In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.  Chronic lymphocytic leukemia (CLL), the most prevalent leukemia among individuals of European descent, is a clonal disease of human B lymphocytes expressing membrane CD5 , 2. The Consistent with CLL being a disease of B lymphocytes, the structure and antigen-binding properties of the immunoglobulins (Igs) on the leukemia cell membrane are fundamental for disease development, evolution, and response to therapy . In thisAdditionally, CLL Igs bind a diverse spectrum of antigens, spanning foreign and autologous molecules , 16. It Despite the accumulated knowledge regarding the unique features and clinical importance of the BCR in CLL, much less is known about the mechanism(s) whereby BCRs influence disease development and progression. For example, although most CLL B cells express both IgM and IgD, the physiologic relevance of the 2 isotypes is unclear. Indeed, since the IgM and IgD BCR isotypes bind the same antigen and the loss of function does not drastically perturb B cell development , 22, theUsing samples from CLL patients whose in vivo leukemia cell birth rates were directly measured , 30, we n = 44) and U-CLL (n = 21) types (https://doi.org/10.1172/JCI149308DS1). In agreement with D’Avola et al.  of CLL patients grouped by high and low IgM levels based on a receiver-operating-characteristic (ROC) curve, defining an empirical MFI cutoff of 70 . Notablylo cases , IgDhi a in TTFT . Using t in TTFT . Similar in TTFT , E and F+ CLLs, a subset of patients with enhanced disease aggressiveness . This revealed significantly earlier treatment for patients with DvM of 1.15 or lower, in contrast to those with DvM greater than 1.15 , G and Hsiveness , 33, whed ZAP70– . Collectd ZAP70–  and sugg2H incorporated into newly synthesized DNA of CD19+CD5+ cells  to study the Ig membrane features of the CLL cells in the 2 cohorts , A–G. IFhenotype  and collhenotype . Based ohenotype , C and Dhenotype . Moreovehenotype  and meashenotype .+CD5– B cells displayed comparable IgM and IgD spot counts and spot areas  among CLL clones grouped based on their leukemic cell BRs, the latter being an indicator of disease aggressiveness , 36. EacTogether, the above data suggest the density and the topography of IgM associate better with CLL B cell proliferation in vivo than IgD.2 fragments of goat anti–human IgM and IgD], we measured the absolute number of IgM and IgD BCRs on CLL B cell membranes embranes , A–G. Quembranes  were incembranes . The IgMembranes . In geneembranes , C and Dembranes . Also coembranes , left. Iembranes , right. rability . Thus, tSince CLL cells express a membrane phenotype resembling chronically stimulated B cells , we askeBecause our data support a link between IgM (not IgD) and BR  and betw2) and largest (>110 μm2) cells for each case. Notably, neither MCS nor the fraction of the smallest cells differed significantly between the BR subgroups  population  revealed a similar significant trend when the Low and High BR groups were compared; the Intermediate (Int) group did not differ significantly from the others, consistent with a progressive, linear relationship between BR and metabolic parameters . This wasubgroup . Hence, ++ influx upon BCR signaling  or heterozygous (IgM+/–) KO animals or mice lacking the TCL1 transgene showed this increase  of the reported IGHVs. In contrast, IgM–/– TCL1 did not express any of the IGHV genes overrepresented in normal and autoimmune B-1 and CLL-like B cells. Finally, IgM–/– mice displayed a polyclonal population and a different IGHV usage with respect to IgM+/+ TCL1 as well  and for IGHV usage. The IgM–/– TCL1 CD19+CD5– B cell expansion is likely due to the genetic pressure of TCL1 overexpression, leading to the formation of other lymphoma/leukemia in these mice. This further supports the notion that lack of IgM expression does not allow CLL-like cell development.When comparing lymphocyte subpopulations in the spleens, we found decreased numbers of CD19CL1 mice . Conversulations . The latIGHV1-55 , 45. Thu–/– mice , finding as well . Thus, t–/– TCL1 mice possess a CD19+CD5+IgD+ B cell population from which the leukemia could initiate. However, despite the presence of these cells, a CLL-like leukemia did not develop in these animals. Since only IgM BCRs from humans and mice with CLL can signal autonomously , effectively blocks BCR signaling in vitro  and in preatment , supportreatment .During in vivo ibrutinib treatment, we did not observe statistically significant changes in Ig features for both IgM and IgD (data not shown). This could be due to the point in the disease at which we tested, at which treatment started, and/or the duration of the treatment. In addition, the role that kinases upstream of BTK play in Ig reorganization is complex and unappreciated. Finally, the number of cases available for study might have been insufficient to define significant effects. Indeed, the changes in IgM and IgD organization are believed to be strongly associated with an “inside-out” Syk activity that modifies actin cytoskeleton and BCR organization upon engagement . Thus, iIn line with this, OCR showed an overall significant reduction in the 2 BR groups for basal respiration, ATP production, and maximal respiration . AdditioThus, inhibition of BCR signaling associates with a regression in cell size and metabolic profile in vivo, and the changes are proportionally greater for the larger and more metabolically active leukemic B cells. Collectively, our findings assign the effects solely to the IgM isotype.The leukemic B lymphocytes in CLL patients are highly dependent on signals delivered by the BCR for survival and growth. Since IgM and IgD initiate these signals, we studied the amount, distribution, and signal transmission capacity of the 2 isotypes, and then associated these with leukemic cell BRs in patients, a measure of disease activity, and with cell size and metabolic activity, consequences of cell activation and division. A strength of these studies is the use of samples from patients for whom clonal in vivo BRs had been measured. Because some patients’ BRs were measured just prior to starting iBTK monotherapy, we were also able to directly associate our findings before and when BCR signaling was inhibited.Using this approach, we documented that IgM and IgD BCRs on the same leukemic B cell are organized and function differently. On unmanipulated ex vivo cells, we found that IgM is present in greater amounts and is organized into more and larger high-density regions/spots than for IgD. Since higher clonal BRs directly associate with poor prognosis , our finWe also found that, despite IgM and IgD bearing identical antigen-binding domains, only IgM molecules mediate autonomous signaling in human and murine CLL B lymphocytes. Thus, in some manner, the IgM constant region leads to or allows Ig-Ig self-association that promotes and appears necessary for autonomous BCR signaling . Support+CD5+IgD+ B cells existed in these animals, suggesting that IgM expression is involved in the biased use of the specific IGHVs in B cells. Indeed, TCL1 mice expressing IgM mainly displayed IGHV11-2, IGHV12-3, and IGHV1-55, which are the most commonly used IGHVs in the restricted repertoires exhibited by normal and autoimmune B-1 cells and by CLL-like murine CD5+ B cells . BCR signaling and OXPHOS are directly linked in CLL, as evidenced by genetic ablation studies knocking out PI3Kδ , as well1 phase of the cell cycle increased IgM BCR levels and assembly into more frequent and larger high-density regions/spots, (b) faster in vivo BRs and enhanced cell size and heightened metabolic activity, and (c) increased IgM BCR levels and faster in vivo BRs with the types of BCR signaling. Regarding the latter, in vivo iBTK therapy, which we showed blocks autonomous as well as extrinsic antigen–mediated BCR signaling, led to significant decreases in cell size and clonal metabolic activity that were proportional with in vivo BRs. While inhibition of autonomous signaling by ibrutinib might be expected, this is the first formal report to our knowledge of such an action by ibrutinib and, thus, has implications for our understanding of CLL biology as well as how the drug might act as a therapeutic agent. These findings indicate that clonal metabolic activity is significantly influenced by cellular activation through the BCR, consistent with the activated membrane phenotype of CLL cells  and the ll cycle . Moreovell cycle , 53. Lasll cycle .Finally, it is crucial to understand the relative contributions of autonomous BCR signaling, mediated by self-association induced by binding to antigens intrinsic to the BCR, and classical BCR signaling induced by Ig binding to antigens extrinsic to the BCR, to the findings reported here and to link these to CLL B cell biology. Regarding autonomous signaling, the structures that allow this are inherent to IgM. Consequently, IgM BCRs always have the opportunity to self-associate and autonomously signal, regardless of the location in the body of an individual cell at any point in time. For classical BCR signaling, since both IgM and IgD engage the same extrinsic antigen, the 2 isotypes together initiate this form of signaling. However, in order to bind Ig-extrinsic antigens and thereby lead to cell stimulation and growth (as opposed to anergy), leukemic B cells should reside in solid tissues, e.g., lymph nodes (LNs) –57, wherThus, we propose the following chronologic scenario. CLL cell proliferation is initiated by engaging BCR-extrinsic antigen(s) (auto or foreign antigens) via IgM/IgD BCRs in proliferation centers of the stromal microenvironment , and thiAlthough this proposal stresses a role for autonomous signaling in the periphery, such stimulation would also occur centrally and likely alter responses and cell fates initiated by classical BCR signaling. In this regard, since productive IgD engagement requires multivalent antigens , which aFurther information can be found in Supplemental Methods.This study was designed to compare the membrane topography of the clonally restricted IgM and IgD BCRs on CLL cells and to assess the contributions their respective signaling played in the development and progression of the disease.n = 12 and 11, respectively; 2H) incorporated into newly synthesized DNA of leukemic cells replicating in vivo after the patients had drunk deuterated water (2H2O). Additional information about the 2 clinical trials and in vivo kinetics of these patients is available in Burger et al. and Messmer et al.  were sacrificed at the same time points.TCL1-transgenic mice , 44 were–/– mice  to obtaiP value) of patient TTFT was calculated using the log-rank test. For mouse populations and cell-size grouped analyses, Mann-Whitney test was used. Wilcoxon’s rank test was used for paired analysis (Ig clusters and cell size before and during treatment). Correlations were calculated with Pearson’s coefficient and lines fit with linear regression analysis. For multiple comparisons, 1-way ANOVA with Tukey’s correction was used. Statistical significance was defined by a P value of less than 0.05 (2-tailed). Data are presented as mean ± SEM, unless otherwise indicated.For statistical analysis, GraphPad Prism software was used. The statistical significance (Human studies were carried out in accordance with the Declaration of Helsinki and approved by the Institutional Review Boards of The Feinstein Institute for Medical Research and Freiburg University. All patients provided written informed consent. Animal experiments were performed according to institutional ethical allowance and in compliance with the guidelines of the German law, license number 1288, regional board Tübingen, Germany, and approved by the Institutional Animal Ethics Committee of Ulm University under permission number 1288.ANM, EGG, MDVM, PCM, PJM, HJ, and NC conceptualized the study. ANM, EGG, and MDVM developed the methodology. ANM, EGG, MDVM, and PCM performed validation studies, conducted formal analyses of the data, and prepared the figures. ANM, EGG, MDVM, PCM, TNT, AN, GF, SV, YL, and DB carried out the investigation. KRR, JAB, PJM, HJ, and NC provided resources. ANM, MDVM, HJ, and NC wrote the original draft of the manuscript. ANM, EGG, PCM, PJM, HJ, and NC reviewed and edited the manuscript. HJ and NC supervised the study. ANM, EGG, HJ, and NC provided project administration. HJ and NC acquired funding.


Introduction: The actions taken by the government to deal with the consequences of the coronavirus diseases 2019 (COVID-19) pandemic caused different levels of restriction on the mobility of the population. The need to continue offering physical exercise to individuals after stroke became an emergency. However, these individuals may have barriers to adhere to the programs delivered remotely. There is a lack of evidence related to adherence, attendance, safety, and satisfaction of remote exercise programs for this population.Objective: The aim was to evaluate adherence and barriers to attend a remote physical exercise program for individuals after stroke. We aimed (a) to identify adherence and attendance rate of the remote physical exercise program ; (b) to identify the safety of a remote physical exercise program ; and (c) to identify the overall experience to participate in a remote program.Materials and methods: This is a longitudinal study, including 36 stroke survivors who already attended a face-to-face physical exercise program prior to the COVID-19 pandemic. The remote physical exercise program included sessions for 2 days/week for a duration of 22 weeks, with a total of 44 sessions, which were delivered asynchrony via recorded video sessions. As outcome measures, we performed two questionnaires  to identify attendance, barriers, safety, and overall experience related to the program.Results: The adherence rate was 86 (9%). The attendance rate was 19, with a total of 8 sessions (108.3 min/week). The main barriers for lower attendance rate were as follows: lack of motor skills and physical fitness to workout in 80 reports (20.6%), followed by no exercise companion in 44 reports (11.3%). The remote physical program has been shown to be safe, and the overall experience was positive from the perspectives of the participants and the family members.Conclusion: Although the adherence rate was high, the attendance rate was low on the remote physical exercise program. The main barriers to attending the program remotely reflect the need of tailoring individually an asynchrony mode of delivering the sessions to individuals after stroke. Our results also indicate how the COVID-19 impacted the health conditions of stroke survivors. The program was safe, and the overall experience indicated a change in the mental, physical, and social health of individuals after stroke and their family members. Social distancing was applied to mitigate the effects of the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [coronavirus disease 2019 (COVID-19)], leading to mobility restrictions and suspending face-to-face services. Physical exercise facilities  were closed, constraining the physical activity for physical conditioning, therapeutic exercise, or leisure purposes. In particular, physical activity programs for individuals with chronic health conditions were suspended or were dramatically reduced. To overcome such limitations, alternatives based on remote-supervised activities emerged  to measure the adherence rate and attendance to a remote physical exercise program ; (b) to describe how safe a remote physical exercise program is ; and (c) to measure the overall experience to participate in a remote physical exercise program for individuals after stroke and their family members.This is a phase-I clinical trial, and the CONSORT checklist is used. It was developed at the Motor Behavior Laboratory of the School of Physical Education and Sport of the University of São Paulo, São Paulo, Brazil. We have the approval of the Human Ethics Committee . All individuals accepted to participate and provided their written informed consent.Individuals with a stroke diagnosis who attended the face-to-face community rehabilitation program at the School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil earlier were invited to join this study after the COVID-19 pandemic had started. The inclusion criteria were as follows: confirmed diagnosis of stroke by image or medical report; either types of stroke (ischemic and hemorrhagic) in chronic phase  of the participants were recorded previously in January and February, during the face-to-face physical exercise program, before the start of the COVID-19 pandemic in Brazil. Physical information from each participant, as a prerequisite to engaging in the face-to-face program, was obtained. The activity-specific balance confidence scale (ABC) Branco,  and the via mobile phone and email to all participants. The videos contained different exercises each day. Initially, the instruction on how to perform the exercise was given, such as execution time, perceived effort (intensity), the number of series, and repetitions given by the trainer. All exercises had different levels of intensity and complexity so that all participants were able to perform the exercises and adapt them to their own level. An exercise protocol was developed for this remote program maintaining the same objective as the face-to-face program: to reduce physical inactivity, to increase physical function, such as aerobic capacity, muscle resistance, mobility, balance, and gait, and to improve balance confidence and cognition  per week of performed exercise was also considered. Furthermore, we aimed to describe the higher and lower attendance frequencies, including the participants who attended more than 80% and <20% of the sessions.The working team for this remote exercise program was composed of a team leader (faculty member), a physical education instructor (faculty member), a health instructor team (four graduate students and an in-charge to supervise the intern team), and an intern team (20 undergraduate students). The entire team was trained and familiarized with how to apply the questionnaires. The intern team was in charge of weekly calls  with each participant, where it was asked how hard it was to perform the proposed activities. Although every participant had the same videos, those weekly calls were designed to adapt the sessions to personal limitations and needs.A digital consent term copy was sent via mobile phone and email to all individuals who were interested in joining this study. The research team called the potential participants to explain all the procedures to them and their family members or their caregivers. Then, those who agreed to participate replied with the informed consent option checked.Every week, the researcher team called each participant to talk about the barriers, how often he/she exercised, and how hard it was to exercise. Through a questionnaire using mostly yes-or-no answers, this tool assessed the perceived barriers . Safety was evaluated based on adverse sensations  during the session, fear, or insecurity to exercise, and adverse events such as falls. Participants also expressed their reasons not to do the remote physical exercise program in an open question. The perceived barriers were based on all the comments about how hard it was to exercise. These barriers were clustered within domains and counted.www.mentimeter.com)  using an 18-item questionnaire created by the authors. Participants answered 12 questions, while family members/caregivers answered the other 6 questions. After the phone call with the participant, the call was directed to the caregiver, who answered the corresponding questions in the questionnaire. The same caregiver answered these questions every month. A 5-point Likert scale was applied based on the positive and negative statements Likert, . The ansMean, mode, and median were used to depict data information. SD and 95% CI were used to describe data variability. We used Origin  for the plots and SPSS  for statistical analysis.The adherence rate was defined as the relative frequency of individuals who accepted to engage in the remote exercise program . Ten (25%) participants attended 80% or more sessions, and 13 (32.5%) participants attended <20% of the sessions.Based on the sessions attended, the active participants utilized 99.1 ± 19.4 min/week of physical exercise of the program.Pandemic refers to the changes in the daily routine due to the home office and the need to move out with the family to assure social isolation. The health-related factors include lack of physical capacity, impairments due to the stroke, and other health problems that may have been described as barriers to performing the remote physical exercise program .To illustrate the most cited health conditions-related barrier , we described the following statements:I have difficulty to perform the exercises using my affected upper limb. Although I see the staff member performing the exercise on the video, I cannot do the same. I have difficulty going to the mat and exercise on the floor. I feel I cannot stand up safely by myself. It is difficult to stand up.”“To illustrate the environmental-related barriers, we have extracted sentences from the reports of the participants:I don't have a companion to exercise with myself because my daughter says she does not have time to assist me during the activities due to her professional commitments.”“Other aspects that were included as environmental-related barriers were “problems with communication and lack of knowledge to use Internet devices and tools” (5.4%). To illustrate this barrier, we extracted the following sentence:I have difficulty accessing the videos because of poor Internet connection.”“The main barrier related to the pandemic construct was “job commitments during the coronavirus pandemic.” To illustrate it, we presented the following statement from the participants:I can't manage my work hours with my need for physical exercise. It seems even harder to do home office during this pandemic situation.”“In 48 sessions, the individuals described 45 reports (5.7%) of pain during the sessions and pain a day after the session in 50 reports (6.3%). Participants just reported one fall (0.1%), dizziness in 30 reports (3.8%), motion sickness in seven reports (0.9%), and fear of exercising in 92 reports (11.6%) during the exercise sessions. In terms of activity, for every 10,000 min, there was 0.2% fall, 9.5% pain reports during the session, 10.5% pain reports a day after the session, 6.3% seizures, 1.5% motion sickness, and 19.4% fear of exercising reports during the sessions.For the personnel construct, the median answer for question 2 was 5 (range 3), and for question 3 the median was 1 (range 4). In question 2, participants agreed with “to feel safe in activities.” In question 3, participants strongly disagreed with the fact that the program interfered negatively with their mood.For the communication construct, the median answer to question 7 was 5 (range 4) and question 10 was 1 (range 4); therefore, participants fully agreed with how easy the communication with the team members was established, and question 10 reflects that they had problems with technology and the Internet connection.For the program construct, the median answer for questions 1 and 8 was 5, but with different ranges (3 for question 1 and 4 for question 8), question 4 was 3 (range 4), and question 5 was 1 (range 4). Thus, participants felt involved (question 1) and supported (question 8) with the program, but neither they felt that they were able to perform all the proposed activities (question 4), nor they think that the remote program should replace the face-to-face program.For the social support construct, the median answer for questions 6 and 11 was 5 (range 4), and question 9 was 1 (range 4); thus, participants felt assisted (questions 6), which positively affected their relationships (question 11), and they disagreed about the fact that they would not do activities with these professionals or teams again (question 9).For the caregiver construct, the median answer for questions 13 and 14 was 5 (ranges 3 and 4), question 15 was 1 (range 1), question 16 was 4 (range 4), and questions 17 and 18 were 5 (ranges 2 and 4). Thus, the family member or caregiver felt comfortable doing exercises with their family members (question 13), and he/she was assisted by the instructions they had (question 14). The family members and caregivers did not feel affected in their routine by supporting the participant (question 15). They thought that monitoring family members did not require much effort (question 16). They had fun doing the exercises with their family member involved in the remote program (question 17) and the overall experience doing the exercises positively affected their life during the pandemic .This study has evaluated adherence, attendance rate, and barriers to attend to a remote physical exercise program for stroke survivors. Our remote physical exercise program presented a high adherence. The barriers to attending the remote exercise program were difficulties related to health conditions (including the use of the most affected side), the pandemic , and environmental issues . These barriers were different from other studies with the same population. In general, barriers to exercise are described to understand why sedentary people do not engage in physical exercise activities. Due to the COVID-19 pandemic, barriers to switching between a face-to-face group exercise program to a remote mode of delivery are mandatory. Our results show that specific barriers have emerged from this condition.The adherence to the remote physical exercise program was higher compared with the literature . Our results must be read through the cultural lenses of a country, highly impacted by the COVID-19 socially and economically  (Bull et al., The main barrier to attending the remote program was the difficulty to do the proposed exercises due to motor impairments. To tailor exercises to personal features was a challenge at this remote model because each participant had different impairments. The asynchronous participation gives opportunities to the attendees to judge how safe it is to do the whole exercise session and also demands self-motivation to engage, family support, and the willingness for a healthy lifestyle after a stroke.The second most related barrier was the lack of someone to aid the exercise at home. Even though they are considered independent for self-care and walking, stroke survivors have limitations of activities, such as physical exercise (Campos et al., In the category of pandemic issues, “job commitments during the pandemic coronavirus” was the most frequent issue. This finding correlates with the present environmental category, a profile of individuals who present a perception that their physical condition is a barrier to physical exercise and they need a family member or caregiver to advise them. However, those family members/caregivers are facing new home office obligations due to the change in routine caused by the COVID-19 pandemic.Most of the participants reported that the remote physical exercise was safe. Just one fall was reported across all the sessions. To the best of our knowledge, this is the first telemonitoring study for stroke survivors to report these data. Individuals tend to experience social discomfort, loss of confidence and personal identity, long-term functional disability, and loss of independence when falls happen (Schmid and Rittman, Pain complaint was one of the most reported issues. The main changes related to physical activity are a high frequency of pain and discomfort (Jones et al., “Physical activity causes me pain or discomfort.”“I think physical activity is very tiring.”“I am afraid to do my physical exercises alone.”Opinions of the participants concerning the overall experience about this remote program suggest that it was safe from the view of the participants, thus allowing a major interaction with the program. The ease of communication between the participants and the team allowed us to be aware of all the issues that harmed the progress of the program and allowed us to correct these flaws.According to reports of the program, it was well-accepted, despite the fact that most of the participants prefer the face-to-face program. Remote programs are well-accepted (Chen et al., Due to the COVID-19 pandemic, our sample was selected by convenience and all participants were already engaged in a face-to-face program. Another limitation was not having a control group with no intervention or face-to-face intervention to contrast our results. Our questionnaires were not previously validated but were developed for this study. It would be important to add other tools to evaluate the psychosocial domains within a telemonitoring program.The remote exercise program has a high adherence rate among stroke survivors to engage and a low attendance rate. The main reported barriers involved the need to individually tailor the exercises to personal impairments. Attending a remote physical exercise program is safe, and the overall experiences of the participants and their family members/caregivers were positive. A remote physical exercise program can reduce sedentary behavior and increase the physical activity level in individuals who had a stroke.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by School of Physical Education and Sport of the University of São Paulo, São Paulo, Brazil. The patients/participants provided their written informed consent to participate in this study.CT-P: an idealizer and coordinator of the physical activity program, coordinated the conversion of the face-to-face model to the remote program, collaborated in the creation of the questionnaires, coordinated the collections, and writing and revision of the text. GP, MM, and BA: conversion of the face-to-face model to the remote program, collaborated in the creation of the questionnaires, monitored the individuals, and writing and revision of the text. AL: creation of the questionnaires and writing and revision of the text. TS, MC, and RJ: performed individuals monitoring and data analysis. VD and TF: conversion face-to-face model to the remote program and collaborated in the creation of the questionnaires. LM: creator of the project, creation of the questionnaires, performed data analysis, text writing, and text review. All authors contributed to the article and approved the submitted version.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.


ARID1A alterations in a cohort of EC, studying the possible associations between DNA mutation (genomic level), RNA expression (transcriptomic level), and protein expression (proteomic level). A total of 50 endometrial carcinomas were characterized for ARID1A mutations (using targeted DNA next-generation sequencing—NGS), ARID1A gene expression (using RNAseq and qRT-PCR), and ARID1A protein expression (using immunohistochemistry—IHC). Moreover, we have investigated if ARID1A mutations may alter the protein structure, using the Protein Data Bank sequence. We found a good correlation between ARID1A mutations and protein immunostaining, even if we did not find statistically significant differences in the ARID1A expression levels. In conclusion, our data demonstrated that the molecular characterization of ARID1A should be associated with IHC analysis, mainly in those cases harboring “novel” ARID1A mutations or in those alterations with “uncertain” pathogenic significance.Since the Cancer Genome Atlas (TCGA) project identified four distinct groups based on molecular alterations, mutation analyses have been integrated into the characterization of endometrial carcinomas (ECs). ARID1A seems to be the subunit more involved in the loss of function of the SWI/SNF complex in ECs. The aim of this study is to define the relevance of  ARID1A gene. On the contrary, ARID1A alterations have not been detected in serous endometrial carcinomas [ARID1A gene may result in the loss of ARID1A protein expression, with consequent alterations in the SWI/SNF functions. This loss of function determines defects in the cell cycle checkpoint activation in response to DNA damage [The annual incidence rates of endometrial cancer (EC) range between 15 and 25 per 100,000 women in western countries ,9,10,11.rcinomas ,13,14. MA damage , and an ARID1A, it is crucial to determine if a detected mutation is disruptive or well-tolerated at the protein level. In fact, some alterations involving the “genomic level” of ARID1A may not lead to the loss of function of ARID1A protein, without affecting the SWI/SNF complex functions.The multigene high-throughput technique, as next-generation sequencing (NGS), is increasingly widespread in tumors’ characterization. This approach has allowed not only to combine multigene analysis with high analytical sensitivity but also to integrate morphological and molecular features of the analyzed samples ,18,19,20ARID1A alterations in a cohort of EC studying the possible association between DNA mutation (genomic level), RNA expression (transcriptomic level), and protein expression (proteomic level), to better define which ARID1A mutations may be considered for EC classification.The aim of this study is to define the relevance of ARID1A mutational status was evaluated using a multigene custom panel that allows for sequencing the whole coding sequence (CDS) of the ARID1A gene [http://software.broadinstitute.org/software/igv/, accessed on 1 October 2021) [https://varsome.com/, accessed on 1 October 2021).NGS analyses was performed on 50 endometrial carcinomas, as previously described . BrieflyD1A gene . The resImmunohistochemistry (IHC) was performed using the Benchmark Ultra immunostainer. ARID1A expression was evaluated using the rabbit anti-ARID1A polyclonal antibody . Cases were independently scored using an immunoreactive scoring system by three pathologists, ADL (Pathologist 1), DS (Pathologist 2), and CC (Pathologist 3), who were blinded to the sequencing results. Abnormal ARID1A IHC was defined as a complete loss of nuclear staining in the tumor. Conversely, normal ARID1A IHC included cases with positive nuclear staining, including cases with weak staining  and strong staining . ARID1A nuclear staining was scored as follows: negative “loss of expression”, “positive” (weak or strong), or as “clonal loss”  ,22. In tARID1A mutant and 10 ARID1A WT.ARID1A expression at the transcript level was evaluated by RNAseq and by qRT-PCR in 24 cases of which 14 were Briefly, for the RNAseq analysis, RNA quality and concentration were evaluated by the Agilent 2100 Bioanalyzer system. RNA sequencing libraries were constructed starting from 100 ng of RNA as input through the QIAseq Stranded Total RNA Lib Kit (Qiagen) following the manufacturer’s protocol. The quality and the size distribution of the libraries were checked using the High Sensitivity DNA Analysis kit . Subsequently, libraries were pooled and sequenced on an Illumina Nexsteq500 using the NextSeq High Output kit v2 (150 cycles) . Obtained sequences were mapped to the human genome (GRCh38) using the algorithm HISAT2  and a prARID1A qRT-PCR was performed on cDNA synthesized from FFPE tumor samples. cDNA was obtained with the SuperScript™ IV VILO™ Master Mix kit (ThermoFisher) and ARID1A expression levels were evaluated using quantitative-PCR on the 7900HT instrument . Fold change was evaluated using the DDCt method, using GAPDH as a housekeeping gene. Primer used were: ARID1A_Fw 5′-CCAGCCGGTTCTTCGTG-3′; ARID1A_Rev 5′-ATCGGTGAAGAAGGGCGAG-3′; GAPDH_Fw 5′-CGGGAAGCTTGTCATCAAT-3′ and GAPDH_Rev 5′-GACTCCACGACGTACTCAGC-3′.The ARID1A mutations were mapped on the structures available in the Protein Data Bank  were missense mutations, 5 (20.8%) frameshift small indels, and 5 (20.8%) stop codon substitutions (n = 8), exon 3 (n = 3), exon 1 (n = 2), exon 5 (n = 2), and exon 18 (n = 2). One mutation was detected in exons 6, 7, 8, 10, 12, 15, 19. In 3 of 22 mutated samples (13.6%), concomitant ARID1A substitutions were observed (Twenty of 50 samples (40.0%) harbored at least one itutions . The mutobserved .According to PolyPhen2, 20 of 24 variants were scored as “damaging” for the protein function, while four were “well-tolerated”. According to the Varsome tool, 9 of 25 alterations (36.0%) were classified as “Benign/Likely benign”, 10 (40.0%) as “Pathogenic/Likely Pathogenic”, and 7 (28.0%) as “Variant of Uncertain Significance—VUS”.Three of four mutations scored as well-tolerated by PolyPhen2 had been predicted as “Benign/Likely benign” according to the Varsome tools . The othARID1A mutant and wildtype patients (p = 0.08), as shown in At the RNAseq level, ARID1A expression was not significantly deregulated between p = 0.06)  .ARID1A was scored as positive in 28 of 50 cases (56.0%), and negative/loss in the remaining 22 (44.0%).ARID1A variant, while in 4 cases (14.3%) at least one mutation was detected  harbored a disruptive mutation according to in silico tools (PolyPhen2), but with uncertain significance according to Varsome; one (#6) had two concomitant mutations, one likely benign and the other one disruptive according to in silico tools (PolyPhen2), but with an uncertain significance according to Varsome  did not harbor any detected . As rega Varsome .ARID1A mutation was observed. Of these 16 mutated cases, 10 (62.5%) harbored a frameshift/stop codon variant, while in 6 (37.5%) a missense mutation was identified  no variants were detected, while in the remaining 16 (72.7%) at least one entified . Of the ARID1A mutations were detected and the IHC staining was positive  the PolyPhen2 tool predicted a damaging effect of the mutation on the protein, the Varsome database provided a “Pathogenic/Likely Pathogenic” result, and IHC staining was negative (protein expression loss)  IHC staining was lost and no 6 cases, ; (ii) IH6 cases, .Results were doubtful when: (i) the IHC result was concordant with only one prediction of in silico tools  2 cases, ; (ii) acOverall, in 36 cases a perfect concordance was found between the prediction of mutation’s effect (PolyPhen2 score), integrated data about the mutation (Varsome database), and IHC staining ; in 7 caARID1A variant was scored as “well-tolerated” . Recently, a new ARID1A structure in the SWI/SNF complex was deposited in the PDB-Dev database (PDB-Dev ID 00000056). In the latter structure, the solved residues were 1663-1740, 1771-1783, 1804-1840, 1904-1941, 1961-2026, and 2049-2285. In other words, no structural information is available for the ARID1A N-terminal region before residue 1000 and for a large portion, comprised between residues 1119 and 1639. Even in the solved regions, there are several parts for which no structure is available. The length of the missing regions and the absence of suitable templates impede the reconstruction through homology modeling and for this reason, only the mutations that can be mapped on the available structures are discussed hereafter.Regarding the mutations, it is possible to suppose that all the “stop” mutations  do not allow the translation of the C-terminal region, and thus the interaction with the other proteins of the SWI/SNF complex is not possible, preventing the correct assembly of the complex itself. The same can be inferred for the “frameshift” mutations . From the frameshift mutation onwards, the protein may not fold correctly unless a second frameshift later in the sequence allows it to fold correctly again.p.Leu2195Arg (6LTH/6LTJ) (Case #5). This residue is located at the buried edge of a partially buried α-helix. Leu2195 forms hydrophobic contacts with ARID1A Val2168 and Leu2241. The observed mutation can cause mild to severe effects on the latter interactions considering the positive charge and the H-bond donor capability of the arginine side chain tail, even if it is difficult to predict their extent.p.Leu1100Phe (1RYU) (Case #12). Leu1100 is located in the ARID1A hydrophobic core and is partially accessible to the solvent. The vicinity of several ARID1A aromatic residues  can cause the formation of new interactions when Leu1100 is mutated in phenylalanine. On the other hand, it is difficult to predict the effect of this mutation on the ARID1A folding.p.Asn1705Ser (6LTH/6LTJ) (Case #14). This residue is located on a solvent-exposed α-helix. Asn1705 is H-bonded to ARID1A Ser1707 and Asn1997. Considering the similar length and the similar H-bond propensity of asparagine and serine, this mutation is not supposed to cause large structural effects.p.Arg1833Cys (6LTH/6LTJ) (Case #16). This residue is located on a solvent-exposed loop. The side chain of Arg1833 is H-bonded to Gln70 and Leu71 backbone oxygen atoms of the SMARCC2 subunit and to ARID1A Glu1853. The observed mutation can damage these interactions considering the different lengths in the side chain of an arginine residue if compared with the length of a cysteine. Moreover, cysteine is not a good H-bond donor as an arginine.p.Arg1906Gln (00000056) (Case #17). The mutation is on a residue that is exposed to the solvent and that does not interact with other residues from ARID1A or from other SWI/SNF proteins. A polar residue is replaced by a similar equally polar residue. Apparently, this mutation is not supposed to cause any effect.For the missense mutations, the following can be observed from the available structures  [ARID1A alterations seem to play an important role in defining the clinical and prognostic features. Sequencing of the entire coding region of ARID1A may lead to detecting “novel” mutations and/or mutations with unknown significance regarding the effect on the protein functions [ARID1A alterations in a cohort of ECs evaluating the possible association between DNA mutation (genomic level), RNA expression (transcriptomic level), and protein expression (proteomic level). The detection of a variant that does not influence the role of ARID1A protein should not lead to the use of this data for the molecular algorithm for classifying ECs. In our study, we have observed that ARID1A mutations do not influence in a statistically significant way the ARID1A expression level in ECs. Several in silico tools are nowadays available for predicting the possible role of gene mutations . We have tried to classify ARID1A mutations identified in our cohort of ECs both using two in silico tools (PolyPhen2 and Varsome) and comparing genetic results with ARID1A protein immunostaining. In the vast majority (72%) there was a good concordance between the different methods used for classifying the detected mutations. The regions covered by the used panel allow analyzing the whole coding sequence (CDS) and 20 nucleotides spanning each exon of the ARID1A gene (including possible splicing sites). Eventual alterations in splicing sites will be then detectable using this panel. It should be considered that some mutations may fall into intronic regions not covered by our panel, but usually, these types of alterations do not affect RNA and protein expression. The integration of molecular data with histopathological features in EC is becoming more and more important since TCGA proposed to stratify EC in different groups, according to their molecular alterations (t group) . Severalt group) ,5,6,7,8.https://www.ncbi.nlm.nih.gov/clinvar/, accessed on 31 January 2022) and the Varsome database . This mutation has been frequently described in endometrial cancer , large intestine tumors (14 samples), pancreatic ductal adenocarcinomas (5 samples), breast carcinomas (4 entries), and gastric cancer (3 cases). This mutation has been reported also in one case of cervical squamous cell carcinoma in the TCGA pan-cancer data . According to data reported on cBioPortal, the ARID1A p.Arg1989Ter mutation is likely oncogenic and will likely induce a loss of ARID1A expression, which may be related to sensitivity to EZH2 and BET inhibitors [The p.Arg1989Ter (c.5965C>T) mutation was the most common substitution found in our cohort. This non-sense mutation is classified as pathogenic according to ClinVar  the same clonal neoplastic cells that harbor one ARID1A mutation on one allele and the other alteration on the other allele; or (ii) tumor heterogeneity, i.e., different clones of neoplastic cells that harbor different ARID1A mutations.The presence of concomitant mutations in the ARID1A sequence. In these samples it is possible that a loss of protein immunostaining is due to epigenetic mechanisms [ARID1A copy-number aberration [ARID1A sequence did not correspond to a loss of protein expression. This discrepancy may be due to missense mutations that did not affect the ARID1A protein expression, and then these cases should be considered as “not ARID1A mutated”. However, it should be considered that acetylation-level changes in specific histone sites in ARID1A mutated samples [ARID1A mutated samples”. Using Protein Data Bank sequences, we have also tried to further investigate if detected mutations may alter the ARID1A protein structure, but only in a few cases do we find the corresponding structures deposited. In those tumors harboring ARID1A mutations but with ARID1A positive immunostaining, it would be very interesting to deeply investigate if these variants may influence the assembling of ARID1A with other subunits of the SWI/SNF complex . In fact, the ARID1A subunit is crucial for the SWI/SNF-mediated chromatin remodeling and for mediating ARID1A-associated SWI/SNF family DNA binding. It has been previously demonstrated that mutations in ARID1A are associated with a worse prognosis, mainly in the No Specific Molecular Profile (NSMP) group [ARID1A [ARID1A mutation [For some variants, the in silico tools were not concordant, or the significance was unclear. In those cases, it is fundamental to test protein expression using IHC. Intriguingly, in six cases ARID1A was lost at IHC, even if no alterations were detectable in the chanisms ,35, inclerration , or the erration . In five samples ; some muP) group . As rega [ARID1A . The ARI [ARID1A . Moreove [ARID1A ,33. The mutation ,41,42.ARID1A mutations or with “uncertain” pathogenic significance. Unfortunately, due to the small number of the series and the low number of recurrences, it was not possible to evaluate the prognostic significance of ARID1A at the mutational, RNA, and protein levels. Further information about the possible effect of the mutation on ARID1A functions should be also investigated, using for example, in silico structure analysis. Moreover, using both ARID1A sequencing and IHC may help to also detect those cases in which loss of ARID1A functions is due to different mechanisms than DNA alterations.In conclusion, our data demonstrated that the molecular characterization of ARID1A should be associated with IHC analysis, mainly in those cases with “novel”


In-person relationship education classes funded by the federal government tend to experience relatively high attrition rates and have only a limited effect on relationships. In contrast, low-income couples tend to report meaningful gains from web-based relationship education when provided with individualized coach contact. However, little is known about the method and intensity of practitioner contact that a couple requires to complete the web-based program and receive the intended benefit.within 3 different levels of coaching, and examine the most powerful predictors of treatment adherence and gains in relationship satisfaction among the 3 levels of coach contact.The aim of this study is to use within-group models to create an algorithm to assign future couples to different programs and levels of coach contact, identify the most powerful predictors of treatment adherence and gains in relationship satisfaction To accomplish these goals, this project intends to use data from a web-based Sequential Multiple Assignment Randomized Trial of the OurRelationship and web-based Prevention and Relationship Enhancement programs, in which the method and type of coach contact were randomly varied across 1248 couples , with the hope of advancing theory in this area and generating accurate predictions. This study was funded by the US Department of Health and Human Services, Administration for Children and Families (grant number 90PD0309).Data collection from the Sequential Multiple Assignment Randomized Trial of the OurRelationship and web-based Prevention and Relationship Enhancement Program was completed in October of 2020.Some of the direct benefits of this study include benefits to social services program administrators, tailoring of more effective relationship education, and effective delivery of evidence- and web-based relationship health interventions.DERR1-10.2196/33047 Individ06-0.09)  and not  d≥0.20) .Attrition problems are not unique to the field of relationship education and appear to be an inevitable outcome of web-based randomized controlled trials (RCTs) . This haOthers have argued that the law of attrition could be enhanced by the inclusion of user characteristics . User chThus, determining who is likely to drop out of the program and who is likely to complete the program at the program outset would be a major benefit to administering effective web-based relationship education. Focusing on completion of the program (and its specific activities) rather than aiming to increase the desired outcome  not only decreases attrition but may also increase the desired behavioral changes . The addRecently, strides have been made in machine learning to mirror these ideas, allowing researchers to predict and compare the predicted treatment effects for couples ,14. Thisd=0.15-0.69) in key areas of individual and relationship well-being compared with a waitlist control group [This precision medicine and machine learning approach is relatively new to the field of relationship education and web-based health interventions. Thus, starting a precision medicine approach with programs that have already been shown to be evidence-based and beneficial would be worthwhile. Arguably, OurRelationship and web-based Prevention and Relationship Enhancement Program (ePREP) are the 2 web-based relationship education programs with the largest evidence base. OurRelationship and ePREP contain only 6 to 10 hours of content—substantially less content than comprehensive programs previously delivered by the federal government. The OurRelationship program is a web-based, self-help adaptation of integrative behavioral couple therapy that emphasizes acceptance and change . The prool group . Furtherol group . The Ourol group .The ePREP program is a web-based adaptation of the Prevention and Relationship Enhancement Program, which emphasizes skill building ,21. AccodOurRelationship=0.53; Cohen dePREP=0.42) and emotional support , along with decreases in breakup potential , communication conflict , and intimate partner violence  [d=−0.24) [Recently, both OurRelationship and ePREP were compared with one another and to a waitlist control condition in a large Administration for Children and Families (ACF)–funded RCT with low-income couples (N=742) . When deP=−0.08) . FurtherP=−0.08) . When thd=−0.24) . These fd=−0.24) .The literature examining general predictors of adherence and treatment gains in web-based interventions is filled with a mix of inconclusive findings. Regularly, demographic variables have been examined as predictors of adherence to and treatment gains in web-based protocols. Gender and education, for instance, were significant predictors of treatment adherence in some studies but nonsignificant predictors in others -32. Findlower levels of depression and anxiety predict better adherence to web-based programs for mood disorders [higher baseline levels of alcohol use predicted better adherence to web-based alcohol intervention programs [Equally, the literature examining the role that baseline levels of psychopathology or symptomology play in adherence to web-based protocols usually yields inconsistent findings, even among reviews and meta-analyses. For instance, in a recent meta-analysis of self-guided web-based programs  for depression, levels of depression did not significantly predict treatment adherence . Howeverisorders , higher programs . A possiprograms ,32, wherprograms . In all,Despite the inconsistencies in this area, 2 predictors yield relatively consistent findings. First, much like in-person relationship education, high levels of external stress seem to be an important barrier. Specifically, participants who reported that the treatment was too demanding, who reported greater perceived external barriers, more time constraints, more pressure to complete the program, the presence of a physical illness, a family history of mental illness, and those who reported that school or work got in the way were all less likely to adhere to web-based protocols and reported less improvement ,30,39,40Although research suggests that brief web-based relationship education yields promising completion rates and relationship outcomes, one area of interest is not well understood: the type and amount of coaching needed for couples to complete the web-based material. Coaches in the OurRelationship and ePREP programs are usually master’s-level clinicians with a degree in psychology that help reinforce the content taught in web-based curricula using telephone or video chat. The supportive accountability model argues that adherence to web-based interventions is primarily predicted by a couple’s accountability to complete the program, that is, the implicit or explicit expectation that the couple is required to complete the material . AccountThe lack of motivation to complete the program may prevent a couple from completing the agreed upon material . IdeallyThe method of communication can also serve as a moderator for the effect of accountability on material completion. For example, some couples may enjoy the additional connection video conference meetings that their coach provides, whereas others might find regular email contact to be less intrusive. This variability highlights the need to tailor coaching to meet the needs of specific couples. Thus, identifying the best way to determine the amount of coaching a couple needs as well as the right method of communication is critical to the success of relationship education .The effect of a coach has garnered significant attention in the field of web-based interventions. Several studies of individual interventions have investigated the effect of not having a coach , conditional coach support , some coach support , and full-coach support . The definitions of coach contact vary widely across studies and have, unfortunately, yielded a series of mixed results. Studies of web-based interventions for both couples and individuals have found that more intensive coach support yields (1) better adherence rates, program completion, and treatment outcomes ,41; (2) Some studies have compared web-based content coupled with coach support with only web-based content. For instance, when completion rates from a no-coach version of the OurRelationship program were compared with those of a trial with full coaching , 6.1% of individuals in the no-coach trial completed the program compared with 66.1% in the full-coach version of the program . FurtherOther studies have varied the intensity and type of coaching that couples and individuals can receive and its effects on program outcomes and adherence. When comparing the OurRelationship program with 4 calls to the OurRelationship program with one call, those who received 4 calls were more likely to complete the program  and saw greater reductions in anxiety symptoms than those who had one call . HoweverFew studies have investigated whether baseline characteristics moderate the effect of different coaching levels. Of the 20 baseline moderators tested in the OurRelationship program, only 2 significant interactions with coaching level emerged. First, those with higher baseline levels of depressive symptoms were actually less likely to complete the program when receiving 4 coach calls compared with the stand-alone version of the program . Second,Given past research, this study has 3 aims. The first aim is to investigate how well we can predict the completion rates and changes in relationship satisfaction. As model evaluation has rarely been used in this area of research, no specific hypotheses have been posited. The second aim of this study is to document the most powerful predictors of treatment adherence and gains in relationship satisfaction within 3 different levels of coaching. More specifically, what are the most powerful predictors of treatment adherence and gains in relationship satisfaction in a full-coach condition , automated coach condition (where couples only receive emails), and a contingent coach condition ? Although many of the findings in this area of research are inconclusive, 2 findings are relatively consistent: lower external stress and higher intrinsic motivation both predict treatment adherence and gains. Thus, we hypothesize that, regardless of coach assignment, lower external stress and higher intrinsic motivation will emerge as reliable predictors of better adherence to the web-based program and subsequent treatment gains. In the final aim of the study, we intend to use the information gathered in the first 2 steps to determine the most powerful predictors of between-group differences. More specifically, what baseline characteristics are indicative of a given couple’s adherence or gains in relationship satisfaction to couples assigned to the full-coach program compared with automated coaching, contingent coaching, or the waitlist control condition? As this area of research is relatively underdeveloped, no specific hypotheses will be posited.This study was funded by the US Department of Health and Human Services, ACF , did not have access to highspeed internet, or had previously participated in an ePREP or OurRelationship program.A total of 226 couples were randomly assigned to the OurRelationship coach condition, 145 couples were assigned to the OurRelationship contingent coach condition, and 145 couples were assigned to the OurRelationship automated  condition. Similarly, 222 couples were assigned to the ePREP coach condition, 143 couples were assigned to the ePREP contingent coach condition, and 143 couples were assigned to the ePREP automated  condition. A total of 224 couples were assigned to the waitlist control condition. In total, 4 individuals from the OurRelationship coach condition, 2 from the OurRelationship contingent coach condition, and 2 from the OurRelationship automated condition asked to discontinue and have their data removed from the study. Furthermore, 3 individuals from the ePREP coach condition, 2 from the ePREP contingent coach condition, and 3 from the ePREP automated condition asked to discontinue and have their data removed from the study.In total, 47.5% (1178/2480) of the sample identified as male, and 52.5% (1302/2480) of the sample identified as female. The average length of the relationship was 5.74 years (SD 5.18). Most of the participants reported belonging to an opposite-gender relationship , and a smaller percentage were in same-gender relationships . Most participants identified as White non-Hispanic , fewer identified as Black , White Hispanic , Black Hispanic , American Indian or Alaskan Native , Asian , Hawaiian or Pacific Islander , and Biracial ; and 3.59% (89/2480) of participants belonged to a race that was not listed. Furthermore, 6.81% (169/2480) of participants did not have a degree or diploma, 12.3% (305/2480) had earned a general education diploma, 14.23% (353/2480) had completed high school, 8.23% (204/2480) had a technical or vocational certification, 22.54% (559/2480) had some college degree, 8.83% (219/2480) had graduated with an associate’s degree, 10.69% (265/2480) had a bachelor’s degree, and 2.38% (59/2480) had a master’s or advanced degree.The first outcome of interest in this study is whether a couple completes all the required activities in the program to which they are assigned. Couples that complete all (100%) the activities to which they are assigned will be coded as 1, whereas couples that do not complete all the program activities will be coded as 0.r>.78) and positive communication (r>.75), providing evidence its validity [Relationship satisfaction will be measured using the Couples Satisfaction Index–4 . The Couvalidity .Although support for demographic variables and their relationship to treatment adherence varies, a host of demographic variables will be included as candidate predictor variables. Predictor variables include race, ethnicity, household income, age, gender, as well as identifying as a same-gender couple.In line with the consistencies in previous research and in addition to relationship satisfaction, several measures of relationship symptomology will be used as possible candidates to predict program completion and gains in relationship satisfaction.The thought of ending my relationship has crossed my mind), which has good internal consistency (Cronbach α=.83) in past RCTs involving low-income couples [Breakup potential will be assessed using a three-item Likert-style measure adapted from the Marital Instability Index .To assess relationship commitment, this study plans to use a single-item Likert-style measure developed by the ACF  in your relationship?).The intensity of a couple’s biggest relationship problem could be a powerful predictor of nonadherence. This construct was measured using a single item on a Likert-style scale . Past RCTs using this measure have reported good internal consistency (Cronbach α=.89) [This form of negative communication was measured using a Likert-style scale developed by the ACF  .As another measure of baseline relationship symptomology, emotional support was measured using a five-item measure developed by the ACF. Past RCTs using this measure have reported good internal consistency (Cronbach α=.83) .Minor levels of IPV were assessed using 7 items created in consultation with the National Domestic Violence Hotline . Among oIn addition to including predictors of relationship well-being, the inclusion of measures assessing external stress has been shown to be a consistent predictor of treatment adherence. In addition, as this is measured at the individual level, this may highlight within-relationship differences that may aid in the prediction of treatment nonadherence and satisfaction gains. This study intends to measure psychological distress and general health using the following measures:During the past 30 days, how often have you felt nervous) [To assess individual distress, this study intends to use the Kessler Psychological Distress Scale (nervous) . This isnervous) . Furthernervous) .In the last 30 days, how often have you felt that you were unable to control the important things in your life?) [To assess perceived distress, this study intends to use the Perceived Stress Scale, a 4-item Likert-style measure (r life?) . A past r life?) .the following are a list of events that may cause stress for you and your partner. Check each thing (or similar thing) that has happened to you in the past month. Some of the answers from this item include changes in work schedule, changes in childcare, changes in family structure, or changes in finances.Another measure of external stress will include a single-item measure of chaotic events that an individual may experience in their relationship. This will be assessed using a check-all-that-apply item developed by the ACF; that is, In general, how would you describe your health).Impacts on the physical well-being  of one individual may prevent both members of the dyad from completing the program. To assess general health, this study intends to use a single-item measure of general well-being on a Likert-style scale developed by the ACF , providing evidence of the scale’s reliability and validity.One measure of motivation is grit or passion and perseverance for long-term goals under challenging circumstances . DespiteWhich of the following statements best describes your view of your current relationship problems [I don’t think I have relationship problems and therefore nothing should be done about it (coded as 0) to I know I have relationship problems, and I am here to take action to work on them now.Participants’ motivation to change will also be measured with the following item adapted for this study: problems ? ParticiIn an effort to reduce computational complexity, rather than using individual responses, responses from both members of the couple will be combined in the average score for the couple for each of the continuous predictor variables listed above as well as the continuous outcome variable .0: RMSEModel 1 = RMSEModel 2), program assignment will be ignored resulting in 3 conditions , which will be used in the proceeding aims. If the interactions account for a significant portion of the variance (Ha = RMSEModel 1 ≠ RMSEModel 2), independent models will be built by treatment  and coach condition  resulting in 6 models: OurRelationship full coach, OurRelationship automated coach, OurRelationship contingent coach, ePREP full coach, ePREP automated coach, and ePREP contingent coach, which will be used in the proceeding aims.The first research question asks: how well can one predict program completion and anticipated gains within (1) the full-coach condition, (2) the automated coach condition, and (3) the contingent coach condition? To ensure that the different programs do not account for a substantial portion of the variance when predicting program completion or changes in relationship satisfaction, a test will be performed where 2 models will be created and compared. In the first model, all predictors will be interacted with the level of coaching  and the program assignment  resulting in Predictor×Treatment×Coaching interactions. However, the second model will maintain all predictors by coaching interactions but drop all program assignment interactions . Next, the root mean square error (RMSE) between the 2 models will be compared. If the null hypothesis does not get rejected (H. For binary outcomes (program completion), the model characteristics used to evaluate the validity of the model include sensitivity, specificity, positive predictive value, and negative predictive value of the model built on the test data set  the full-coach condition, (2) the automated coach condition, and (3) the contingent coach condition? If, in aim 1, the interaction terms result in a lower RMSE, better sensitivity, or better specificity, models will be constructed based on treatment assignment  and coach assignment . One statistically powerful way to test the most potent predictors of treatment adherence and gains is through the use of variable importance (VIMP). VIMP is a nonparametric approach within the random forest algorithm, which can help investigators identify which variables play a key role in predicting a binary  or continuous  variable . VIMP haAssuming that the models from the previous aim have levels of prediction accuracy, each of the within-group models will be used to generate predicted outcomes for each couple’s likelihood of adhering to the program as well as their treatment gains, creating counterfactual estimates of program completion and treatment gains as if each couple in the data set completed each intensity and method of coach contact .To do this, the within-group models built in aim 1 will be used to generate potential outcomes for each couple’s likelihood of adhering as well as their gains in a (1) full coaching, (2) automated coaching, and (3) contingent coach conditions. If, in aim 1, the interaction terms result in a better RMSE, models will be constructed based on treatment assignment  and coach assignment . The estimates between the conditions for each couple’s between-group treatment differences between the full and automated coaching conditions can be understood as: is the between-group likelihood of treatment adherence or gains for couple x, Ŷ is the within-group likelihood of treatment adherence or gains for couple x in the full-coach condition, and Ŷ is the within-group likelihood of treatment adherence or gains for couple x in the contingent coach condition [where ondition . These hFinally, because missing data are anticipated, all missing data will be imputed using the miss-forest algorithm of Ishwaran and Kogalur randomForestSRC package in R  . Miss-foData collection was completed in October 2020 and data are being prepared to be analyzed. Overall, 63.8% (286/448) of individuals completed the material in the OurRelationship coach condition, 53.8% (155/288) of couples completed the OurRelationship contingent coach condition, and 54.5% (157/288) of individuals completed the OurRelationship automated  condition. Similarly, 69.4% (306/441) of couples completed the content in the ePREP coach condition, 74.2% (210/283) completed the content in the ePREP contingent coach condition, and 66.2% (188/284) of couples completed content in the ePREP automated  condition. Currently, no other outcomes except for completion rates have been analyzed. Given the large sample size within and between conditions, the current sample is large enough to identify potential predictor variables and evaluate their prediction accuracy. This study is expected to conclude in the summer of 2022.One of the direct benefits of this study will accrue to social services programs and program administrators of web-based relationship education. The results of this study will allow for more effective tailoring of coach contact to better meet the needs of unique low-income couples experiencing relationship distress. A second benefit is the improvement of web and evidence-based interventions. The federal government is increasing emphasis on delivering evidence-based interventions and, given the social distancing regulations put in place because of COVID-19, participation in web-based programs will likely increase either as an initial intervention or as a backup intervention in cases where in-person services are suspended. Thus, it is important that web-based federal services are as effective and accessible as possible. Overall, this study hopes to help practitioners by generating accurate predictions to match unique couples to the level of web-based programming that will help them to obtain the maximal benefit.


Public safety personnel  are at risk of developing mental health problems due to experiencing potentially psychologically traumatic events during their career. Research examining evidence-based treatments for psychological injuries resulting from operational duties  has not yielded robust results that would indicate ongoing interventions as the best solution for managing PSP mental health injuries; as such, proactive psychological interventions designed to bolster resilience are being considered potentially beneficial for mitigating the impact of occupational stress on PSP. Despite the growing popularity of resilience programs, most are delivered in a single session after an event deemed particularly problematic with no follow-up. Longer interventions may better support sustained resiliency, mitigate the impact of operational stress, and increase positive PSP workplace outcomes. The current article introduces the Before Operational Stress (BOS) program, which was designed for delivery early in a PSP career to enhance self-awareness and healthy relationships. The year-long program is derived from cognitive behavior therapy and group therapeutic techniques to meet program objectives. The current BOS program evaluation demonstrated small, statistically significant improvements in symptoms of PTSD, quality of life, stigma, and perceived social support from baseline (Time 1) to 6 months (Time 4). There were also non-significant improvements observed in symptoms of depression, anxiety, stress, alcohol use, as well as in emotional regulation and resilience. Qualitative results indicated participants positively perceived the BOS program, with participants reporting specific improvements in self-awareness, avoidant behaviors, and relationships with family and colleagues. The BOS program content  and processes  appear unique relative to other PSP resilience programs, with promising initial results in support of PSP mental health. Recommendations for future research and program development are provided. Public safety personnel  appear to experience higher prevalence rates of mental disorders than the general population  program . The independent evaluation is part of a transition WWC supports for all programming to move from being evidence-informed to evidence-based. There were two groups of PSP in Alberta (firefighters) and Ontario (paramedics) recruited to receive BOS training and participate in the pilot program evaluation. The group participants provided valuable feedback on the program and BOS in its current form was officially launched in October 2018.BOS was not designed to be, and is not intended to be, a PPTE-focused treatment program. BOS was designed as a program to increase self-awareness and encourage authentic, healthy relationships specifically for PSP. Participants become more aware of spiritual , physical, emotional, and mental aspects of themselves during the first half of the eight-week program. The first six BOS content modules are based on cognitive behavior therapy Beck, , which iThe BOS program content focuses on the current impact of operational stressors on the individual PSP. Although organizational stressors may also be part of the PSP experience, BOS specifically focuses on how PSP react to operational stress. There are at least four aspects of BOS that are currently unique relative to other available programming. First, BOS is designed to provide substantial amounts of time for participants to receive, reflect upon, and practice the educational component within a supportive learning structure. The learning is facilitated by an interactive mental health clinician to make the information relevant and applicable to individual participants' daily experiences. Approximately half of each session is dedicated to interactive engagement tailoring the material to one or more specific participant experiences. Participants are encouraged to talk about their struggles and successes when working with the information learned in BOS. Participants are also supported in problem solving how to incorporate the BOS content into their operational roles. The interactive experiences within the BOS program are believed to be at least as significant a mechanism for change as the actual content. The image of a stereotypical, stoic PSP maintains a common assumption that PSP would be unwilling to discuss their diverse vocational challenges  than mental health professionals  and developing communication strategies with loved ones regarding the level of nervous system arousal before arriving home. Throughout the BOS program, PSP are taught skills that can help distinguish operational function from their personal lives and encouraged to develop consistent patterns of re-entry from work to home . The group is facilitated by one clinician if there will be fewer than seven participants or co-facilitated by two clinicians for groups of seven to 12 participants. All certified facilitators have completed a mandatory two-day training program provided by Dr. Megan McElheran and Dr. Milena Spasojevic of Wayfound Mental Health Group. Following the 2-day training, BOS clinicians must be supervised when facilitating their first two BOS groups. Supervision by an experienced BOS clinician is provided remotely or in-person, depending on the geographical location of the clinician and supervisor. Facilitators complete a fidelity checklist at the end of each session reporting on the extent to which the respective module content was covered. Facilitators also complete a rating of group cohesion. The fidelity check list and group cohesion report are used during supervision and to ensure consistent delivery across facilitators.A significant component of the BOS clinician training program involves enhancing the cultural competency of clinicians who will be working with PSP. Most clinicians who have sought out BOS training have had at least some experience serving clients impacted by PPTE exposures and many have experience specifically with PSP or military populations. Clinicians who participated in the pilot BOS program underscored the importance of ensuring future facilitators understand the daily occupational experiences of PSP. Appreciating PSP experiences is likely critical for developing and maintaining rapport with PSP, which is crucial for participants' group engagement and maximization of potential benefits.BOS is a 16-h program divided into 8 weekly modules delivered in a group setting of up to 12 participants . Each 2-h session is balanced between the presentation of didactic content (psychoeducation) and group processing time. The group processing allows participants to reflect on the presented psychoeducation materials and consider how to make the information personally relevant and applicable. Participants are encouraged to talk about the struggles and successes they have had relative to the psychoeducation content and generate ideas for ways to incorporate the psychoeducation content into their daily lives . For example, during the third module (see below), participants turn their window of tolerance Siegel,  into a 1Following the initial eight-session active phase of BOS, 10 monthly follow-up sessions are scheduled. The choice to include follow-up sessions came from client feedback regarding classroom-based programs  that did not include follow-up or check-in sessions. Clients reported a lack of opportunity to implement knowledge or receive peer feedback on skills. Skill loss is known to occur without consistent practice or use  can negatively impact communication. Key communication skills are presented to enhance interpersonal understanding.Empathy and FD/FR. The eighth and final session is designed to introduce how empathy can be helpful to the individual and the community they serve, but can also be a vulnerability during the PSP career. FD/FR is revisited as a strategy to balance the paradox of empathy. PSP are encouraged to recognize they can draw on different skills and values when engaged in occupational functions as compared to when they are engaged in their personal roles. Self-awareness is necessary to differentiate their roles through an intentional, actionable process. PSP are encouraged throughout the final module to identify specific strategies they will employ to facilitate FD/FR.The group schedules 10 monthly meetings following the last module . No specific content is scheduled for the monthly maintenance session. Instead, the maintenance sessions allow members opportunities for continued support from the group as well as ongoing opportunities for interactive problem-solving after the active training phase. The maintenance phase sessions are designed to further enhance integration of BOS training into members' working and personal lives. Facilitators support in-depth processing of ongoing challenges that members are experiencing as they incorporate BOS ideas into their daily routines. For example, one participant shared newly acquired ideas from the BOS group in the workplace and was criticized by peers for talking about mental health. The following session, the participant was able to ask for feedback about how other participants maintain a focus on self and stay healthy in a toxic work environment. The follow-up sessions also allow for ongoing benefits of group support, opportunities for self-reflection, and earlier support for referral to assessment or therapy where needed.BOS can be made available through individual or PSP organizational referral. A trained mental health clinician conducts a structured clinical intake interview with each potential member to ensure suitability for the group. The intake interview is completed shortly before the start of the program and includes discussion of the participant's experience with operational stress, previous treatment history, group expectations, informed consent, and current symptoms. Persons who are suffering from severe symptoms of any psychological condition that would interfere with the ability to tolerate being in a group , or experiencing active psychosis, are ineligible to participate in BOS.The current study was designed to assess individual and group changes in several mental health symptoms commonly reported among PSP , as well as changes in emotion regulation, guilt and shame, resilience, social support, and stigma from baseline (Time 1) to 10 months after the intervention (Time 6). Consistent with results from previous studies evaluating training programs delivered to PSP  expressed interest in participating in the research. Data was collected at six time points: pre-BOS , post-BOS , and at 1 month (Time 3), 4 months (Time 4), 7 months (Time 5), and 10 months (Time 6) post-BOS and immediately following the corresponding monthly maintenance session. Open-ended questions were included at the end of the Time 2 and Time 6 surveys to elicit qualitative data that would assist in understanding the participant's overall experience and perception of the program. The open-ended questions were also designed to obtain suggestions for improving the program components and delivery. The study was approved by the University of Regina Institutional Research Ethics Board (File #2018-191).Participants were recruited Outcome measures were chosen based on the BOS module objectives. Each module has a specific topic focus, but concepts build throughout the program and are regularly revisited. BOS is designed to reduce active mental disorder symptoms and stress through awareness of cognitions, emotions, and behaviors across all sessions. Mental disorder symptoms were measured using well-validated tools, including the Depression Anxiety Stress Scale-21  What has been the most helpful aspect of BOS for you?; (2) What has been the least helpful aspect of BOS for you?; (3) Has anything gotten better for you as a result of BOS? If so, please describe; (4) Has anything gotten worse for you as a result of BOS? If so, please describe; and (5) Please use the space below to provide any other comments you would like about your participation in BOS.Table 2). Some participants did not complete consecutive surveys and there were large amounts of missing data. As such, we were only able to analyze changes up to Time 4. We were not able to analyze for group differences  because of the small sample size and missing data. Qualitative data were obtained through open-ended survey questions at the end of the Time 2 and Time 6 surveys, rather than through focus groups. Group cohesion was assessed using the Therapeutic Factors Inventory  estimates were calculated to assess the size of treatment effects over time. We interpreted the magnitude of effect size according to Cohen's d guidelines with 0.20 representing a small effect; 0.50 a medium effect; and 0.80 a large effect. Intraclass correlation (ICC) were used to measure the proportion of the variance due to differences between individuals.Multilevel modeling (MLM) was performed to assess effects over time. Unbalanced longitudinal data sets  can be analyzed using MLMs . I am fortunate to have access to help.” The results suggest the importance of ensuring that subsequent psychological support is available to those who complete the program. Other behavioral changes included: better coping, more effective response to stressful situations, ability to recognize the emotions of others and better support others, and increased prevention and resiliency. One participant noted that, “I have been avoiding arguments when I am hyper-aroused and so far so good.”The reported benefits appeared to extend to participants' family and work lives. There were six participants who mentioned benefits to their relationships with partners or children. “I am better toward my family,” one noted. There were two participants who reported better relationships with colleagues, including a “tighter bond” and “more support from colleagues,” and a third identified a generally improved work life. The improved relationships were likely connected, in part, to more effective communication practices—another reported outcome of the BOS program—with five total references.After increased emotional awareness, the second most frequently coded theme was “Feeling Normal/Not Alone” (13 references). Participants often spoke about the benefits of feeling connected to others with a similar experience. Participants reported appreciating “knowing I'm not alone in how I feel,” “realizing the rest of the group has similar issues,” and “realizing that my issues are common amongst my peers.” In a few cases, knowledge of the science behind OSIs was helpful and contributed to the feeling of normalcy. There was a general appreciation for the educational component, including access to psychologists, psychological and biological data, and other scientific information.In contrast to the benefits reported from BOS content, views of the group component were mixed. Some participants reported believing they would have been “more open” in a one-on-one setting. Disclosure was a challenge for some participants, who reported a discomfort with group members who over-shared personal stories or a dislike for “airing personal aspects of home life.” There were two participants who reported not enjoying the virtual online sessions, which they felt were less effective or desirable. There was one participant who also suggested more content on physiological aspects of OSIs would help, which reflects the previously mentioned benefits of understanding the science behind mental health problems.BOS tends to facilitate participants experiencing potentially challenging emotions or recalling past PPTE exposures; as such, there was one participant suggested placing the coping skills early in the program. Several participants reported wanting to see the group move to biweekly meetings rather than monthly, as the frequency was preferred. Some participants reported having difficulties finding time away from work to complete the program, but participants overwhelmingly described the program as worthwhile. Timing was considered an important area for future change; specifically, several participants suggested the importance of attending the program early in one's career. There was one participant who self-identified as an “old dog” who described “the BOS program is integral for every new recruit, prior to operational stress.”The current study was designed to evaluate the Before Operational Stress (BOS) program, an evidence-informed program to proactively support the psychological health of PSP. Overall, participants' perceptions of the program were positive. As expected, improvements in symptoms of PTSD, depression, anxiety, and stress, and alcohol use over time were observed in participants who attended the program. Similarly, improvements were observed in measures of quality of life, perceived social support, resilience, emotional regulation, and stigma. Small, statistically significant changes were only found for improvements in PTSD, quality of life, perceived social support, and stigma from Time 1 to Time 4.There have been few evaluations of mental health training programs for PSP  ensuring that psychological support is available to those who complete the program; (2) offering different delivery modalities ; (3) introducing coping skills earlier in the program; (4) biweekly maintenance sessions (instead of monthly meetings); (5) emphasizing participation in the program early in PSP careers; and (6) having the program offered at varied times to accommodate PSP shift work. The exploratory qualitative analysis suggested that the improvements that participants attributed to BOS may not have been adequately captured by the outcome measures used in the evaluation. For example, participants reported changes in self-awareness and self-monitoring of feelings and thoughts; behavioral changes ; and improved relationships with family and colleagues. The outcomes identified for BOS were not directly measured as part of the current evaluation. Inclusion of such measures in ongoing evaluations of the program may be beneficial in highlighting potential benefits of attending the BOS program.The current study results contribute to a large gap in understanding the effectiveness of programs for PSP, and the current limitations provide important directions for future research. A significant study limitation is participant attrition. Research participation was not mandatory for BOS registrants and 155 of 203 (76.4%) registrants expressed interest in research participation; however, only 81 participants completed the survey at Time 1, 66 completed the survey at Time 2, 29 completed the survey at Time 3, and 19 completed the survey at Time 4. Survey responses were also inconsistent; for example, some participants completed the first survey and one other survey throughout the data collection period resulting in incomplete data sets for participants. We are unable to assess why participants did not complete certain studies, but future studies investigating demographic variables associated with non-completion see  appear wPSP are at risk of developing mental health problems. Participation in proactive programs focused on protecting PSP mental health may reduce the risk and encourage early help-seeking. The BOS program is a clinically- and evidence-informed 8-week, group-based mental health training program combining theoretical and experiential learning to mitigate the impacts of operational stress. BOS is also delivered by PSP culturally competent facilitators who have received special training and have expertise in working within the context of PSP environments.Early evidence suggests that participation in the BOS program supports small, statistically significant improvement in PTSD symptoms, quality of life, perceived social support, and stigma until at least 6 months from baseline. Qualitative responses revealed other areas of improvement, including self-awareness, behavioral changes, and improved relationships with family and colleagues, which were not measured quantitatively. Further evaluation and research are needed to better understand how long outcomes are sustained, and to assess for sociodemographic differences. BOS appears to be a promising training program for the proactive protection of PSP mental health and is one of few such programs with any empirical support. All training programs intended to provide proactive protection for PSP mental health must develop an empirical base to support widespread adoption, continuous improvement, and culturally-appropriate options for clinicians working with PSP.andrea.stelnicki@uregina.ca.The datasets presented in this article are not readily available because of potentially identifiable information that could compromise participant confidentiality. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by University of Regina Institutional Research Ethics Board (File #2018-191). The patients/participants provided their written informed consent to participate in this study.AS and RNC managed research design, recruitment, and data collection. AS, LJ, and AF were responsible for data analysis. All authors contributed to the first manuscript draft, manuscript revision, and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.