assess the primaryoutcome of psychosocial well-being at 12 months post-stroke. Due to the complexityof the final model, we did not use the dichotomized “normal mood” (GHQ-28 < 5)end-point, as it resulted in convergence issues when fitting the binary logisticmixed model. The continuous sum-score based on the Likert-type-scoring of GHQ-28 wasused as the dependent variable. The other factors of the model remained the same asin the predetermined statistical analysis plan. The details of the linear mixedmodel are supplied in the online supplementary files .Analyses of the primary and secondary outcomes were performed using logisticregression for binary outcomes and independent and paired samples 37 All statistical tests were performed as two-sided tests with a significancelevel of α = 0.05.Statistical tests were performed with SPSS, version 25.0 for Windows.19 Between consent and the baseline assessment, 31 (8.8%) participants droppedout. Thus, 322 participants were assessed at baseline and subsequently allocated tothe intervention group (n = 166) or the control group(n = 156).The CONSORT flow diagram is presented in Baseline demographics and clinical characteristics are presented in Results from the between-group comparisons at 12 months post-stroke for primary andsecondary outcomes are presented in The secondary outcomes showed no statistically significant between-group differencein depression, sense of coherence, or health-related quality of life at 12 months.Self-reThe results of the linear mixed model analysis are displayed in Higher scores on sense of coherence were associated with lower GHQ-28 scores,indicating that higher sense of coherence scores were associated with higher levelsof psychosocial well-being. Reporting additional comorbidities, caringresponsibilities, fatigue, and depression was associated with higher GHQ-28 scores,which indicated lower psychosocial well-being. Adjusted for all factors in thelinear mixed model, the intervention group scored lower  on GHQ-28 compared to the control group; however, thebetween-group differences were not statistically significant .Contrary to our hypotheses, the results of this trial did not demonstrate at thespecified statistical significance level that the participants in the interventiongroup experienced higher levels of psychosocial well-being and lower levels ofdepressive symptoms and anxiety than participants in the control group at 12 monthspost-stroke. Nor did the secondary outcomes show statistically significantly higherlevels of sense of coherence or higher levels of health-related quality of life inthe intervention group compared with the control group at 12 months post-stroke.38 Plausible explanations may include flaws in the underlyingtheoretical assumptions or characteristics of the intervention, the timing of theintervention, the standard care provided to the intervention and control groups, thesample of participants enrolled, or the outcome measures.In the following, we will highlight possible reasons for the statisticallynon-significant results in this RCT, drawing on the results of a comprehensiveprocess evaluation of the RCT and existing research to interpret the outcomes of thetrial.22 we assumed that an important active ingredient in the intervention would beto support the participants’ perceptions of their lives as comprehensible,manageable, and meaningful. We anticipated that the intervention would fosterunderstanding and re-creation of meaning through narrative dialogue and that theintervention provider could support the participants’ coping efforts and developmentof new life skills through the guided self-determination problem-solvingapproach.14Based on Antonovsky’s theory of sense of coherence,22 Others have shown that sense of coherence is less stable over time thanAntonovsky assumed.39 We hypothesized that the intervention would be able to influence theparticipants’ sense of coherence after a life-threatening event such as stroke andthat a higher sense of coherence would lead to higher levels of psychosocialwell-being.Antonovsky framed sense of coherence as a stable trait that may to some degree bedynamic with fluctuations in periods of threatening life events.This twofold hypothesis was only supported in part. The lack of differences withingroups over time and between the intervention and control group does not support thenotion that the intervention succeeded in influencing the levels of sense ofcoherence. The results of the study suggest that this intervention did not influencesense of coherence and that it is a stable construct. However, the results supportthe notion that a higher sense of coherence is important in the promotion ofpsychosocial well-being.This knowledge may be important to clinicians who need to be able to identify strokepatients who need extra attention with regard to promoting psychosocial well-being.It may be advisable to screen for sense of coherence during the early post-strokephase to identify those with lower sense of coherence, who may be more vulnerable tolower psychosocial well-being.3 For some participants, the intervention may have led to decreasing stress andfor some to potentially increasing it, depending on their existing stress levels. Ifthe participants did not experience increased stress or challenges in coping in thisphase of their adjustment process, we need to consider if the focus on psychosocialchallenges in the intervention may have increased rather than decreased theirstress. In the future, screening for distress at baseline may be advisable in orderto explore whether the intervention may be more appropriate for those with somelevel of existing stress/distress.Another assumption made in this intervention was that it would be possible to preventdepression that manifested after stroke due to the increased stress and chaos oftrying to cope with the post-stroke changes.13 the intervention in this trial was designed to be deliveredover a period of five months starting four to six weeks post-stroke and concludingwithin six months post-stroke.15 The intervention periodcoincides with a period in which spontaneous functional recovery may peak41 and overlapswith a period of comprehensive physical rehabilitation within Norwegian stroke services.31 The psychosocial intervention provided to the intervention group may not havemade a discernable impact in this context with substantial rehabilitation effortswithin the regular healthcare services.Based on assumptions that early rehabilitation efforts are important to promotepsychosocial well-being,42 Therefore, despite high baseline scores, there was still room for improvementin health-related quality of life in this group of participants.At baseline, participants in both groups reported high scores on the Stroke andAphasia Quality of Life Scale-39g . AlthougThe participants received substantial rehabilitation services as part of theirstandard stroke treatment. At baseline, 114 (68.7%) participants in the interventiongroup and 99 (63.5%) participants in the control group received one or morerehabilitation services, most frequently physical therapy. At 12 months, theproportion was still high: 70 (42.3%) participants in the intervention group and 66(42.1%) participants in the control group.41 However, the focus on physical recovery is more pronounced in the beginning,while the focus on psychosocial issues such as biographical adjustment gainsemphasis later in the trajectory. Introducing this intervention on top of thenatural recovery and rehabilitation processes may not have added to the adjustmentprocess, or the participants may have been more focused on other parts of theiradjustment than that of a psychosocial nature.Earlier theoretical work has shown that the physical recovery, daily life adaptation,and normalization, as well as biographical adjustment, occurs simultaneouslythroughout the first 12 months of the adjustment process after a stroke.11It is important to consider whether the extra attention given to the psychosocialissues in the intervention group came at an inappropriate time in the participants’stroke recovery and whether we may have increased the awareness on psychosocialdifficulties rather than prevented them. Other studies have shown successful resultsin promoting normal moods when introducing early psychosocial support by providingmotivational interviewing to support and build patients’ motivation to adjust andadapt to having had a stroke.16 motivational interviewingaimed to promote self-efficacy. The patients raised the issues they wanted todiscuss themselves instead of having topics outlined for each meeting.16 Compared to the theoretical assumptions of anticipated active ingredients ofthe intervention tested in this RCT, motivational interviewing may have been morealigned with the patients’ phase of adjustment and more aligned with their focus on,that is, getting well or frustration in this early adjustment phase.43 The focus on patient-initiated discussion themes rather than thepre-specified themes related to psychosocial issues may have supported theiradjustment to a greater degree than in the intervention tested in this RCT.In the study of Watkins and colleagues,45 Wide inclusion criteria were applied in the RCT, which may haveinadvertently resulted in the enrolment of participants who did not particularlyneed this kind of intervention. The process evaluation that was conducted alongsidethe trial showed that not all participants expected a personal benefit and that akey motivation to participate was to contribute to research and to help other stroke survivors.38 Despite this observation, the majority of the participants who participatedin the qualitative interviews as part of the process evaluation found theintervention useful and found that it facilitated their post-stroke adjustments.38The feasibility work done during the development of the intervention showed thatparticipants found the intervention helpful; however, it failed to clearly identifyspecific patient groups who would potentially benefit from thisintervention.19Some participants in the control group reported that the assessment interviewsfacilitated reflection and adjustment, and some indicated that allocation to thecontrol group and the themes raised in the assessment interviews influenced theirhelp-seeking behavior outside the trial.46It is still important to identify subgroups of the stroke population who mightbenefit from a psychosocial intervention to promote psychosocial well-being.Patients who reported depressive symptoms, fatigue, comorbidities, and caringresponsibilities were prone to lower levels of psychosocial well-being in thisstudy. Earlier studies have shown that emotional distress at one month post-stroke,higher stroke severity, and communication impairments predict emotional distressduring the first six months post-stroke.47 Conversely dependency in activities of daily living (i.e. toileting) predictemotional distress two to five years post-stroke.47 Identifying patients with the characteristics identified in this and otherstudies may be especially important in clinical settings to identify those who mayneed closer attention and follow-up with regard to psychosocial well-being.Studies exploring predictors of emotional distress and well-being in a longerpost-stroke perspective have found that higher age (>65 years), independence inmobility, having social support, and being employed are important predictors of well-being.16 However, the sensitivity ofthe GHQ-28 does not necessarily mean it was the most suitable outcome measure inthis study.There is a need to consider whether the chosen outcome measures were appropriate todetect the kind of change the intervention targeted. The change in emotionaldistress in both the intervention and control groups across the trajectory indicatedthat the GHQ-28 was sensitive to change. There was a substantial increase in theproportion of participants with GHQ-28 scores <5 in both groups. Furthermore, thelevel of improvement exceeded the findings in a similar study in which motivationalinterviewing was provided post-stroke.48The intervention was aimed at promoting psychosocial well-being. Thus, using aninstrument that measured breaks in normal function and presence of emotionaldistress and reduction in depressive symptoms to enable comparison with similarstudies may not have been an ideal choice. Including a measure that targeted thepositive concept of well-being more directly, such as the Warwick–Edinburgh MentalWell-being Scales, could have strengthened the study. This scale was developed toenable the measuring of mental well-being in the general population and to enablethe evaluation of interventions that aim to improve mental well-being.Based on the definition of psychosocial well-being used in the development of thisintervention, including outcome measures that assess participation in meaningfulactivities may have added important data to evaluate the outcomes of theintervention. The lack of such an outcome measure was a limitation to this study.Additional outcome measures for participation and well-being should be explored infuture research.14 The trial wasconducted in a rigorous manner following the Consolidated Standards of ReportingTrials (CONSORT) statement.49A strength in this study was the systematic development and feasibility testing ofthe intervention prior to full-scale effectiveness tests in this RCT.32 was an important advantage in documenting the trial implementation and inunderstanding trial outcomes.In addition, the comprehensive process evaluation, including the evaluation ofimplementation fidelity that was conducted alongside the trial,All intervention providers and assessors participating in the study were required tocomplete training prior to their participation, which was important in establishinguniform delivery of the intervention and the assessment interviews. Completingintervention sessions with parallel goals of individualization and uniform deliverymay, however, have been a limitation in this study. Participating in supervisionsessions was voluntary for intervention providers, and the follow-ups of theassessors were also based on a voluntary and as-needed basis. In retrospect,mandatory follow-up and supervision may have been warranted to assure uniformdelivery of the intervention and uniform assessment across the study trajectory.19 The nurses and occupational therapists who enrolled participants reportedthat it was difficult to assess whether patients with aphasia were able to consent.Ensuring an informed consent was perceived to be too time-consuming in the clinicalsetting, resulting in few participants with aphasia.Another limitation in this study was the difficulties in enrolling patients with moresevere stroke symptoms and aphasia who were presumably more vulnerable topsychosocial problems. However, the sample included in this study represents thelargest group of stroke patients admitted to hospitals in Norway.Furthermore, enrolment personnel found it difficult to approach the patients withmore severe stroke during the short time that they were treated in the stroke unit.These challenges emphasized the need for dedicated personnel who were not involvedin other clinical duties while simultaneously enrolling patients to the trial. Forfuture studies, it may be advisable to enroll patients directly from the communityand from rehabilitation units providing subacute care to reach a broader group ofpatients with more severe impairments.This study showed that certain subgroups  were prone to lower levels ofpsychosocial well-being. The results also support the notion that a higher sense ofcoherence is important in the promotion of psychosocial well-being. This may informinclusion criteria and screening for certain vulnerabilities when enrollingparticipants in future research.The results in this study suggest that more research is needed to explore therelationships between psychosocial well-being, sense of coherence, and the processof meaning-making and adjustment following an acute stroke. Additional mechanisms,such as the impact of resilience should be taken into account. Furthermore,exploring these relationships must include the use of more adequate instruments tomeasure psychosocial well-being.With respect to clinical practice, there is insufficient evidence to support theimplementation of the intervention in its current form based on the outcome measuresused in this RCT. However, the inclusion criteria in this study may have been toowide, and further research is needed to confirm whether certain subgroups of strokepatients may benefit from such a psychosocial intervention and at what timepost-stroke such an intervention may be appropriate.The dialogue-based intervention implemented in this RCT did not lead tolower levels of emotional distress and anxiety at 12 monthspost-stroke.The intervention did not lead to higher levels of health-related qualityof life or higher sense of coherence at 12 months post-stroke.Based on the outcome measures used in this study, there is insufficientevidence to support implementation of the intervention in its currentform.Click here for additional data file.Supplemental material, SupplementalFile-1-ScoringGHQ-28 for The effects of adialogue-based intervention to promote psychosocial well-being after stroke: arandomized controlled trial by Line Kildal Bragstad, Ellen Gabrielsen Hjelle,Manuela Zucknick, Unni Sveen, Bente Thommessen, Berit Arnesveen Bronken, RandiMartinsen, Gabriele Kitzmüller, Margrete Mangset, Kari Johanne Kvigne, KaterinaHilari, C Elizabeth Lightbody and Marit Kirkevold in Clinical RehabilitationClick here for additional data file.Supplemental material, SupplementalFile-2-Imputation for The effects of adialogue-based intervention to promote psychosocial well-being after stroke: arandomized controlled trial by Line Kildal Bragstad, Ellen Gabrielsen Hjelle,Manuela Zucknick, Unni Sveen, Bente Thommessen, Berit Arnesveen Bronken, RandiMartinsen, Gabriele Kitzmüller, Margrete Mangset, Kari Johanne Kvigne, KaterinaHilari, C Elizabeth Lightbody and Marit Kirkevold in Clinical RehabilitationClick here for additional data file.Supplemental material, SupplementalFile-3-LMM for The effects of a dialogue-basedintervention to promote psychosocial well-being after stroke: a randomizedcontrolled trial by Line Kildal Bragstad, Ellen Gabrielsen Hjelle, ManuelaZucknick, Unni Sveen, Bente Thommessen, Berit Arnesveen Bronken, RandiMartinsen, Gabriele Kitzmüller, Margrete Mangset, Kari Johanne Kvigne, KaterinaHilari, C Elizabeth Lightbody and Marit Kirkevold in Clinical RehabilitationClick here for additional data file.Supplemental material, Suppl_Table_S1 for The effects of a dialogue-basedintervention to promote psychosocial well-being after stroke: a randomizedcontrolled trial by Line Kildal Bragstad, Ellen Gabrielsen Hjelle, ManuelaZucknick, Unni Sveen, Bente Thommessen, Berit Arnesveen Bronken, RandiMartinsen, Gabriele Kitzmüller, Margrete Mangset, Kari Johanne Kvigne, KaterinaHilari, C Elizabeth Lightbody and Marit Kirkevold in Clinical Rehabilitation


In this paper, we study the optical trapping of anti-reflection core-shell microspheres by regular Gaussian beam and several structured beams including radially polarized Gaussian, petal, and hard-aperture-truncated circular Airy beams. We show that using an appropriate anti-reflection core-shell microsphere for the optical trapping by several structured light beams can dramatically enhance the strength of the trap compared to the trapping by the common Gaussian beam. The optimal core-shell thickness ratio that minimizes the scattering force is obtained for polystyrene-silica and anatase-amorphous titania microspheres, such that the core-shells act as anti-reflection coated microspheres. We show that the trapping strength of the anti-reflection coated microparticles trapped by the common Gaussian beam is enhanced up to 2-fold compared to that of trapped uncoated microparticles, while the trapping of anti-reflection coated microparticles, by the radially polarized beam, is strengthened up to 4-fold in comparison to that of the trapped uncoated microparticles by the Gaussian beam. Our results indicate that for anatase-amorphous titania microparticles highest trap strength is obtained by radially polarized beam, while for the polystyrene-silica microparticles, the strongest trapping is achieved by the petal beam. Common OTs is a single Gaussian laser beam that is highly focused at the diffraction-limited spot size by utilizing a high numerical aperture (NA) objective lens. A steep potential well of light intensity at the focus pushes the micro-, nano-particles towards the focal spot, and leads to the formation of a stable optical trap. This radiation pressure exerted on a particle is the consequence of the momentum-transfer of light to the particle15. The force experienced by particles is Hookian force  is easier than obtaining adequately strong axial optical traps, and therefore the latter has been a challenging issue in the optical trapping investigations18.Optical tweezers (OTs)23. Examples include using circular Airy beam for enhanced trapping efficiency of Rayleigh particles24, optical trapping with Laguerre–Gaussian modes with improved axial optical forces exerted on the dielectric particles25, optical trapping with cylindrical vector beams21, creation of enhanced trapping forces with complex-valued Elegant Hermitte- and Laguerre-Gaussian laser beams compared to standard Gaussian beams26, and effective multiple optical trapping with petal beams27 . On the other hand, the optical and physical properties of the intended particle play an effective role in optical trap enhancement30. For instance, using hollow gold and silver nanoparticles as handles in the OTs experience stronger optical forces31, the effect of resonant mode interference on trapping of gold core-silicon shell nanoparticles have been investigated32, optical forces exerted on graphene-coated dielectric particle under the illumination of an arbitrary optical beam have been investigated, theoretically33, and plasmonic effects of gold-coated black silicon have shown enhancement of the efficiency of OTs34.Optical trap stiffness can be enhanced by either using specific beam shapes or appropriate beads with engineered optical properties, in such a manner to boost the gradient force or to suppress the scattering force. On the one hand, the use of shaped laser beams enhances the trapping forces via minimizing the light scattering35, viscosity measurements36, thermo optical switches37, and biocompatible drug delivery38. Coated microspheres are shown to result in stronger traps40, and therefore they have been used as handles in OTs40. A core-shell microbead may gain anti-reflection property, with proper choices of materials, and a suitable core-shell diameter ratio, such that the scattering field from the particle is dramatically decreased, and hence a rather strong optical trap is formed41. So far, anti-reflection coated polystyrene (PS)-silica and titania (anatase and amorphous) core-shells have been utilized in optical trapping by linearly polarized Gaussian beam, that yielded a few nano-newton optical trap forces44.Dielectric microspheres have intriguing applications in sensing46, petal beam (PB)47, and hard-aperture-truncated circular Airy beam (CAB)50, and compare them with those of a Gaussian beam commonly used in OTs. Titania core-shell microspheres in two phases (anatase core and amorphous shell), are great candidates to be used in OTs to achieve nano-Newton forces with trap stiffness greater than 43, we use a 51 along with the generalized Lorenz–Mie theory (GLMT), and Maxwell stress tensor52 to numerically calculate the optical forces on the anti-reflection-coated microspheres.In this paper, we bring together both structured beams and anti-reflection coated microspheres to be used in OTs. We calculate optical trapping forces exerted on core-shell microspheres of PS-silica and anatase-amorphous titania, under illumination by several structured beams such as radially polarized beam (RPB)In this section, we briefly discuss the properties of the anti-reflection-coated microspheres. Additionally, the mathematical formulations of the intended structured beams are introduced. Finally, we present the physical and mathematical basis for the calculations of the optical forces under the formalism of GLMT.R, respectively. we can use the refractive index of the anti-reflection material for a single anti-reflection layer of a planar surface for a shell in spherical geometry44 which is:43.Structure of anti-reflection core-shell microspheres of PS-silica and titania are illustrated in Fig. mentclass2pt{minim45x, y are the Cartesian coordinates in the transverse plane.Radially polarized beam (RPB) is categorized as a cylindrical vector beam in which the polarization direction is radially symmetric in a lateral plane of a cylindrical coordinate system. This symmetrical polarization distribution results in a polarization singularity on the beam axis which leads to a dark spot on the axis. RPB can be expressed as a superposition of two orthogonal lowest order Hermite–Gaussian modes with orthogonal polarizations (l) and zero radial topological charge (47) because of the petal-like field distribution in the lateral plane  is categorized in a new family of beams known as abruptly autofocusing beams, possessing a special capability of autofocusing without an external element. The electric field envelope of CAB at an initial propagation distance ; In OTs setups, an optical beam is tightly focused by a high numerical aperture (NA) oil-immersion objective lens. The beam passes through a lam (coverglass) and enters the trapping medium (water), as shown in Fig. entclass1pt{minimaentclass1pt{minima by Eqs. , transforlambda for GB (first column), RPB (second column), PB (third column), and CAB (fourth column), respectively, in the presence of the aberration. In the lateral direction, on the other hand, the intensity distributions are less affected by the spherical aberration. The lateral FWHMs are approximately Figure 58:58:59:k is the wave number in the ambient medium).To calculate the net optical force exerted on a particle, it is necessary to calculate scattered fields from the particle. The GLMT is used to calculate the scattered fields from the trapped particle. In this method, the incoming and scattered fields are expanded in terms of vector spherical harmonics57:S represents a surface encircling the particle, and S, respectively. c and The optical force exerted on the particle can be calculated by numerical integration over time-averaged Maxwell stress tensorHere, we summarize the results of our calculations based on above mentioned theoretical foundations for uncoated uniform and anti-reflection-coated microparticles. In our calculations, the GB, RPB, PB and, CAB are used for optical trapping. The trapping wavelength and the power in the entrance pupil are assumed to be 60.First, we report the calculation results for uniform microspheres of PS, silica, amorphous-, and anatase-titania with refractive indices outlined in Table see Fig. i. In thesee Fig. j. Among Anti-reflection coating on the microspheres reduces the scattering fields that destabilize the optical traps. Figure The most pronounced message of Fig. Although, Fig. Figure 43. In order to compare our results, the maximum reported lateral and axial trap stiffnesses in their work are Looking at Fig. In conclusion, it is shown that the core-shell microspheres have a great potential to enhance the optical trapping strength. With a proper core-shell thickness ratio, microparticles exhibit an anti-reflection property that dramatically reduces the scattering force and leads to stronger optical traps. Moreover, utilizing the structured light beams in place of the Gaussian beam to trap the core-shell particles result in rather stronger optical traps. We showed that the titania anti-reflection-coated microspheres provide stronger trap stiffness than the PS-silica particles in both axial and lateral directions. Additionally, using a proper core-shell ratio for both PS-silica and anatase-amorphous titania microspheres, the structured light beams provide stronger traps in the axial direction compared to the GB. For instance, for the PS-silica microsphere, the axial trapping stiffness of the PB is two times greater than that of the GB, while for the titania core-shell microspheres, the axial trapping stiffness of the RPB is


Expansion microscopy (ExM) is an emerging super-resolution imaging technology. ExM works by infusing a biological specimen with a superabsorbent hydrogel, followed by mechanical homogenization and isotropical expansion of the specimen in water. The unique and cost-effective process of ExM enables super-resolution optical imaging of sample of interest without the need to invest and use of a sophisticated microscope instrument. Here, we demonstrate that a nearly 3-fold isotropic physical expansion of E.coli fixed cells can be achieved in PBS, and the cell morphology during binary fission is clearly resolved in the expanded state, using a diffraction-limited microscope. The fundamental of the physical expansion of biological specimens is to infuse fixed samples with the gelling solution consisting of monomers, followed by in situ polymerization for the formation of a tissue hydrogel hybrid. The gelling solution consists of sodium acrylate, a monomer that confers superabsorbent properties, along with co-monomer acrylamide, cross-linker N-N′-methylene-bisacrylamide, accelerator tetramethylethyl-enediamine, and initiator ammonium persulfate, to trigger the free-radical polymerization. After homogenizing the mechanical properties of the sample-gel composite, isotropic expansion can be achieved after iterative washes with pure water. The extent of expansion, which is defined as expansion factor, can be controlled by varying the salt concentration of the solution, where the maximum expansion factor is reached with pure water.Expansion microscopy (ExM) is a newly emerged imaging technique that seeks to overcome the everlasting hindrance of the optical imaging system, the diffraction limit. Different from the other super-resolution microscopy types (for example Structured illumination microscopy (SIM), Stochastic optical reconstruction Microscopy (STORM) or Stimulated emission depletion microscopy (STED)), ExM provides the solution to achieve nano-scale resolution (~70 nm) through the physical expansion of biological samples, independent of an intricate imaging system2. The protein-retention ExM (proExM) later managed to preserve antigens in the gel via modification of the amines on proteins with an acrylamide functional group that participate in the free-radical polymerization for anchoring3. Successful expansions have been performed among a wide range of cell lineages, and tissue types4, showing the potential of ExM to facilitate the basic science research and clinical sample diagnosis.Parameters in the steps of gelation and homogenization and the strategies for presenting target molecules have been continually improving to accommodate a broader range of applications. The first version of ExM requires the unique design of the fluorescent labels that can directly incorporate to the polymer network to survive the homogenization with protease KE. coli) is one of the most studied gram-negative bacteria. E. coli possesses a cell wall composed of a thin peptidoglycan layer (2-4 nm) and an outer membrane layer. The peptidoglycan polysaccharide chain is composed alternatively from N-acetylglucosamine and N-acetylmuramic acids and is further crosslinked by tetrapeptides, exhibiting rigidity for maintaining cell shape and providing protection from osmotic lysis5. In a recent study, application of ExM on imaging E. coli cells has been reported in combination with a miniaturized microscope6, using the first generation ExM2.Owing to the small size, microbial cells, such as bacteria and fungi, are challenging to study using conventional optical microscope. Nanoscale optical imaging of microbial cells can potentially open new doors to experimental observation of novel biological structure with molecular contrast, which is not available with electron microscopy. Escherichia coli  and stained with DAPI  and a lectin wheat germ agglutinin (WGA) to visualize its nucleus and polysaccharide-containing cell structure, respectively . Images were allacquired with a Nikon Ti2 eclipse fluorescence microscope, with a CSU-W1 confocal module under a long working distance water immersion 40× objective  = 1.33, confocal microscope). Without expansion it is hard to clearly distinguish single cells and both DAPI and WGA stains overlapped within a rod shape . The treatment of enzymatic homogenization allows isotropic expansion of the gelled E. coli cells. Approximately 3-fold expansion factor (2.8) was achieved in PBS . After expansion, the cell morphology during binary fission is clearly resolved while the overall distortion compared to pre-expansion state is minimal . Further expansion is achievable with pure water, if additional resolving power is needed.Here, we demonstrate the application of ExM on imaging


The rapid advancement of the COVID-19 pandemic has prompted an accelerated pursuit to identify effective therapeutics. Stages of the disease course have been defined by viral burden, lung pathology, and progression through phases of the immune response. Immunological factors including inflammatory cell infiltration and cytokine storm have been associated with severe disease and death. Many immunomodulatory therapies for COVID-19 are currently being investigated, and preliminary results support the premise of targeting the immune response. However, because suppressing immune mechanisms could also impact the clearance of the virus in the early stages of infection, therapeutic success is likely to depend on timing with respect to the disease course. Azithromycin is an immunomodulatory drug that has been shown to have antiviral effects and potential benefit in patients with COVID-19. Multiple immunomodulatory effects have been defined for azithromycin which could provide efficacy during the late stages of the disease, including inhibition of pro-inflammatory cytokine production, inhibition of neutrophil influx, induction of regulatory functions of macrophages, and alterations in autophagy. Here we review the published evidence of these mechanisms along with the current clinical use of azithromycin as an immunomodulatory therapeutic. We then discuss the potential impact of azithromycin on the immune response to COVID-19, as well as caution against immunosuppressive and off-target effects including cardiotoxicity in these patients. While azithromycin has the potential to contribute efficacy, its impact on the COVID-19 immune response requires additional characterization so as to better define its role in individualized therapy. Azithromycin is administered to over 40 million patients annually for its antibacterial activity , but chaThe emergence of severe acute respiratory syndrome (SARS)-coronavirus 2 (CoV-2) has thrust azithromycin into the spotlight due to early reports of improved outcomes in patients treated with azithromycin and hydroxychloroquine . The immThe clinical observations and data from COVID-19 patients support this premise. Many therapies are being investigated that suppress the overactive immune response , but theCoronaviruses (CoV) are enveloped, single stranded RNA viruses capable of infecting a range of hosts including humans, with the novel CoV's resulting in potentially fatal lower respiratory tract infection . The thrCOVID-19 has loosely been characterized to be comprised of 3 immunological stages. In the first stage, an interferon response coordinates the control of viral replication. The second stage is characterized by suppression of interferons by the virus, leading to lung damage and progressive disease. Some patients progress to a third stage of hyperinflammation coordinated by excessive macrophage activation and coagulation . In lateThe host response is therefore implicated as a pathologic factor of disease progression. Animal models of SARS infection demonstrate that lung inflammation worsens after viral clearance and peaks several days later . This suMultiple clinical trials are currently being conducted that include agents that suppress immune function, with some being used clinically despite the lack safety or efficacy data. Many of the inflammatory mechanisms being targeted in these trials are associated with macrophage activation or macrophage effector functions . These tin vitro . Patients with CF suffer from chronic lung inflammation due to immune dysregulation and thickened mucus in the lungs caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene that impacts proper chloride ion transport . A seriepatients .Staphylococcus aureus and P. aeruginosa infections, and with the exception that use of the drug was associated with a higher number of infections with macrolide-resistant S. aureus, the risk for acquiring all other infections was not impacted  , 125 andhromycin . Similarhromycin , 133.Clinical studies also reflect the impact of azithromycin therapy on macrophage polarization in patients with inflammatory diseases. Azithromycin administration to patients with COPD led to an increase in expression of the M2-associated protein MR, along with reduced airway inflammatory cytokine concentrations and improved phagocytic function of alveolar macrophages . And in In vitro azithromycin exposure of human neutrophils modulates neutrophil extracellular trap (NET) release  cells. NK cells are innate effector lymphocytes that are important in the response to viral infections. ell line . AlthougAzithromycin also can dampen the lymphocytic airway inflammation associated with BOS after lung transplantation. IL-17 is important in the pathophysiology of allograft rejection, during which production of the cytokine by CD8+ T cells drives a lymphocytic airway inflammation , 150. CoWhen azithromycin polarizes macrophages to an alternative-like phenotype, this includes a dramatic increase in the production of the M2 protein arginase-1 , 57. ArgAutophagy, the catabolic process that regulates the elimination of defective intracellular proteins and components , also plUpon exposure to CoV, the innate immune response is activated through standard anti-viral signaling cascades initiated through toll-like receptors (TLR), NOD-like receptors (NLR), and others, which result in signaling through pathways to activate downstream transcription factors including IRF3 and NF-κB . TypicalSome patients infected with novel coronaviruses, however, fail to mount a robust initial response. This is thought to be due to evasive mechanisms of SARS-CoV, MERS, and SARS-CoV-2 that suppress early type I interferon production. SARS-CoV suppresses the activation of TNF receptor-associated factors (TRAF) 3 and 6, which suppresses early type I interferons and pro-inflammatory effectors including IL-6 and TNFα . The virAdditionally, activated CD8+ cytotoxic T lymphocytes and NK cells are important for viral clearance, and both of these cell types have been found to be significantly decreased in patients with more severe COVID-19 , 185. AlThe established ability of azithromycin to up-regulate the production of type I interferons in response to rhinovirus and Zika virus provides a foundation for investigating this mechanism in SARS-CoV-2 infection –44. InteThe pathologic drivers of disease in patients infected with SARS-CoV-2 are not completely understood, but it is clear that severe disease is not only related to viral load. The dysregulated, excessive inflammatory response is thought to be a major driver of disease severity and death in patients with COVID-19 –29, 190.Pathologic inflammation in patients infected with novel coronaviruses is driven by high numbers of neutrophils, monocytes, and macrophages in the airways . Common The role of regulatory macrophage functions in response to SARS-CoV-2 is not well-understood, although data is beginning to emerge. A subset of macrophages from COVID-19 patients has been described as expressing a gene signature associated with tissue repair . WhetherHowever, animal studies of SARS-CoV demonstrate that M2 polarization and increased arginase-1 activity could be detrimental. In a mouse model of SARS-CoV infection, investigators demonstrated that alternatively activated macrophages were responsible for enhancing the pulmonary pathology . PreviouThe autophagy-lysosomal system plays a central role during infection with SARS-CoV , 208. HoThe ability of azithromycin to blunt macrophage-driven neutrophil influx lends promise to the drug's potential impact on patients infected with SARS-CoV-2. Recent reports concerning the immune response in COVID-19 have characterized extensive neutrophil infiltration into diseased lung tissue as well as significant evidence of NET release in the serum , 212. MuDespite their depletion in COVID-19, lymphocytes appear to contribute to the macrophage hyperactivation that leads to the development of cytokine storm, a state in which pro-inflammatory cytokines drive excessive, damaging inflammation. Systemic cytokine profiles in patients with COVID-19 are similar to those observed in cytokine release syndromes often driven by macrophages, including high levels of circulating IL-6, IL-17, TNFα, CCL2, and CXCL10. These cytokines will induce T cells and NK cells to produce additional cytokines including GM-CSF and IFNγ. An abundance of peripheral blood CD14+CD16+ monocytes has also been described in COVID-19 patients with severe disease to produce high amounts of IL-6 , 221. Inin vitro, which links lymphopenia to hyperinflammation (The severity of disease for MERS-CoV, SARS-CoV, and SARS-CoV-2 has also been shown to positively correlate with levels of IL-17 and other Th17 cell-related pro-inflammatory cytokines , 224. A ammation . As discammation . If azitammation , 52, 61.Based on the antiviral and immunomodulatory mechanisms presented, and based on the limited clinical evidence of its impact on viral clearance, the thorough evaluation of azithromycin as a possible treatment for patients with COVID-19 is warranted. The ability of azithromycin to impact the production of inflammatory mediators through its inhibition of NF-κB and other pro-inflammatory signaling pathways suggests the potential for benefit in counteracting the hyperinflammatory state that manifests through neutrophil influx, lung inflammation, cytokine storm, and hypercoagulation. It is likely that these immunomodulatory effects will be beneficial in patients infected with COVID-19, but careful evaluation of when to utilize the drug based upon current viral burden and immune status is critical. This approach has also been proposed in a recent communication published in The Lancet in which the authors recommend that patients with COVID-19 should be screened for hyperinflammation in order to identify the subgroup that may benefit from immunomodulatory or immunosuppressive therapies .In conclusion, the immunomodulatory effects of azithromycin are complex and multifactorial. Impacting macrophage polarization, autophagy, and cytokine release will likely be beneficial in a subgroup of patients, but any treatment that impacts immune function should be used with caution in patients with an active infection. Until regulatory aspects of macrophage function are better defined in the setting of COVID-19, and until azithromycin and other therapies are properly evaluated in randomized clinical trials in defined patient populations, extreme caution should be exercised when utilizing azithromycin in these patients.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.VV, AA-L, JG, and DF have a patent pending for an azithromycin formulation to modulate immune responses. The remaining 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.


Scientific Reportshttps://doi.org/10.1038/s41598-019-57330-w, published online 15 January 2020.Correction to: This Article contains an error where the Discussion section has been inadvertently omitted. As a result of this, 19 references were also omitted. The correct Discussion section appears below.1, we demonstrated that PD images corrupted by motion could be noticeably improved by motion correction. However, this is feasible only if the thyroid motion in-plane that can be reliably tracked using ultrasound speckle tracking techniques. Anatomically, thyroids glands are intrinsically bound by a rigid trachea and a pulsating carotid artery at opposite ends, which influences its motion characteristics and its scope for correction across different imaging planes. The goal of this pilot study is to investigated the efficacy of motion correction for thyroid microvascular imaging across the transverse and longitudinal cross-sections.Carotid pulsations can significantly degrade the visualization of vascular pathway in the thyroid, especially of small blood vessels. In a recent publication2. However, in-plane motion can be reliably tracked in terms of axial and lateral components, and subsequently corrected. Whereas, elevational motion leads to out-of-plane excursions that cannot be accounted using 2D ultrasound. This was also observed in the CNR and SNR barplots in Fig. 9, the improvements in signal quality was higher in the longitudinal direction compared to transverse. Although absolute CNR and SNR varied across patients and cross-sections depending on flow intensity of the chosen vessel, attenuation at the imaging depth, and size of the acquired Doppler ensemble, however, improvement in PD image quality was more prominent in the acquisitions obtained from the longitudinal view. This is consistent with the observation that ensemble correlation coefficient was also higher in the longitudinal plane, suggesting motion was reliably tracked for subsequent correction. Ideally, in the absence of motion, we don’t expect one imaging cross-section to display higher microvascular signal quality than other. However, in this paper, comparison of absolute CNR and SNR of the same vessel across the two imaging planes was not possible without real-time 2D imaging capabilities for vessel registration or 3D volumetric imaging. Therefore direct comparison of absolute estimates of CNR and SNR across longitudinal and transverse cross-section may not be appropriate.The preliminary results obtained on 24 patients, involving 48 image acquisitions demonstrate that tracking of thyroid motion in the longitudinal view was more reliable than than in the transverse. In-plane thyroid motion was primarily visible in the longitudinal view , owing to the distal presence of rigid trachea that restricted elevational motion. Further, thyroid motion in the longitudinal plane was associated with a strong lateral component of displacement . Reciprocally in the transverse view, presence of trachea limited lateral motion  but increased out-of-plane incursions [Figs. 5–7]Cross-correlation coefficient has been commonly used in strain elastography to detect speckle decorrelation, which is typically occurs due to (1) out-of-plane motion or (2) at very large strains (> 10%). The correlation coefficient associated with frame-to-frame tracking was consistently > 0.97, regardless of the imaging cross-section, which was specifically due to high frame-rate of imaging. However, results obtained from matching the reference frame with other ensemble frames displayed a steep decay in cross-correlation coefficient in the transverse view than in the longitudinal view [Fig. 5]. This observation was consistent across all the 48 acquisitions [Fig. 5], and the pooled distribution demonstrated that the normalized ensemble cross-correlation—which is indicative increased speckle decorrelation—was significantly higher (p < 0.05) in the longitudinal view than in the transverse, in the later.3. This is based on the anatomical orientation of the thyroid with respect to the rigid trachea and the pulsating carotid artery, and its surrounding tissue. With recent developments in motion-robust clutter-filtering6, a suitable investigation to assess the impact of imaging cross-section on feasibility of thyroid microvasculature imaging can be useful in successfully translating it to the clinic for non-invasive diagnosis. An important question is: What are the disastrous consequences of imaging thyroid microvasculature with increased out-of-plane motion? Three obvious consequences are that this could (i) substantially hamper any efforts in motion tracking and correction, in the absence of 3D ultrasound imaging that is currently at a very nascent stage of development and implementation7; (ii) would require advanced performance metrics for real-time assessment of the coherence of the acquired data to identify frames subjected to out-of-plane motion, which must be discarded prior to slow-time PD integration8. Further, discarding frames from the Doppler ensemble will inadvertently hamper the quality of the PD image, especially since flow signal in ultrasound images are only marginally higher than the noise floor1; (iii) would complicate implementation of real-time imaging—a crucial feature of 2D ultrasound—for comprehensive assessment of the thyroid microvasculature in the entire nodule9.In strain elastography, WFUMB guidelines recommend imaging of the thyroid in the longitudinal view10. Further, the angular configurations of compounding plane wave imaging is also an important factor that determines the efficacy of the motion tracking, especially in the lateral direction10. Additionally, the capability of ultrafast imaging is currently available in limited high-end ultrasound systems. The motion corrected PD microvasculature imaging technique can be valuable in improving the performance of mid-range clinical scanners that multiplex repeated acquisitions due to reduced number of receive channels, which can affect imaging frame-rate. Specifically, this study was conducted using an Alipinion E-Cube 12R clinical ultrasound system, which acquired received data of 128 channels with only 64 elements at a time, therefore reducing the imaging frame-rate by half. Even at high imaging frame-rate, motion correction can be valuable in acquiring and concatenating Doppler ensembles to produce an ultra-long, coherent ensemble than can substantially boost the signal strength of small vessels at depth, which may get degraded due to ultrasound attenuation.High frame-rate imaging is expected to reduce the impact of motion on thyroid microvasculature imaging. However, size and location of the tumor can impact depth of imaging, which is inversely proportional to frame-rate. Further, multi-angle plane-wave insonifications required for compounded plane wave imaging also is an important factor that decides the image quality, and influences the frame-rate. Increasing the number of angular insonification can considerably reduce the impact of grating lobes, and improve the signal-to-noise ratio of the received signal14. The in-plane axial and lateral displacements estimated across the 48 cross-sections varied across subjects depending on factors such as the size and stiffness of the nodule and its bonding with the surrounding, it location in the thyroid and its proximity to the carotid artery [Fig. 8]. Further, the amplitude of the carotid pulsation that depends on pulse pressure and the stiffness of the artery, will also determine the displacements incurred by the thyroid nodule15. Further, the axial and lateral motion estimated in the thyroid nodule using the 2D normalized cross-correlation were similar to those reported in carotid elastography studies17.Conventionally, lack of phase information in the direction perpendicular to ultrasound beam propagation makes estimation of lateral motion very challenging. Several studies have been conducted to advance tracking and beam-forming techniques to improve the robustness of lateral motion tracking using ultrasound6, including contrast-enhanced ultrasound blood flow imaging19. In our future work, we will address this limitation by performing motion correction adaptively in local overlapping windows that can be reasonably assumed to have uniform displacements.A limitation of our current study is that a first-order global motion correction approach was used for correcting thyroid motion, under the assumption of rigid body translations, which may not be accurate. However, since the variance in frameto- frame displacements obtained in the target nodules [Figs. 3  and 4 ] were observed to be low, we used such an approach for this pilot investigation. Similar motion correction techniques based on rigid-body assumption have also been used in previous studies