ARID1A were detected at a high frequency in cholangiocarcinoma (CCA). Growing evidence indicates that the loss of ARID1A expression leads to activation of the PI3K/AKT pathway and increasing sensitivity of ARID1A-deficient cells for treatment with the PI3K/AKT inhibitor. Therefore, we investigated the association between genetic alterations of ARID1A and the PI3K/AKT pathway and evaluated the effect of AKT inhibition on ARID1A-deficient CCA cells.Genetic alterations in ARID1A, PI3K/AKT pathway-related genes, clinicopathological data and overall survival of 795 CCA patients were retrieved from cBio Cancer Genomics Portal  databases. The association between genetic alterations and clinical data were analyzed. The effect of the AKT inhibitor (MK-2206) on ARID1A-deficient CCA cell lines and stable ARID1A-knockdown cell lines was investigated. Cell viability, apoptosis, and expression of AKT signaling were analyzed using an MTT assay, flow cytometry, and Western blots, respectively.Alterations of ARID1A alterations significantly co-occurred with mutations of EPHA2 (p < 0.001), PIK3CA (p = 0.047), and LAMA1 (p = 0.024). Among the EPHA2 mutant CCA tumors, 82% of EPHA2 mutant tumors co-occurred with ARID1A truncating mutations. CCA tumors with ARID1A and EPHA2 mutations correlated with better survival compared to tumors with ARID1A mutations alone. We detected that 30% of patients with PIK3CA driver missense mutations harbored ARID1A-truncated mutations and 60% of LAMA1-mutated CCA co-occurred with truncating mutations of ARID1A. Interestingly, ARID1A-deficient CCA cell lines and ARID1A-knockdown CCA cells led to increased sensitivity to treatment with MK-2206 compared to the control. Treatment with MK-2206 induced apoptosis in ARID1A-knockdown KKU-213A and HUCCT1 cell lines and decreased the expression of pAKTS473 and mTOR.The analysis of a total of 795 CCA samples revealed that These findings suggest a dependency of ARID1A-deficient CCA tumors with the activation of the PI3K/AKT-pathway, and that they may be more vulnerable to selective AKT pathway inhibitors which can be used therapeutically. Opisthorchis viverrini (Ov) (FGFR), isocitrate dehydrogenase (IDH1/2), human epidermal growth factor receptor (HER), neurotropic tyrosine kinase receptor (NTRK) fusions, and BRAF mutations have been identified in CCA tumors (Cholangiocarcinoma (CCA) is a malignancy arising from the epithelial cells along the biliary tree. The highest incidence rates of CCA have been reported in northeast Thailand, which is the endemic area of the group 1 carcinogen, the liver fluke, ini (Ov) . Cholangini (Ov) . Althougini (Ov) . Thus, mA tumors .ARID1A is commonly inactivated in tumors including CCA (ARID1A has resulted in a significant increase in proliferation in vitro (ARID1A mutations is involved in carcinogenesis via activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway  is a crucial non-catalytic subunit of human switch/sucrose nonfermentable (SWI/SNF) complex . It playding CCA . Silenciin vitro . In CCA in vitro . Recent  pathway . Co-occuc cancer . Silenciand PI3K , suggestt tumors .ARID1A mutations and the depletion of ARID1A protein expression sensitized cancer cells to PI3K/AKT inhibitors. In breast and gastric cancer, ARID1A-depleted cells showed an increased sensitivity to PI3K and AKT inhibitor compared to wild-type cells  , KKU-055S473 , AKT , mTOR , Bax , Bcl-2 , and β-actin .The following reagents and antibodies were used: MK-2206  was dissolved in DMSO at a concentration of 10 mM as a stock solution, ARID1A , phospho-AKThttp://cbioportal.org), a web-based, open-access resource for the analysis of cancer genomics data from The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC). In the present study, somatic mutations of ARID1A, genes in RAS/PI3K/AKT pathways , clinicopathological data and patient survival of 795 CCA patients/798 samples were analyzed. Collectively, the six data sets included a TCGA data portal (Firehose Legacy) and a ICGC data portal , ARID1A#2  and non-targeted shRNA  using polybrene . The following shRNA sequences were used: non-targeted shRNA: sense: CCGGGCGCGATAGCGCTAATAATTTCTC, shARID1A#1: CCGTTGATGAACTCATTGGTT, and shARID1A#2: GCCTGATCTATCTGGTTCAAT.The cholangiocarcinoma cell lines were infected with MISSIONAfter 24-hours of infection, cells were selected using 1–2 µg/ml of puromycin . Expression levels of ARID1A were confirmed by real time-PCR and Western blot.Cell viability was determined by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay . Cells were seeded into 96-well plates . After 24-hour exposure of inhibitor, 100 µl of 0.5 mg/ml MTT reagent was added and incubated for 2 h. After adding DMSO for 15 min, the absorbance was measured using a microplate reader at a wavelength of 570 nm. Each experiment was performed in triplicate and the results were given as means ± SD. The percentage of cell viability was calculated using the formula: % cell viability= (Nt/Nc) x100. Nt and Nc refer to the absorbance of the treated and control groups, respectively.Cells were lysed in RIPA lysis buffer. Protein lysates were centrifuge at 14,000g for 20 mins at 4 °C. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit . Protein lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk or BSA in 1xTBS  for 1 h at room temperature. Membranes were subsequently incubated with primary antibodies overnight at 4 °C. After washing, the secondary goat anti-Rabbit IgG-HRP  or rabbit anti-Mouse IgG-HRP  was used. The immunoreactive signals were visualized using Amersham™ ECL™ Prime Western Blotting Detection Reagent .Cell apoptosis was detected using an Annexin V-FITC and propidium iodide (PI) Kit  according to the manufacturer’s protocol. Briefly, cells were seeded into 6-well culture plates overnight. Cells were then exposed to MK-2206 at designated concentrations and 0.3% DMSO was used as the control. After 24 h, cells were trypsinized, washed with ice-cold PBS, and resuspended in binding buffer containing Annexin V-FITC, whereupon, Annexin V/PI was added. Cells were resuspended in reaction buffer containing PI and immediately analyzed by BD FACSCanto II Flow cytometry  to detect the rate of apoptosis.® Reagent  according to the manufacturer’s protocol. Subsequently, 2 µg of total RNA was converted to cDNA using High-Capacity cDNA Reverse Transcription Kit . Real-time reverse transcription polymerase chain reaction  was performed using TaqMan gene expression assay: TaqMan probes  to detected mRNA levels of ARID1A and β-actin. Real time PCR was performed using the ABI real-time PCR system, Quantstudio™ 6 Flex . β-actin was used as the housekeeping gene.Total RNA was isolated from cell pellets using TRIzol50) values were calculated by dose–response curves (Y = 100/(1 + X/IC50)). A two-tailed unpaired t-test was used for two-group comparisons. For multiple group comparisons, one-way analysis of variance, Kruskal-Wallis test, and Fisher’s Least Significant Difference Test (LSD Test) test were used. A two-sided p<0.05 was considered statistically significant. Adjusted p-values were calculated using Benjamini–Hochberg correction.All experiments were repeated at least two times. Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 23.0 software  or GraphPad Prism v.8.0  software. Overall survival (OS) curves were constructed according to the Kaplan–Meier estimator and differences between curves were tested for significance by means of log-rank tests. The half inhibitory concentration  in CCA (ARID1A were found in 19% (137/711) of CCA patients. Truncating ARID1A mutations were the most prevalent in ARID1A mutated CCA  , III  and IV , ARID1A mutation were predominantly in CCA with high tumor stage and may involve in CCA progression. We then investigated if ARID1A mutations co-occurred with mutations in the kinase-related pathway (ARID1A mutations were significantly correlated with mutations of EPHA2 (adjusted p<0.001), PIK3CA (adjusted p = 0.047), and LAMA1 (adjusted p = 0.024) (EPHA2 was mutated in 9% (46/516) of CCA patients   harbored ARID1A mutations (EPHA2 mutant tumors co-occurred with ARID1A truncating mutations (PIK3CA mutations was 6% (41/711), and 80% (33/41) of PIK3CA-mutated CCA tumors were driver missense mutations   of CCA with PIK3CA driver missense mutations harbored ARID1A-truncated mutations (LAMA1 was mutated in 4% (20/516) of CCA  in CCA . Somatic116/137) –2, 3A. T pathway . Interes= 0.024) . EPHA2 wpatients . Truncat0Rfs*33) –2, 3A. Autations , 3B. Of utations . The fre H1047L) . We founutations . LAMA1 w) of CCA . LAMA1 mA tumors , 3A and utations , Fig. 3Bf ARID1A . We did  pathway .ARID1A, EPHA2, PIK3CA and LAMA1 in CCA, we further performed survival analysis in CCA tumors with or without mutations of ARID1A, EPHA2, PIK3CA and LAMA1 , while co-occurrence of ARID1A-EPHA2 mutations was significantly correlated with poor overall survival .To examine the prognostic survival values of mutational co-occurrence between mutations of nd LAMA1 . As showARID1A truncating mutations exhibited the loss or reduction of ARID1A protein expression  when compared to the control  when compared to the control . Even though, ARID1A-silencing in CCA cell lines minimally increased phosphorylation of AKT at Ser-473 (pAKTS473), treatment with MK-2206 (30 µM) significantly reduced pAKTS473 level in ARID1A-knockdown KKU-213A and HUCCT-1 cell lines compared to the non-targeting control shRNA . Of note, in vitro and in vivo experiments revealed that EPHA2 mutations led to ligand-independent phosphorylation of Ser897 and were associated with lymph node metastasis of ICC  mutations harbored ARID1A-truncated mutations. This evidence suggests a synergistic mode of ARID1A mutations and PIK3CA activation, which resulted in the activation of the PI3K/AKT pathway. Importantly, our results have shown for the first time the association between mutations in ARID1A and LAMA1. LAMA1 is a subunit of laminins family. Laminins are the main component of the basement membrane, and they can promote tumor growth and metastasis . The effectiveness of AKT inhibitor (MK-2206) in inducing apoptosis was reported as a monotherapy in CCA cell lines in vitro . A phasehibitors . Here, wparlisib . Moreoveer cells . Lee et SK690693 . Likewisrcinomas , the effo models . A previo models , suggestARID1A mutations and PI3K/AKT pathway, its exact mechanism and function in human cancer cells has yet to be fully elucidated. We also acknowledge that a limitation of our study is the lack of in vivo experiments. Future work should investigate MK-2206 properties in vivo and center on developing more effective combination therapies to improve treatment efficacy. Further assessment of the mechanism of action could shed light on the clinical utility of using AKT inhibitors to treat CCA patients harboring ARID1A alterations.There were some limitations in our study. Although our data provide in silico information regarding the association between in vitro. Additionally, our results have shown the co-occurrence of genetic alterations of ARID1A with the PI3K/AKT pathway in CCA tumors. The findings suggest a synthetic lethal interaction between the loss of ARID1A and the inhibition of the PI3K/AKT pathway. Furthermore, results from our study provide a sound basis and a unique opportunity for predicting favorable treatment responses to small molecule inhibitors of the PI3K/AKT pathway on ARID1A-mutated CCA which can improve treatment outcomes.Our results have demonstrated that depletion of ARID1A leads to a significantly increased sensitivity towards AKT-inhibition in CCA cells 10.7717/peerj.12750/supp-1Supplemental Information 1https://www.cbioportal.org).The Oncoplot was generated on cBioPortal . Densitometric quantification of the relative Bax/Bcl-2 expression in CCA cell lines were obtained using ImageJ . Cells were treated with MK-2206 for 12 h (12 h) or 24 h (24 h) and 0.3% DMSO was used as the control (Ctl). KKU-213A, ∗p = 0.032 and HUCCT1, ∗p = 0.025 and 0.018 respectively, unpaired t-test.Relative Bax/Bcl-2 levels were increased in Click here for additional data file.10.7717/peerj.12750/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12750/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.12750/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.12750/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.12750/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12750/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.12750/supp-9Supplemental Information 9Click here for additional data file.


Plant-based nanotechnology programs using virus-like particles (VLPs) and virus nanoparticles (VNPs) are emerging platforms that are increasingly used for a variety of applications in biotechnology and medicine. Tobacco mosaic virus (TMV) and potato virus X (PVX), by virtue of having high aspect ratios, make ideal platforms for drug delivery. TMV and PVX both possess rod-shaped structures and single-stranded RNA genomes encapsidated by their respective capsid proteins and have shown great promise as drug delivery systems. Cowpea mosaic virus (CPMV) has an icosahedral structure, and thus brings unique benefits as a nanoparticle. The uses of these three plant viruses as either nanostructures or expression vectors for high value pharmaceutical proteins such as vaccines and antibodies are discussed extensively in the following review. In addition, the potential uses of geminiviruses in medical biotechnology are explored. The uses of these expression vectors in plant biotechnology applications are also discussed. Finally, in this review, we project future prospects for plant viruses in the fields of medicine, human health, prophylaxis, and therapy of human diseases. Over the past few years, plant viruses have increased in visibility for a wide range of applications in biotechnology. Plant viruses are highly suitable for production of vaccines as they are recognized by the innate immune system through the pathogen associated molecular pattern (PAMP) receptors  while bePlant virus genomes have been engineered to express heterologous open reading frames. For example, deconstructed virus vectors  were genPlant viruses have also been developed as VLPs and VNPs in order to present them as epitope display systems for vaccine production and as scaffolds for the conjugation of drugs or molecules used in diagnostics . VNPs anVLPs are a subset of the VNPs but bereft of any nucleic acid genome, thus, making them noninfectious. VLPs are powerful vaccine candidates as they simulate the conformations of native viruses, utilizing their intrinsic immunogenicity while not compromising their safety. They accomplish this by having no viral genome, and therefore being unable to replicate . Hence, Knowledge and insight into the molecular structure of TMV  and PVX The following review describes many of the uses of plant viruses in biotechnology, with examples based on TMV, PVX, CPMV, and geminiviruses. In the last section, we conclude with a future projection of the significance of plant viruses in the fields of medicine and engineering. TMV was initially characterized in the 19th century and has since become a paradigm for our current perspective on the morphogenesis of self-assembling viral particle structures . TMV is TMV is also simple and well-characterized with respect to particle structure and genome organization. Thus, it is well suited as a highly amenable experimental system for different applications. The rod-shaped virus particle measures 300 nm in length and 18 nm in diameter, and contains a 6.7 kb viral RNA genome that is encapsidated by 2130 identical copies of the capsid protein assembled in a helical arrangement. The crystal structure of the 158 amino acid capsid protein has been determined . The genDifferent strategies can be used to modify TMV, such as the modification of the interior or exterior surface of the capsid through genetic engineering, chemical conjugation, or a combination of both processes. The interaction and transport of heterologous cargo within the virus inner cavity or generation of multivalent structures by particle integration have thus been adopted. The conformation of the TMV CP facilitates the insertion of foreign peptides at both its N- and C-termini. In addition to this, the loop formed from CP amino acids 59–66 can be used towards surface display of foreign peptides on intact virions or on CP assemblies . The location of C-terminus of the TMV CP on the exterior surface of assembled TMV virions makes it the most used site for insertion of foreign peptides. TMV particles have been exploited for active enzyme display, with wide-ranging uses in biodetection, sensor development, medicine, and enzymatic conversion. Enzymes such as penicillinase ,63, horsTMV membranes have been engineered that could be recruited as tissue engineering frameworks by sequentially altered layering of two TMV variants with different charges. Recently, these TMV-based carrier templates have been used to prepare surfaces that promote cellular attachment and differentiation ,67,68. Some cells have been cultivated on TMV-covered culture supports and peptide ligands have been presented in a spatially defined manner over nanometric scales. Arginine–glycine–aspartic acid peptide associated TMV layers have been used for osteogenesis of stem cells from bone marrow ,68. TMV Colletotrichum acutatum, a phytopathogenic fungus which proved to efficiently assemble virus particles inside hyphal cells . VLPs can be engineered to display on their surface functional groups such as ligands for targeting, epitopes, imaging dyes, and drug payloads. The VLPs by virtue of their size and shape facilitate vascular transport, active cellular uptake, and molecular interactions. VLPs can tolerate harsh environments while being biocompatible. In addition, high doses of VLPs are mostly well tolerated and the VLPs are completely and rapidly cleared by proteolytic degradation to enable diminished side effects. Moreover, the characteristic ability of the VLPs to self-assemble coupled with novel molecular design using chemical biology technologies enable the production of functionalized hybrid VLP nanomaterials. The field of VNP- and VLP-based technologies for drug delivery applications continues to evolve with several candidates in clinical trials that should lead to advanced therapeutics, in the near future. In the future, it is very much likely that more plant viruses would be genetically engineered and repurposed for further use in biotechnology and medicine.


Durign recrutiment and the study period, there were approximately 1000–2000 new cases of COVID-19 daily in central Pennsylvania . The overall adherence rate to the sessions was 91%, and no participants reported new or existing COVID-19 symptoms or diagnoses during the two-week study period or 10-days following the final session. Despite enforcing mandatory COVID-19 safety measures , the participants reported enjoying the program and feeling socially connected with one another, while perceiving the program to be safe. Additionally, all participants expressed a desire to participate again, if the program were offered in the future. These results suggest that an outdoor group sport play program for adults can be feasible, acceptable, and well-adhered to despite enforcing mandatory COVID-19 safety precautions. Physical activity programs similar to the one presented here may provide an easily-adaptable approach to outdoor physical activity during the current and potential future pandemics that have viral transmission characteristics similar to COVID-19.The purpose of this study was to investigate the feasibility, acceptability, and short-term adherence to an adult outdoor group sport play program designed to maximize enjoyment that was modified to incorporate COVID-19 risk mitigation strategies. We enrolled 17 healthy, but sedentary, central Pennsylvania adults ( One highly apparent disruption to health-related behavior due to the pandemic is a reduction in physical activity (PA), driven by factors such as mandatory stay-at-home orders, quarantines, and fearfulness of contracting and/or transmitting the disease. Several large-scale studies have reported decreased PA along with increased sitting and screen time, especially among those who were most physically active prior to the pandemic . The resOther recent data suggest that the mental health of many people around the world has deteriorated over the course of the pandemic. Specifically, people report feeling less connected to others, more stressed, and more likely to report symptoms of depression and anxiety . Given tThe most recent evidence suggests COVID-19 is an airborne virus transmitted from person-to-person through respiratory droplets released into the air when talking, coughing, or sneezing . When peIn the rapidly changing landscape of the pandemic, transmission prevention is paramount to decrease risks to individuals in those communities most likely to experience severe illness symptoms or death . These transmission prevention efforts are also crucial for athletes and physically active individuals to minimize interruptions in training and possible short-and long-term adverse effects on an infected individual’s respiratory and cardiovascular systems, such as myocarditis . Spread a priori hypotheses.Despite the accumulating evidence of how to safely “return to sport” during the COVID-19 pandemic, it is unclear how the PA experience of individuals may be effected by mandatory safety protocols or whether people actually have interest in returning to group PA during the pandemic. For example, recent evidence has shown that masking during incremental PA can lead to mild hypercapnea . For som2This study is part of a larger parent randomized-controlled trial of adult PA funded by the National Heart, Lung, and Blood Institute that was indefinitely suspended with the onset of the coronavirus pandemic, in March 2020 . The par2.1Given the growing body of evidence suggesting that people who perceive PA as enjoyable are more likely to both adopt and adhere to it , this adPlayFit incorporates several tenets of self-determination theory , hedonic2.22 . During a condensed two-week recruitment period, we mailed approximately 5,100 letters to adults living in the same county as the research site. Of those who received letters, 98 (1.9%) responded with interest, and 52 were further screened for eligibility (see n = 15) or reported a BMI ≥ 30 (n = 13). Of the 21 eligible individuals, 17  provided informed consent and enrolled in the study. This rate of interest in participating in the study was similar to the rates we observed for our parent trial that recruited prior to the COVID-19 pandemic, where, of approximately 21,000 letters mailed, 3% contacted our team expressing interest. However, given these more stringent eligibility criteria, it is inappropriate to compare actual rates of enrollment.We recruited adults between 18 and 50 years of age, healthy enough for moderate-to vigorous-intensity physical activity , sedentary , and not participating in another PA or weight loss program. Additionally, because of the emerging risk-factors associated with severe COVID-19 symptoms, we implemented additional exclusion criteria . These i2.3Program attendance. Attendance was recorded at each session by the group leader.Perceptions of the program. During each debriefing phone conversation following the end of the study, the participants were asked several questions regarding their perceptions of the PA program and the COVID-19 safety modifications. Enjoyment was assessed using a single, face-valid item: “On a scale of 0–10, with 0 being no enjoyment and 10 being the highest amount of enjoyment: How much did you enjoy the exercise program?” We also used 0–10 scales to assess how likely each participant was to recommend the program to a friend, family member, or colleague , instead providing general comments to the entire group . Individual positive feedback was reserved for private conversations. Celebrations between participants and the leader were limited to verbal interactions . Once the COVID-19 screening protocol was completed, a trained PlayFit leader paired participants to play five-minutes of “catch” using the sport implement of the day as a warm-up prior to gameplay. The participants were randomly assigned to teams each day and wore colored mesh jerseys to differentiate one another. During the 60-minute play session, individual periods were eight-minutes long, and each period was followed by a five-minute socialization, water, and sanitization break.The PlayFit sessions were held outdoors on a level, grass soccer field in central Pennsylvania during October 2020, from 5:30 pm-6:30 pm, on four weekday evenings with at least one day between sessions, over two consecutive weeks. Because PlayFit is designed to be easier to play, especially for individuals with low motor skillfulness and who are sedentary, overweight, and/or unfit, the field size was 180′ x 90′ and demarcated using cones. We used small (4′ x 6′) pop-up nets for soccer and handball. A slightly under-inflated volleyball  was used for soccer, to reduce the distance and speed at which it could travel along with making it lower impact if it struck the body. A soft, flexible Frisbee was used for ultimate Frisbee to reduce flight distances and difficulty with catching. For similar reasons, a foam football was used for ultimate football. Finally, a rubberized dodgeball was used for handball. The rulesets for each game were also minimized. For example, we did not keep score. Participants could stand and attempt to score from anywhere , drops or out-of-bounds were automatically turned-over to the other team, and there were no goaltenders. During ultimate Frisbee, football, and handball, a player could take up to 2-steps before passing to an open teammate using any type of pass or attempting to score. The players could take a break at any time by “switching out” with a teammate or could simply reduce their effort without repercussion. To avoid singling out individuals, when providing public feedback to participants, the session leader avoided specific comments , and BMI category . In addition, we examined relationships between attendance rate and sex, age group, and BMI category using chi-squares tests. Because we conducted 15 tests of probability, our Bonferroni-corrected alpha was set to 0.003 , to reduce the likelihood of Type I error.3COVID-19 symptoms and/or diagnoses. No participants reported or were screened by research staff to have COVID-19 symptoms before, during, or after each of the PA sessions. In addition, no participants reported new COVID-19 symptoms or diagnoses during debriefing interviews 10-days following the final session.Program attendance. The session attendance rate during the four sessions was 94%, 100%, 94%, and 76% . The first three sessions were held in clear, sunny, weather and the final session was overcast and cooler with light rain. There were no significant relationships between program attendance and sex, age group, and BMI category.Program perception. Overall, the participants reported a mean enjoyment score of 8.9/10 , a mean score on the likelihood to recommend the program to others of 9.4/10 , and all participants (n = 17) responded that they would be willing to continue the program if offered in the future. The mean score for perceptions of safety was 8.6/10  and, despite the inherent limitations created by compulsory masking and social distancing, the mean score for perceptions of social connectedness to others was 7.9/10 . Finally, there were no significant differences in the mean scores on these variables based on sex, age, or BMI .Qualitative program perception. A selection of program qualitative responses are presented in 4The purpose of this study was to examine whether an adult outdoor group sport play program designed to maximize enjoyment that incorporated COVID-19 risk mitigation strategies was feasible, acceptable, and well-adhered to, while remaining enjoyable. Our results suggest that the program was feasible, well-accepted, and likely to be adhered to over the short-term among healthy, but sedentary adults, albeit those without obesity. Despite enforcing safety precautions, such as universal masking and social distancing, the participants, in general, reported enjoying the program, as well as experiencing feelings of social connectedness with the other participants. In addition, the overall attendance rate was 91% and no participants reported new COVID-19 symptoms or positive test results during the two-weeks of activity sessions and over the 10-days following the final PA program session. While not as overwhelming as post-Thanksgiving  2020, COVID-19 was circulating actively at that time in the Pennsylvania community where the PA program sessions were held .any program that allowed for socialization because of the negative social and mental health impacts of mandatory stay-at-home orders and quarantine requirements. Indeed, several participants expressed their happiness with having the opportunity to socialize during the pandemic, without specifically alluding to PA. These individuals may not have been as motivated to join this specific PA program if other options were available in a non-pandemic setting. In addition, the PA sessions were conducted outdoors and safe group PA programming may be more difficult and sometimes infeasible indoors, as the poor ventilation often present indoors could be attributed to increased spread of COVID-19 , as some researchers suggest the likelihood of future pandemics is growing due to increased globalization, urbanization, climate change, human-animal contact, and health care worker shortages .Matthew A. Ladwig: Conceptualization, Methodology, Data curation, Writing - original draft, Investigation, Supervision, Formal analysis. Christopher N. Sciamanna: Conceptualization, Methodology, Writing - review & editing, Supervision. Kayla Rutt: Methodology, Investigation, Writing - review & editing. Joshua Blaker: Methodology, Investigation, Writing - review & editing. Kalen Kearcher: Methodology, Investigation, Writing - review & editing. Brandon J. Auer: Methodology, Writing - review & editing. Liza S. Rovniak: Methodology, Writing - review & editing. David E. Conroy: Methodology, Writing - review & editing. Jinger S. Gottschall: Methodology, Writing - review & editing. Matthew L. Silvis: Methodology, Supervision, Writing - review & editing. Joshua M. Smyth: Methodology, Writing - review & editing. Ming Wang: Methodology, Writing - review & editing.The authors declare the following financial interests/personal relationships which may be considered: Christopher Sciamanna has an investment, such as stock, in a company which has begun to investigate the possibility of creating a business that provides exercise programs.


We discuss how to assess computationally the aesthetic value of “small” objects, namely those that have short digital descriptions. Such small objects still matter: they include headlines, poems, song lyrics, short musical scripts and other culturally crucial items. Yet, small objects are a confounding case for our recent work adapting ideas from algorithmic information theory (AIT) to the domain of computational creativity, as they cannot be either logically deep or sophisticated following the traditional definitions of AIT. We show how restricting the class of models under analysis can make it the case that we can still separate high-quality small objects from ordinary ones, and discuss the strengths and limitations of our adaptation. Creative objects with small complete combinatorial representations  can be of extraordinary influence in the creative world. In some cases, the small object may be a member of a class of other small objects, but an exemplary member of that class. For example, very short poems, such as Basho’s haiku , or hymnOften, these short works can be, at least in part, reactions to earlier, larger objects. William Carlos Williams’s very short poems  and symbolic music , while the continuous set includes live performances, photography, movies, and paintings.Producers of these various art forms probably do not spend much time thinking about whether they are creating a fundamentally digital object or not, but in a digital era, the difference is significant for a number of reasons. For example, there is no such thing as a higher-resolution version of a novel; a proper encoding of the complete text is a full representation of the creative artifact. Given that novels in English tend to be fewer than 100,000 words, this means that a novel can be represented in ASCII encoding in a few megabits, and transmitted in less than a second. Adding more storage to the representation does not make for a better reading experience. Obviously, one could include historical versions of the text, annotations, criticism, contextual information and other augmentations of that sort. However, the core text can be stored at a scale of over a thousand novels per gigabyte; even the entire text of the King James Bible requires only a few megabytes.By contrast, advances in digital photography technology, for production, storage, and compression, give viewers increasingly accurate representations of the image that the artist sought to generate. The 320 × 200-pixel images in sixteen colors that a Commodore 64 computer had in the 1980s could be stored in 32 kilobytes, but offered poor detail, no ability to properly indicate shadow or dimensionality, and (obviously) low resolution and color reconstruction. A 2019 Mac has the resolution of 5120 × 2880 pixels in over a billion colors, requiring roughly 2000 times as much video memory and an astonishing amount of computation to show a user images. However, it is also able to represent artwork in vastly greater detail; further, super-high-resolution images of famous artworks can require gigabytes of space, and curators and art historians can use them as primary research tools , given tThis striking contrast in file size may seem like a curiosity, particularly now that we are far enough removed from the Commodore 64 era that image (and audio and video) quality issues are rarely major concerns, and most of us do not even store audio files locally anymore in the streaming music era.Yet the problem of “smallness” creates real challenges for computational creativity: with many fast algorithms, we can easily generate hundreds of thousands of poems or short stories or song lyrics or musical segments in a second, and this creates the need to also evaluate their quality in real time. This is not easy: authors often identify a collection of desiderata that characterize good examples of the genre they are creating and then score the generated pieces against computationally efficiently measurable analogues of each desideratum, highlighting the most successful piece. Ensuring novelty can be a real challenge in this context.We explore a variety of desiderata for creative objects in a recent paper , using tx is the length of the shortest input p to a universal Turing machine U, where on input p, U computes x and halts. Often, we ignore the details of this universal Turing machine U, but in our case, we will spend a fair amount of time limiting the possible inputs to U and restricting the possible behavior of the machine U, or of the machines it simulates. The conditional Kolmogorov complexity of y given the string x, U for which U outputs y, given that U is also provided with a read-only tape on which the string x is pre-loaded. If x provides a great deal of information in planning to later compute y, while if x and y are largely unrelated.To be more specific, we define some of the basic concepts of algorithmic information theory here that apply to our recent work. The Kolmogorov complexity x: a program p that describes the regularity of x and how to compute it, combined with a number of bits of random information y needed to describe the remaining details in x. In this formulation, if we use U to simulate the running of the program p on input y, then U again halts with output x. The usefulness of this idea is that it allows us to explore other inputs to the program p; naturally, this approach is most effective if p does not output x on all inputs. In this formulation, a model for x is a program p which, on some input data y outputs x, and the pair two-part code encoding of x.One way of understanding U, p and y is to see U as running with two (read-only) input tapes that are initialized with p and y, and a work tape; then, we say that U simulates that x on the work tape. To make this domain much more tractable, we require that U computes only on a prefix-free set, that is, if U halts on y, then U goes to an error state on the input y; similarly if U halts on p, then U goes to an error state for the input p. The prefix-free property can seem a bit unnatural, but in the domain of computer programs, this is equivalent to having an END statement at the end of the program p, and for inputs, it can represent a special end-of-file character. An alternative easy way to impose the prefix-free property is to represent a binary object z of length n by the string x have a unique p to be a partial function where U halts on input A clean way of describing the relationships among x that have short data-to-model codes U than those with only longer encodings. This is best described by the algorithmic probability of x: given a universal machine U with the prefix-free property on the inputs it accepts, the algorithmic probability of x isx and U’s second input tape. This probability is the likelihood of x being the outcome of p, a program of interest. Since the smallest x has length x, which is the largest contribution. Let the language of the model p be the set x that p generates.In this framework, strings equality ; we do np we permit, and will do this extensively in later sections of this paper. For example, we can require that p always accepts , that p computes an injective function, that c, and even make restrictions on the size or resource use (space or time) of p. We can require that Q to guarantee that all possible objects of a type are output. Some of these restrictions correspond to substantial demands on the space of valid models and dramatically expand the computational requirements for a valid model; it is possible that under these restrictions, the shortest model for x might be quite a bit longer than x might no longer be a computable string at all with the restrictions. It is also possible that testing whether a program p satisfies the restrictions is itself uncomputable: for example, the requirement that p always accept is untestable, due to the uncomputability of the halting problem. For any property p, p exists, then U (the representations of p and its input y) may be required to be prefix-free, we can consider p and y to be extracted from that prefix-free representation, so by the time the universal Turing machine simulates P on y, the input to p can be assumed to be of length We can further restrict the class of valid models x. The first program is the “print” program, which requires that its inputs all are of the form x on the work tape and halts. This program is of constant size , and for any valid input, x in runtime There are two programs that form special cases that we most likely do not want to have as optimal models for the string x” program, which stores the string x in full detail in its structure, and on every input y, simply prints x on the output tape and halts. This program is of length c, and requires x, which it outputs with probability 1.The second pathological program is even simpler: it is the “print deterministic and non-probabilistic. As such, actions such as “print out a random string of length n” cannot be encoded for this model.Note that the Turing machines we consider in this article are We now define several of the key concepts in the evaluation of creative agents and explain how these concepts integrate with advanced ideas from algorithmic information theory, as defined in our recent paper . A somewtypicality that we give in our recent paper is that, given an “inspiring set” S of objects in our class, and a good model M whose language is a superset of S, the typicality of a new object x is the negative of randomness deficiency of x with respect to M, or M if M generates it with parameterizations that cannot be better represented by changing the model itself: the most typical examples of M are those with a typicality of zero.The definition of c, and the typicality of x with respect to the model M is as defined in the previous paragraph.Of course, as alluded to in the previous section, we must do a good job of defining the class of models we are considering. In our previous paper, we restrict the class of valid models to those for which the model complexity along with the data-to-model code of each member x in two related ways. The first approach uses logical depth to model the inherent computation of the object itself: if all of the short programs for x require substantial computation to generate x, then the best explanation for the creation of x is one that involves substantial effort, and hence, x is of value. To be specific, let b, where x. Again, in this frame, if x is logically deep, it is valuable since it attests to the likely effort that the creator engaged in while building the object x. In the algorithmic probability sense, a logically deep string is a string for which the most likely explanations all require substantial computational effort; while U generates x with some long, quick programs, the bulk of the probability mass is on the shorter, slower programs.Similarly, we define the value of an object sophistication. The sophistication of an object x is the length of the shortest program for which x is a typical output. Formally, let c, p; the model represents the complexity of the creator itself, and the object is satisfying because it would only come from a simple creator in a surprising outcome. To consider our two pathological examples, if x” program is a good model for x since the former requires a too-long argument and the latter a too-long program. However, if x is a random string, then the “print” program is a good model for x. By contrast, if x is still a typical output of a simple program that reads in a binary representation of n and prints out that many 1 s. So, despite being highly-compressible, x is still unsophisticated. Said in a different way, with a small value of c, the tolerance constant in the definition of sophistication, the minimum total program for x is a trivial program that reads in a c-length value (the length of x) and then outputs a string of 1 s of that length.The second approach for the value of an object is its x with a short input, then the execution of that program will be lengthy. See Antunes et al. [It is an important theorem that sophisticated objects are all of high logical depth: that is, if it requires a complex program to compress a non-random string s et al.  for moreS of small objects, it will be hard to beat the trivial model M that just lists off those elements, and then simply prints off the new object, with no real computation involved in any of the explanations. The sophistication of a small object x is going to be small, because the “print” program has x as a typical output, and yet, it is a small program. Compressibility, and the effort found in compression, is not sufficient to explain small objects.Unfortunately for us, this overall approach does not work immediately with small objects. Short objects all have trivial short descriptions; including enough background information to describe the class that an object belongs to makes the overall program much, much larger than any small object. As such, the logical depth of any small object is itself small, and distinguishing among these objects is impossible in this manner. Similarly, for any finite inspiring set x, where x is complete; that is, there exists no string k, how can we identify if x is of high value, or of high novelty or typicality with respect to an inspiring set S, relative to some other small object y?Now, we at last can present the problem of small objects. We know that small objects x is a haiku. To build a program p to generate a good-quality English language haiku, we would need to represent English grammar and vocabulary, syllable counts and so on, all as part of the program p. Thus, p will presumably require megabytes of code and data; after doing so, we might be able to identify x by an input y that is a bit shorter than x, and might even be able to express some aesthetic judgment in prioritizing “good” haiku.The reason why this problem is serious comes from the low value of p is an invalid model for x since p is not allowed. Similarly, x” program is short enough to be a valid answer to the question “how was x generated?”, and its runtime is approximately n; the “print” program run with the input x also generates x in approximately n time, and the pair n. It is possible in some rare cases that a small object might have a very short program that compresses it even more, but it would be very surprising if the best very short program allowed the expansion of x in only very long runtimes indicative of much logical depth. Finally, x cannot be sophisticated because parameterizing the “print” program to output x will be a valid choice.However, by our definition of typicality, the program How can we use algorithmic information theory to differentiate the value and typicality of small objects?x for which not only x, but the set of all objects of a size smaller or equal to x is itself a small object) cannot be subjected to our theory; they are handled in In the previous section, we defined the problem of small objects. Here, we address this problem, though our solution does not yield a full answer to the problem. In the affirmative, we present a couple of ways of solving the problem for small objects that are part of a large set, and in particular, focus on ranking as an approach that can help us with the problem. In the negative, we note that tiny objects . Since x is small, it can also be represented as a member of Q in small space.In all of these examples, we assume that S is no longer small, and we can use AIT to assess whether the corpus is itself logically deep or sophisticated, for example, by assessing if a program that substantially compresses the corpus requires long runtime, or if only long programs can be parameterized so that the corpus is a typical output. To be specific, let S be an inspiring set of small objects; if S is sufficiently large and complex, that S is logically deep or sophisticated by our previous definition. Then, we can say that the objects in S demonstrate substantial internal structure; in particular, this means that there are programs that are substantially shorter than S much more slowly than just enumerating every example of S. At a certain point, complex language models can be used to better generate S, with shorter parameterizations than the actual haiku themselves.One simple way to get started at solving the problem of small objects is to prime the pump with a large set of them. If we are given a corpus S is a corpus of non-random haiku, but instead of haiku satisfying a specific aesthetic property  or creation process, then once we have sufficiently many of them, we will be able to compress them substantially. Given our previous claim that the value of creative objects is assigned to objects generated as typical examples of the output of sophisticated creators, who require substantial computation to generate them, we can then say the following: small objects generated only slowly by the programs for which they are part of a large class of similar objects are themselves small objects of high value.In some sense, this is not surprising. Even if the only modeling we do is to supply a dictionary of valid English words and their syllable counts, then a new haiku can be identified by the indices of its words in that dictionary. If we use the CMUdict dictionary , then wowhich haiku of S are the good ones, just that the corpus overall is good and indeed improves as the complexity of the encoding machine grows.Unfortunately, this approach does not allow us to assess S set off as a corpus is to restrict to models for which p has S as typical outputs. For this approach, instead of asking p to output the entire set S as a single output, we use p as a program, which, upon input y, generates members of S as output. Let Q: that is, given an input d, the model p outputs a set S is a typical output of p, then p as being a sophisticated creator of a corpora of haiku if S is a sophisticated object, and hence p, its likely producer, is a sophisticated creator).One step up from just having the set S is well described by p, and as such, a different typical output from p  will also be of comparable quality and complexity to S. It is possible that the new corpus may be of different size than S, for example, if we develop a compression strategy in p that does not individually compress each example from S. This is not a routine for coming up with better corpora than S. If S is a good collection of largely mediocre objects and accumulates its sophistication as a whole, the same will be true of any other typical output. Another concern is to make sure that p has a panoply of diverse outputs, but this is actually not a live worry: if S can be compressed into a two-part code d as the uncompressible bits of input to p to generate S, then there cannot exist another uncompressible input In this framework, S along with a promise that all examples found in S are good members of Q, we can then compute a model whose job it is to generate members of S alongside new members. In this frame, we can require that a valid model p is injective and total, except when its inputs are not from the valid prefix-free set , and then we specialize to requiring that the members of S are again typical outputs of p. This requires a change to our definition of “typical”: instead of requiring that p to output x is not much longer than the optimal way of using S to assist in encoding x. We may need to make the constant c a bit larger than before . However, with this frame satisfied, if p is again large , then the optimal program p will again output members of Q that have this regularity as their typical outputs. As S grows and the optimal model becomes increasingly sophisticated, the model will generate more and more complex small objects, with substantial code execution underlying them.An alternative framework requires us to start with external evidence of quality. If we are given a large corpus x, we can attempt inputs to p until we obtain a haiku x that we are interested in; if x comes as the output of p to an atypical input y , then x is not a typical output of p, and we cannot immediately assess its quality. If, on the other hand, x does come from a typical input, then its quality is comparable to that of S.To assess a single haiku S comes not just as a set, but as a ranking. Suppose that S is a known set of excellent examples of the class under consideration, and that there exists a quality ranking S, if discovers not only the underlying information found in S, but it also discovers the inherent properties that differentiate good examples of the genre from bad ones. If we again limit p so that it must output all members of Q eventually, then we can use the algorithmic probability of a string x as a measure of the goodness implied by generalizing the precedence order ≺. As the sophistication of p  grows, we can say that we are progressively learning more and more serious descriptions of what makes for good small objects of the type being analyzed. This is similar to the notion of uniform Kolmogorov complexity, wherein given an infinite-length string x, we identify the length n-letter prefix of x, which monotonically increases as n grows; the difference is that we require that p treats the individual members of S as typical outputs, and we move in increments of single objects, not bits.We can also play a different game with enumeration: we can require that x. If we are given a wide set of experiences, it is possible that broad background knowledge can compress x even more effectively than just using examples of the same type as x. For example, learning about narrative might be useful for a haiku generator, even though it only generates seventeen-syllable poems. In this frame, we allow the prior knowledge to come in two parts, S and T, where T is any binary string representing prior background experience of the producer, and then the project of identifying the good-quality examples of Q starts off with a program that can also generate T. Since we are not concerned that the information of T is typical, we do not have to ensure that p outputs its results on ordinary outputs; instead, we can compress T and restrict to models p, where p outputs the members of S on typical inputs. If p is substantially larger for the prior knowledge T, then p has, in fact, built up more sophistication in the analysis of the type of objects found in S than in just T, and its typical outputs, again, will inherit the sophistication  found in p.In the previous subsections, our language model was based only on a corpus of objects of the same type as x, an object we would like to assess. In particular, the “print x” program is a short program that prints out the object, and which does not print any invalid haiku (since it only prints x). Similarly, given a small set of haiku, the regularity detected will not be sophisticated: it is essential to have a sufficiently substantial set S so that even if the best compression is fairly modest, the model can be smaller than the total size of S.One trick that does not work is to just describe the set of valid haiku and then demand that we build a Turing machine whose language is a subset of that language that includes S to be enumerated as the first p, or for S. Neither of these is appropriate, though, as they encourage the model that is generated to overtrain on the members of S: what is needed instead is that S, or its members, are typical outputs of p.Another natural trick is to want the members of We started this paper by considering small objects of minimalism, and in particular, the piece “4tiny object as an object x for which the set of all objects of size at most We define a x to the valid set of objects in Q makes the problem that we can add x to p in constant space that is below the constant threshold c for sophistication or b for logical depth, meaning that we can make p output x for arbitrarily many inputs. . The tiny object x will then be indistinguishable from when it would be output as a specific example of an object of high quality. In fact, it is easier to explain that a tiny x has arisen as the outcome of a bug  than as the result of a program that generates x through a large amount of computation. We can think of this as the computational equivalent of the common “anyone could have done that” dismissal of much conceptual art: even in the frame where a creator has a wide oeuvre and a consistent artistic practice, and decides, as a result of much serious computation, to exhibit a truly minimalist result, it will be very challenging for the community to take it seriously as the outcome of substantive work and not just a trivial provocation.Adding a tiny object In this manuscript, we have given the beginning ideas to extend our recent work on algorithmic information theory as a way of assessing the creativity of digital objects to the domain of small objects, those with very short representations. While the theory does not immediately adapt, we show that by using large corpora of small objects, or by using rankings of small objects known to be of good quality, we can adapt our original ideas to this new domain.In general, the direct practical application of our ideas is challenging, in no small part because of the uncomputability of computing most of the measures in our paper, but also because getting enough of a corpus of “good” examples of the small objects under consideration to allow us to build a model capable of identifying good examples as “typical” ones is itself a significant challenge. Modern language models, such as GPT-2 , are traFuture work in this domain includes identifying other contexts in which we can use external information as a component of the assessment of the quality of digital objects, most notably cultural or other background information, or external biographical data about creators in the model-creation process. Applying algorithmic information theory to questions of quality is a challenge, but it is an interesting challenge indeed.


ARID1A mutations are frequently observed in colorectal cancers (CRCs) and have been reported to be associated with high mutational burden and tumor PD‐L1 expression in vitro.ARID1A is a component of the SWI/SNF complex, which controls the accessibility of proteins to DNA. To clarify the role of ARID1A mutation in CRC.p = 0.01), and the trend was stronger for right‐sided CRCs than left‐sided. TCGA data confirmed these findings (p < 0.01). BRAF V600E and ATM mutations were also found at higher frequencies. Immunohistochemistry showed that 30% of MSI‐H CRCs had ARID1A loss, while this was true in only 6% of MSS CRCs. In both MSI‐H and MSS, PD‐L1 expression by stromal cells was enhanced in the ARID1A‐mutant groups .We used next generation sequencing (NGS) and immunohistochemistry on clinically obtained samples. A total of 201 CRC tissues from Niigata University and Niigata Center Hospital were processed by NGS using the CANCERPLEX panel. Immunohistochemistry for ARID1A, PD‐L1, MLH1, and MSH2 was performed on 66 propensity‐matched (33 microsatellite instability‐high [MSI‐H] and 33 microsatellite‐stable [MSS]) cases among 499 cases from Kyushu University. TCGA data were downloaded from cBioPortal. NGS showed significantly more mutations in ARID1A mutated CRCs (ARID1A‐mutant CRCs, and IHC study showed that ARID1A loss was correlated with high PD‐L1 expression in stromal cells regardless of MSI status. These data support the idea that mutant ARID1A is a potential biomarker for CRCs.We found a higher mutational burden in  Formalin‐fixed, paraffin‐embedded tissues were sectioned into 4‐μm slices, and then antigen retrieval was performed by boiling the slides in 10 mM sodium citrate (pH 6.0) or Target Retrieval Solution (Dako‐Japan). The primary antibodies and staining conditions are summarized in Supplementary Table The expression of MMR proteins (MLH1 and MSH2) was judged as “loss” when there was a complete absence of nuclear staining in neoplastic cells, while the surrounding non‐neoplastic cells consistently showed preserved nuclear staining .ARID1A mutation . As a control, the presence of homogenous strong nuclear staining in background stromal fibroblasts, inflammatory cells, vascular endothelial cells, and normal epithelial cells was a prerequisite for assessing staining in the tumor.In our assessment of ARID1A, clear absence of staining in the nuclei of viable tumor tissue (away from necrotic areas) was considered “loss.” There were two types of ARID1A staining loss, total and focal loss. Both types were regarded as loss because at least part of the tumor was presumed to have PD‐1 was considered positive in tumor cells when membranous staining was evident in 1% or more of these cells; likewise, PD‐L1 was considered positive in stromal cells when membranous staining was observed in 1% or more of these cells . The 1% cutoff values are based on a previous study by Takada et al.2.5U‐test, the chi‐squared test, or Fisher's exact test. All calculations were performed using JMP software v13.0 . p‐Values <0.05 were considered significant.We assessed statistical differences between groups using the Mann‐Whitney 33.1ARID1A mutations (10%) similar to previous reports.ARID1A‐mutated and wild‐type groups. Recently, the laterality of CRCs has been reported to determine their mutational featuresARID1A mutation status in patients with right‐sided CRC, while this difference was not observed in left‐sided CRC patients. The mutational status of ARID1A and mutations that were observed more frequently in ARID1A‐mutant cases are listed in Supplementary Tables 4 and 5. ARID1A mutations were scattered throughout the ARID1A locus and there was no hotspot. Patients with ARID1A mutations were likely to have ATM (25%) or BRAF V600E (24%) mutations, which was similar to a previous report.BRAF V600E and ARID1A mutations were right‐sided CRCs. The rare mutations observed in ARID1A‐mutant samples were thought to be due to the high mutation frequency in these patients. The most noteworthy findings from the sequencings results were the differences in TMB, which are depicted in Figure ARID1A mutations had a significantly greater number of mutations in the cancer‐associated sequenced by CANCERPLEX. This trend was also confirmed with TCGA database, which included 276 cases of whole exome sequencing  and a higher TMB, which was compatible with previous results from in vitro studies.Clinicopathological features of the NGS group are summarized in Table g Figure . In TCGA3.2Clinicopathological features and IHC results of the IHC group are summarized in Table IHC results showed that ARID1A loss was much more prevalent among the MSI‐H group 30%) than the MSS group (6%). Most of the MSI‐H cases were due to impaired MLH1 (82%) and fewer showed MSH2 loss (9%) according to MMR protein IHC. The MSS group showed no irregular staining of either MMR proteins. PD‐L1 expression in both tumor 24%) and stromal (55%) cells was significantly enhanced in the MSI‐H group compared with the MSS group . Detailed clinicopathological and IHC data of each group are shown in Tables 0% than t% and str4ARID1A mutations on CRC and found that ARID1A‐mutated CRCs tended to have higher mutational loads and that ARID1A‐deficient CRCs were more likely to be accompanied by enhanced PD‐L1 expression by stromal cells. We used the CANCERPLEX panel, which can detect as many as 415 cancer‐related genes, to analyze the NGS group. This revealed two primary conclusions. One was that CRCs with ARID1A mutations were likely to co‐occur with BRAF V600E and ATM mutations, which are both clinically targetable with molecular therapies.ARID1A mutations and the BRAF V600E mutation has already been reported,ATM mutations are a new feature of ARID1A‐mutant CRCs. The BRAF V600E mutation is well known for its role in melanoma, and BRAF inhibitors such as dabrafenib have already been shown to be fairly effective in advanced CRCs with the BRAF V600E mutation in clinical studies.ATM is also a well‐known oncogene that regulates cell proliferation and DNA double strand break repair.We used two different cohorts to explore the impacts of ARID1A‐mutant CRCs had a greater number of mutations compared with ARID1A‐wild type CRCs, and this relationship was stronger in right‐sided CRCs. We examined the number of co‐occurring mutations rather than mutational burden due to the lack of mutational burden data in our cohort. This is not a standard evaluation, and is one of the limitations of our study. However, we confirmed the same trend in TCGA cohort, and there are other reports in a large cohort study with over 40 000 cases that ARID1A‐mutated cancers including CRCs had higher mutational burdens than cancers without ARID1A mutations.ARID1A‐mutant CRCs in our cohort. High TMB is a good predictor for tumors that express high amounts of neoantigens, which recruit inflammatory cells including CD8‐positive T lymphocytes into the tumor microenvironment.The second conclusion from this analysis was that ARID1A mutations have a higher TMB, which leads to the recruitment of PD‐L1‐expressing immune cells  to the invasive front, enabling the cancer cells to escape immune response. If PD‐1 or PD‐L1 blockade can break this signaling, ICIs should show efficacy in ARID1A‐mutant CRCs.We evaluated 66 cases in the IHC group, of which 33 were MSI‐H and 33 were matched MSS cases. MSI‐H cases have been reported to have tendency toward being diagnosed in women and at earlier stages, so we matched these factors in the MSS group using propensity scoring. Increased proportions of older age, right‐sidedness, and high histological grade were seen, which have been repetitively reported for MSI‐H CRCs, ensuring the validity of our MSI‐H cohort. Loss of ARID1A staining was seen in 30% and 6% of MSI‐H and MSS cases, respectively. In both groups, loss of ARID1A staining was significantly correlated with elevated PD‐L1 expression in stromal cells. Several studies have investigated the consequences of PD‐L1 expression by stromal cells in CRC. Liu et al. reported that PD‐L1 expression was mainly observed in the stromal cells at the invasive front of MSI‐H CRCs.ARID1A mutation and loss of its IHC staining was discussed in depth in a previous report,ARID1A mutations, which supports the value of the IHC analysis in this study. The second is selection bias of the IHC group. We applied the propensity matching method to eliminate confounding factors . CRCs with ARID1A mutations tended to have a higher histological grade and older age, which may interfere with the IHC results. The small sample size of our study is another limitation. A verification study using a validation cohort with a larger sample size will be necessary. A lack of standardized methods for PD‐L1 staining in CRCs is another limitation. We used the PD‐L1 clone 28‐8, which is a universally accepted antibody.This study had several limitations. First, we applied different methods to two totally different cohorts. However, the association between BRAF V600E and ATM mutations in ARID1A‐mutant CRCs. Furthermore, ARID1A mutations were correlated with a higher mutation number, which may partly account for the enhanced PD‐L1 expression by stromal cells. Although this is a preliminary study, we have provided good evidence to indicate that ARID1A mutations in CRCs could be used to indicate ICI treatment. Recently, an association between ARID1A mutations and increased immune activity in gastrointestinal cancer was reported,ARID1A mutations can be biomarkers for using ICIs in CRC patients.In conclusion, this is the first report to reveal the close relationship between ARID1A and PD‐L1 expression by IHC in CRCs. We also confirmed co‐occurring Eiji Oki reports receiving honoraria for lecturing from Chugai Pharmaceutical Co., Ltd. Taiho Pharmaceutical Co., Ltd., Bayer Yakuhin Japan, Eli Lilly, Yakult Honsha Co., Ltd., Takeda Pharmaceutical Co., Ltd., ONO Pharmaceutical Co., Ltd. and Merck Biopharma Co., Ltd., outside the submitted work.Conceptualization, T.K., E.O., Y.S., Q.H., Y.H., K.A., T.W., Y.O. and M.M.; Methodology, T.K., E.O., Y.S., T.W. and Y.O.; Investigation, T.K., E.O., and S.Y.; Formal Analysis, T.K., E.O., Q.H, Y.H., K.A. and M.S.; Resources, T.K., E.O., Y.S., T.W., Y.O. and M.M.; Writing—Original Draft, T.K. and E.O.; Writing—Review & Editing, T.K., E.O., T.W., Y.O. and M.M.; Supervision, T.W, Y.O and M.M.; Project administration, M.M.This study was approved by the institutional review board of Kyushu University Fukuoka and Niigata University Niigata, Japan (permission number: 30‐367) and conformed to the tenets of the Declaration of Helsinki. Informed consent was waived by the institutional review board owing to the retrospective nature of the study.Supplementary Figure 1 Case selection charts for the (a) NGS and (b) IHC cohortsClick here for additional data file.Supplementary Table 1 Clones, companies, and dilution conditions for the primary antibodies used in this studySupplementary Table 2. Clinicopathological features of right‐sided CRCs with or without ARID1A mutations in the NGS cohort. *Statistically significant difference (p < 0.05)Supplementary Table 3. Clinicopathological features of left‐sided CRCs with or without ARID1A mutations in the NGS cohortSupplementary Table 4. Detailed mutational status of ARID1A in the NGS cohortSupplementary Table 5. Co‐occurring mutations that were seen at significantly higher frequencies in the ARID1A‐mutant NGS cohort. *Statistically significant difference (p < 0.05)Click here for additional data file.


As the COVID-19 pandemic spread to the United States, it was followed by unprecedented changes. These changes did not spare undergraduate and graduate medical students. Specifically, medical students applying for residency programs were faced with a novel challenge. In March 2020, as the pandemic became increasingly severe, the Association of American Medical Colleges (AAMC) recommended pulling medical students from in-person clinical rotations. By May 2020, the AAMC recommended that all residency interviews be conducted online for the 2020-2021 residency application cycle. These unprecedented modifications to the interview season required programs to quickly adapt and find ways to utilize online tools to convey what their program offered to applicants. In this paper, we will outline the adaptations, tools, and resources that residencies and applicants have used to navigate through the 2020/2021 interview cycle. Dear EditorAs the COVID-19 pandemic spread to the United States, it was followed by unprecedented changes. These changes did not spare undergraduate and graduate medical education. In March 2020,Although the pandemic accelerated the need for electronic residency program resources targeted to applicants, many of these changes will likely remain post-pandemic. It is probable that many programs will return to in-person interviews; however, research has shown that prospective residents are increasingly in favor of utilizing online resources to learn about residency programs. A 2014 studyTraditionally, the residency interview season has involved medical students traveling to programs across the country, often completing sub-internships, attending pre-interview dinners with residents, hospital tours, and in-person interviews. These experiences allowed applicants to learn a great deal about the program, and importantly, to get a feel for the culture. A 2012 study found that “many of the factors that were ‘very important’ (to applicants) are not tangible but, instead, speak to the culture of an institution.” and that “Applicants appear to care most about collegiality and potential relationships with other residents and faculty.”Different electronic resources can effectively communicate different qualities of a program. Webpages offer an excellent mode to display the program curriculum, faculty, achievements, research, education, application requirements, and more. Social media has different advantages. Its dynamic and interactive nature is helpful in communicating residency culture. Teleconferencing offers prospective applicants the chance to speak directly with residents and faculty to further identify culture and have individual questions answered.A crucial component of building a package of electronic resources is to create a unified brand identity. Programs that successfully engage in brand identification and translate it into an integrated online presence will have an easier time differentiating themselves from other programs. As discussed in a 2018 paper by Shappell et al.,Program websites are one of the most universally used modes of an online presence. However, a 2018 study by Hansberry et al., analyzed 179 radiology program websites and found that very few offered a comprehensive set of content.Social media is another valuable resource in the branding and recruitment toolbox and offers unique advantages over a webpage. Because social media is more dynamic than a webpage, it provides a medium for providing frequent updates, more personalized interactions, and a window into the daily life of residents. This collectively provides prospective residents with a view of the program culture. A 2008 studyTeleconferencing offers a final electronic resource for engaging with residents and furthering the program brand image. Many programs throughout the 2020-2021 application season offered virtual “meet and greet” events with residents and faculty. These online meetings give applicants the chance to experience the culture more personally and have their specific questions answered. Some programs even allowed applicants to view educational conferences. These events give applicants a window into the interactions between faculty and residents, furthering their brand image, and helping applicants imagine themselves at the program.These modalities -- websites, social media, and teleconferencing -- are valuable to the resident recruitment process. Each offers different components in the recruitment toolbox, and when employed appropriately, can bolster the image that programs project and, concomitantly, the size and quality of their applicant pool. One element of paramount importance in an online strategy is the creation of a consistent brand identity that is true to the program and helps applicants to understand its culture and unique features.


Realist evaluation offers a valuable way to understand how interventions function and thus how they can be improved and locally adapted. Consequently, realist evaluation is increasingly conducted in parallel with intervention trials. It comprises a clear philosophical foundation and view of causality, pragmatic mixed data collection methods, and a theory-driven approach in which hypothesised program theories are tested and refined. However, detailed methods for data analysis are seldom well-described in realist studies and no clear method for analysing and presenting realist evaluation data has yet emerged. In this methodological paper we use the worked example of our realist process evaluation of the SAGE yoga trial to illustrate an applied process of data analysis and presentation of findings. We show how we drew on other realist studies for ideas, provide examples of six key tasks involved in conducting a realist process evaluation (including coding data and structuring results) and describe strategies that did not work and our rationale for rejecting them. This detailed account of the decisions and methods that worked for us is intended to provide a practical and informed point of departure for researchers conducting a realist evaluation. Realist evaluation is a theory-driven approach to evaluation that aims to explain why policies and programs work (or not), for whom, and under which circumstances ,2. The uThis methodological paper adds to the literature on analysis in realist evaluation by providing a worked example of a realist process evaluation conducted in parallel with a randomised controlled trial of yoga classes for older adults aimed at preventing falls. We provide a stepwise overview of a realist process evaluation for researchers who may have limited experience of qualitative methods and who are interested in realist evaluation but have yet to use it as a method.Realist evaluation seeks to identify causal patterns comprising three inter-related concepts: “context”, “mechanisms” and “outcomes”  can reveal richer, more comprehensive insights into how interventions function ,14,15. Qpro forma. However, greater attention to the practical detail of conducting realist evaluation in published studies would support transparency and the applicability of this valuable approach [The Realist And Meta-narrative Evidence Syntheses: Evolving Standards (RAMESES) group provide a range of materials to support realist evaluation, including valuable book chapters and papers on realism’s philosophical underpinnings and data collection strategies e.g., ,19,20). ,20. 19,2approach .This worked example of a realist evaluation centres on a current randomised controlled trial involving 560 community-dwelling adults aged 60+ years. Participants are randomised to either: (1) the Successful AGEing (SAGE) yoga exercise program in which they attend 40 weeks of twice-weekly yoga-based exercise classes designed to prevent falls or (2) a seated yoga relaxation program. The primary outcome is rate of falls in the 12 months post randomisation. Secondary outcomes include mental well-being, physical activity, health-related quality of life, balance self-confidence, physical function, pain, goal attainment, and sleep quality. An economic analysis is comparing the cost-effectiveness and cost-utility of the two yoga programs. The SAGE yoga exercise program was designed to be delivered in yoga studios but, following social restrictions in response to the COVID-19 pandemic, the four groups that started classes in a studio were moved online and delivered via Zoom. All but 3 of the 46 participants at that time continued to attend classes online, and all but four of those who remained completed the full program. A realist process evaluation was conducted in parallel with the trial , focusing on the SAGE yoga exercise program. Process evaluation seeks to shed light on why an intervention was or was not effective, and how it (and similar interventions) can be improved for better contextual fit. This is particularly useful for new innovative trials such as SAGE where causal mechanisms are not yet understood . This process evaluation sought to understand how SAGE functioned as both a studio-based program and an online program, and to explain why it worked well for most participants. Given these explanatory aims, we chose a realist approach because of its utility for surfacing and testing program theories. Our two-part research question was: In the SAGE yoga trial, what is working, for whom, and under what circumstances? What aided the transition from studio-based to online yoga classes?Identify outcomes of interestDevelop initial program theoriesCollect data to test these theoriesCode data to capture causal relationshipsEngage with the literaturePresent findings in ICMO configurationsThe conduct of this realist process evaluation centred on the following six tasks:We now explore each of these tasks in turn, using examples from our evaluation to illustrate how they were tackled. This process is illustrated in Engagement with yoga program: attendance (including across transition to online classes), expressed commitment, motivation, and enjoyment.Self-reported health impacts: self-reported improved balance, flexibility, strength, mobility, stress reduction, sleep quality, sense of wellbeing, sense of achievement.Intention to continue yoga .Realist evaluation usually starts by identifying outcomes of interest and seeks to explain how they were produced. Westhorp argues that in order to examine ‘what works for whom?’ it is necessary to define what constitutes ‘working’, i.e., program success . The sucd.Habit formation: routine practice of yoga as part of everyday life ,37.e.Physical literacy: motivation, confidence, physical competence, knowledge of and engagement in physical activity ,39,40.Data sources for these outcomes included routine process measures which indicated high levels of program completion, and post-intervention participant feedback forms in which 87% of our targeted participants reported some degree of improved physical health or functioning, 87% reported improved mental health, and 78% reported improved balance. The qualitative data included comments about additional positive impacts such as improved flexibility, mobility, relaxation and/or sleep quality. Two further process outcomes were identified inductively as data collection progressed, and were refined with guidance from the literature:Discussion with the SAGE trial leaders and implementers about the underlying causal hypotheses that had informed the intervention design. These hypotheses derived from their research and professional expertise in the field of fall prevention, physical activity, and behaviour change, but had also evolved in response to information generated by the unfolding trial, including formal, and informal participant feedback and discussions with the yoga instructors.The literature on falls and older people’s engagement in yoga and yoga-based exercise, and physical activity more broadly. This provided ideas about possible program theories including likely mechanisms. As a form of theory-driven evaluation, the realist evaluation develops tentative initial program theories about how the program functions and uses these to guide data collection and analysis. We developed five initial theories  derived These rough working theories were intended to explain the three outcomes of interest we had identified at that point, and they were configured around intervention strategies and possible mechanisms . Punton et al.  recommenOur hypothesis was that the mechanisms identified in these theories were activated in both studio-based classes and online classes, hence the high rates of program continuation despite the disruption and challenges of transition.We targeted participants who had been randomised to and enrolled in the four studio-based SAGE classes that later transitioned online and had thus experienced both types of classes. Sampling was intended to be purposive to support the realist approach to intra-group comparison in program evaluation , aiming Semi-structured interviews were conducted by phone with trial participants and two of the yoga instructors. The third instructor chose to be interviewed via video on Zoom. All interviews were conducted by a researcher with experience in qualitative research and realist evaluation (AH) who was not involved in the design or implementation of the SAGE trial.Participant interviewees were encouraged to reflect on their experience of the hybrid program, including the differences between studio and online classes. They were asked to expand on scores and comments returned in their feedback form, and to explore their explanations for any intended or unintended impacts they identified . Instrucpeople stick with the program because they feel health benefits”) we asked interviewees why they enrolled and stuck with the program in the early stages before any health benefits were felt. When discussing initial theory five (“free classes are an incentive to give yoga a go and stick with it”) we mentioned the anecdotal theory that people value services less when they are free [In accordance with the realist approach, we explicitly tested the program theories with participants . In earlare free . If inteare free . In later interviews we adapted questions to take account of the evolution in our initial theories and to include emergent theories. In this phase we continued to prompt interviewees to critique the theories, often by highlighting variation in views from other participants. We also added a question about the home environment because it emerged as an important context for online classes.Interventions-as-delivered often look quite different to interventions-as-theorised , and we how the intervention works), the circumstances in which this occurs (context), and the effects produced by the interaction of those elements (outcomes). In addition to identifying those elements, data analysis must capture the relationship between them, i.e., the whole causal pathway. This presents challenges because the most common form of coding in qualitative analysis is similarity coding which forms categories of data that are like one another  but fragments associations, processes, and causal relationships in the original structure of the data. Realist evaluation requires contiguity coding, which preserves these relationships [Realist data analysis is retroductive, that is, it seeks to explain outcomes by hypothesising causal pathways and testing these hypotheses to identify those that are most plausible ,60. In pionships ,23.Decisions must also be made about which elements of the causal pathway should be included. Traditionally, causal relationships in realist evaluation are expressed as Context-Mechanism-Outcome configurations (CMOs). In this approach the intervention activities are implicit because mechanisms are conceptualised as responses to resources provided by the intervention, thus a mechanism aggregates the intervention resource and people’s response to it. Dalkin et al. propose disaggregation, so that the intervention activity is less likely to be considered a mechanism in itself, and so the role of context as a mediating force between intervention resources and human reasoning is clearer . One wayWe reviewed and experimented with a range of approaches that would enable contiguity coding. Matrices have long been used in qualitative data analysis ,63 and i“any description of perceptions, beliefs, ‘logic in use’, emotional responses to situations, or understanding of a role that is relevant to the intervention may provide a clue”( p. 156).Bergeron et al.  coded trConsequently, we coded for program theories in NVivo using its traditional node function (one node for each theory), and supplemented this with additional nodes for context, mechanisms, and outcomes where likely candidates were identified  both coded a proportion of transcripts independently using preliminary coding frames based on our five initial program theories and had frequent discussions about how each new transcript was reshaping the existing program theories and/or indicating others. See Given that the mode of yoga program delivery changed dramatically during the course of the intervention (transitioning from studio-based classes to online classes), we could not assume that the same mechanisms were activated throughout. Consequently, all data in which interviewees compared the two modes of delivery was double coded to a discrete node (Gains and Losses) as well as any relevant theory nodes. This allowed us to review all the comparative reasoning about what worked (or not) more efficiently. We erred on the side of caution in this task, coding to multiple theory nodes when we felt uncertain, and trying to identify likely contextual factors, mechanisms, and outcomes as we went . We kept a running analytic memo within NVivo to capture questions and ideas, and used annotations freely to link comments to a specific piece of text. Memos and annotations also served as a communication device between the two researchers conducting the analysis. Initial program theories were stored in NVivo in a table with additional rows for other possible theories, columns for notes and queries and specific data sources, plus a column for revisions so that we could see how the theory was evolving. We investigated these initial theories and, later, our emergent theories in the literature in an effort to fine-tune them and to identify corroborating or disconfirming findings from other studies. This involved searching using likely keywords in Google Scholar and opportunistically following citations of theoretical or empirical studies that suggested plausible explanations for our findings. Others have conducted one-off replicable searches e.g., ,75,76), ,76, 75,7Analysis was conducted in parallel with data collection. As interviews progressed and our theories evolved, we merged two initial theories, expanded one and added three additional theories to the final coding frame .child nodes in NVivo) but concluded that this would produce thematic categories rather than greater insights into the program theory suggested by that data. Consequently, we used memos to distil the contents of each theory node and to identify ICMO (Intervention activity-Context-Mechanism-Outcome) configurations. After all transcripts were coded, we examined our theory codes to see if they would benefit from sub-coding  worked on this independently and met to develop concordance. Observational data confirmed that the intervention strategies hypothesised to activate mechanisms were being delivered across different instructors’ classes, and this was corroborated by information from participants and instructors. We held periodic workshops with the wider research team to engage in critical dialogue about the emergent findings and to guard against “theory-induced blindness” in which data is overlooked because it does not fit initial hypotheses . “…at a middle level of abstraction: specific enough to clearly explain the phenomenon, and general enough to apply across cases of the same type”( p. 3).Lastly, we worked across the individual ICMO tables to develop a single table that distilled our entire data set, aiming to express these findingsHowever, even after minimising overlapping concepts and repetition, the table was dense and occupied an entire page of A3 paper, making it hard to read easily and potentially overwhelming. This presentation also lacked coherence in relation to the overarching program theories that had formed the basis of our investigation. Work by Punton and colleagues  suggesteTo strengthen the research rigour, we addressed the tasks in Ronkainen and Whiltshire’s framework for assessing validity in realist research in sports and exercise psychology  Table 3Table 3. Ethical approval for this study was provided by the University of Sydney Human Research Ethics Committee, reference 2019/604. Interviewees gave prospective informed consent to take part in an interview at the trial commencement and confirmed this in their post-trial feedback form. They also gave verbal consent to the trial administrators for their details to be passed on the interviewer and confirmed this consent in the interview itself after information about audio recording and use of deidentified data had been stated. This paper adheres to the reporting standards for realist evaluations developed by the Realist And Meta-narrative Evidence Syntheses: Evolving Standards (RAMESES) group . Twenty-four interviews were conducted: 21 with trial participants and 3 with yoga instructors. Interview duration ranged from 29 to 66 min with an average of 50 min. Fifteen trial participants were female (71%), reflecting the gender ratio in the trial, and aged between 61 and 80, with an average age of 68. This sample included four participants who stopped attending online classes, those who gave negative survey feedback and those with pronounced physical limitations such as osteoarthritis, scoliosis and Haglund’s deformity. The 3 yoga instructors were all female with over 15 years’ experience of delivering Iyengar yoga to groups, including those with older people. Analysis included data from 46 participant post-intervention feedback forms.We identified 16 mechanisms. Contextual factors, especially people’s belief in the efficacy of yoga, their physical health, and domestic circumstances, played a powerful role in determining who the program worked for. The COVID-19 pandemic was a shared context that most likely enhanced engagement with online classes by limiting access to other forms of activity and connection. Our discussion focuses on four challenges in realist evaluation and how we tackled them in our realist process evaluation of the SAGE yoga trial.“messy” and “convoluted” coding process by including both discrete constructs and narrative accounts [Realist evaluation requires an analytical approach that can simultaneously identify patterns of causal relationships and discrete elements within these relationships. The need to code for both contiguity and similarity presents analytic challenges. We described various strategies that have been used to tackle these challenges and present a detailed account of the methods that were identified as most fit-for-purpose for our study. Coding to whole program theories using nodes in qualitative data analysis software provided sufficient structure and flexibility to capture data related to evolving program theories. Others have also described how qualitative data management software can support and lend transparency to the accounts .“involves constant shuttling between theory and empirical data, using both inductive and deductive reasoning”( p. 374).The use of theory presents another challenge. Realist evaluation employs an analytical process (retroduction) in which coding must be sensitive to emergence in the data but is also informed by working hypotheses and wider theories. ThisThe movement between inductive and deductive coding is common in some other research approaches where qualitative data is used, such as thematic analysis, content analysis and case study research; consequently, this aspect of analysis may be familiar to experienced qualitative researchers. However, the identification and critique of plausible theories throughout this process adds a layer of complexity. Despite considerable emphasis in the realist literature on the need to search for and iteratively test program theories against plausible rivals, very little guidance is offered about how to do this . The oneimproved health and wellbeing be categorised as a mechanism or process outcome or summative outcome? We concluded that it plays a vital role in all three categories, but in different forms. Participants’ psychological response to anticipating and experiencing health benefits is a mechanism . Given that improved health and wellbeing functions on a continuum, it is also a process outcome (because it contributes to motivation and adherence) and a final outcome indicating the success of the intervention along specific dimensions of health. Westhorp argues that“this is consistent with realist philosophy in which the same thing can be context, mechanism or outcome depending on the particular analysis being undertaken”( p. 378).Much of the realist evaluation literature draws attention to the challenges of differentiating between intervention activities, context, mechanisms, and outcomes . This is“stitched” together during analysis [“slide” depending on which point in the causal chain we are focusing our attention, thus an outcome or a context in a process evaluation may function as a mechanism when investigating longer-term outcomes [Thus, ICMOs are not self-evident and must be analysis . This inoutcomes ,92.“the end result will be partial knowledge about partial improvements we can make in the delivery and targeting of social interventions – quite an achievement”( p. 112).The purpose of realist evaluation (and synthesis) is to present clear causal explanations that articulate the relationships between context, mechanisms, and outcomes ,31,34. MRealist evaluation—comprising a clear philosophical foundation and view of causality, purposeful mixed data collection methods and retroductive analytical strategies—offers a valuable way to better understand how interventions function and thus how they can be improved and locally adapted. Specific methods for analysing data are seldom well-described in realist studies, but moves are now afoot to address this gap and shine a light on applied analytical strategies. This paper adds to that movement. It draws on other realist studies for ideas, provides examples of key tasks involved in conducting a realist process evaluation, and describes strategies that did not work and our rationale for rejecting them. While this approach is unlikely to be directly transferable—research strategies must be tailored to each study —this det