Objectives: As advancing evidence on modifiable resources to support mental health in persons experiencing physical disabilities is of particular importance, we investigate whether structural and functional social relationships relate to mental health in people with spinal cord injury (SCI).Methods: Data from 12,330 participants of the International SCI community survey (InSCI) from 22 countries were analyzed. Structural  and functional aspects of social relationships  were regressed on the SF-36 mental health index (MHI-5), stratified by countries and for the total sample using multilevel models.Results: Functional aspects of social relationships were consistently related to clinically relevant higher MHI-5 scores and lower risk of mental health disorders (MHI-5 >56). Structural social relationships were inconsistently associated with mental health in our sample.Conclusion: This study provides evidence that functional aspects of social relationships are important resources for mental health. Interventions to establish and maintain high quality relationships should be considered in public health interventions and rehabilitation programs to reduce long-term mental health problems in persons experiencing physical disabilities. Structural aspects reflect quantitative characteristics of social relationships, such as social network size, frequency of social contacts, the living situation or partnership status, while functional aspects include qualitative features of relationships. The most frequently studied construct in the latter category is social support, which describes the functional importance of social relationships in their ability to provide instrumental, emotional or informational resources [Mental health has long been recognized as important public health issue  and pooresources , 9. Otheesources ; and proesources –13, impeAlthough evidence for the beneficial effects of social relationships on mental health from general population samples in higher income settings is far advanced and fairly consistent –6, findiThe present study provides the opportunity for two major contributions to current literature on social relationships and mental health in the setting of physical disability by using a large sample of people with spinal cord injury (SCI) from 22 countries. First, this study not only investigates structural aspects of social relationships  but also functional aspects that have rarely been studied in disability research . Second, the use of international data allows the investigation of associations within different countries to assess whether the expected link between social relationships and mental health is observed universally. We use data from people with SCI as a model for physical disability, as this condition often has severe consequences on functioning and health and increased vulnerability to psychological morbidity . TraumatThe International Spinal Cord Injury community survey (InSCI) is a cross-sectional survey performed in 22 countries between January 2017 and May 2019, covering all six World Health Organization regions. People with traumatic or non-traumatic SCI over 18 years of age who lived in the community and were able to respond to the available questionnaire language version were included. People with neuro-degenerative disorders, congenital etiologies, or Guillain Barré syndrome were excluded from the study given their different rehabilitation paths and disease progressions than persons with acquired SCI . NationaResponse modes included paper-pencil or online questionnaires, telephone or personal interviews and countries were responsible to provide the most convenient response modes. All countries received regulatory approvals by Institutional Review Boards or Ethics Committees and compliance with national laws and conformity with the Helsinki Declaration was guaranteed. Each participant provided informed consent in accordance with national regulations. Further information on the rationale, methodology, recruitment outcomes, and basic sample characteristics of the InSCI community survey are reported elsewhere –23.Structural aspects of social relationships were operationalized by partnership status and living situation. Partnership status was assessed by an item asking participants about their current marital status including the response options single; married; cohabiting or in a partnership; separated or divorced; and widowed. People who reported being married, cohabiting or living in a partnership were categorized as “having a partner” and others as “having no partner”. Information on the living situation was gathered by an item asking participants whether they lived alone, whether and how many other people lived in the same household, or whether they lived in an institution . A three-categorical variable was constructed discriminating between living alone, living with others, and living in an institution.Functional aspects of social relationships were assessed with information on feelings of belongingness, satisfaction with social relationships and problems with social interactions. Belongingness was measured with an item from the General Belongingness Scale [ss Scale  asking pss Scale . Particiss Scale . The iteThe SF-36 5-item Mental Health Index  was used to assess general mental health . The MHIThe approach of directed acyclic graphs (DAG) was used to inform the selection of confounders by exploring correlations between potential confounders, predictors and outcomes . We selen = 89; 9–10 points: n = 98), we pooled people scoring 0 into the lowest (combining scores 0–1) and people scoring 9 or 10 into the highest SEP group (combining scores 8–10). This resulted in an SEP score ranging from 0–8. SCI severity was grouped into four categories  based on information concerning level and completeness of the injury. The completeness of injury was evaluated with an item asking participants whether their injury was complete or incomplete, including respective definitions . Employment status was assessed with a multiple-choice question about the current employment status, whereby those indicating to work for salary with an employer or being self-employed were coded as having paid work, and those not selecting either of those categories were coded as having no paid work. Access to health care was assessed with a binary item asking participants whether they were in need of health care but did not get it during the past 12 months. Secondary health conditions were assessed with 14 items of the SCI-Secondary Conditions Scale (SCI-SCS) [To operationalize socioeconomic position (SEP), we built a sum score based on information about years of education , net-equivalent household income , subjective social position , and financial hardship . Given that the number of people with lowest and highest scores were rare (0 points: SCI-SCS)  with modp-values from likelihood ratio tests were reported for all models. We present unadjusted and adjusted results, controlling adjusted models for relevant confounders described above.Analyses were conducted using STATA version 16.0 for Windows . Unadjusted descriptive analysis of distributions of main variables of interest are presented pooled for the total sample and predictor and outcome variables are additionally given stratified for all countries. Association between structural and functional aspects of social relationships with mental health were investigated in the total sample as well as stratified for countries, to explore whether associations were observed universally. For analysis on the total sample, we ran multilevel regressions with a random intercept for country to account for the clustering of data within countries and to adjust for country affiliation . SecondlMissing values were imputed by multiple imputation (MI), assuming that the missing values were not related to the variable with the missing value itself . To impute different types of variables, MI by chained equations (MICE) was applied . ImputatA description of the total sample is displayed in Results for the total sample are shown in Results from country-specific analysis for the 0–100 general mental health score and for the dichotomized score  are displayed in This study demonstrates pronounced and consistent associations of functional aspects of social relationships with mental health in persons with SCI across different regions of the world. Persons with higher sense of belongingness, higher relationship satisfaction and fewer problems with social interactions reported markedly better general mental health and a lower likelihood for mental health disorders than those with less favorable quality of social relationships, even after adjustment for injury severity, demographic characteristics and secondary health conditions. Notably, previous studies identified a 4- to 5-point difference on the MHI-5 as the minimum clinically important difference , 35 and main-effects model suggests that functional relationships directly affect mental health by enhancing access to various forms of support, such as access to health-relevant information or emotional encouragement, which is beneficial for mental health. In turn, dysfunctional social relationships directly affect mental health by triggering physiological responses with adverse effects on mental health and wellbeing. For example, a recent systematic review comprising 20 studies concluded that social disconnectedness is associated with increased allostatic load [stress-buffering model provides an additional explanation for the observed associations, whereby functional social relationships are thought to modulate responses to adversity [Our findings on the positive association between functional social relationships and mental health are in line with previous evidence from quantitative studies, including general populations and few available findings from populations with disabilities –6, 16. Ttic load , which ptic load . The strdversity , 36, 38.dversity . These pdversity .The quantitative method of our study does, however, only allow detecting associations on a group-level and cannot contribute to disentangle individually different underlying mechanisms linking specific resources provided by social relationships to mental health in our specific sample. The above-mentioned well-established explanations on the health effects of social relationships from general populations should therefore be complemented by qualitative findings on the lived experience of persons with SCI. A meta-synthesis of qualitative findings in persons with SCI revealed for example that social relationships have a particularly important role for re-establishing self-worth after injury, as functional relationships to close persons were relevant resources for recognizing that the injury did not change the essence of their person, that their existence was valuable for others, and that they were still able to contribute to society despite potentially severe physical impairments . It is aOur study further shows weak and inconsistent associations between the structural aspects of relationships  and mental health, a tendency that has been described earlier for populations experiencing physical disabilities , 16. HavBased on our findings emphasizing the importance of social relationships for mental health in persons with SCI, interventions targeting functional aspects of social relationships should be considered in rehabilitation programs to reduce long-term mental health problems in persons experiencing physical disabilities. Results from a qualitative study on persons with SCI and their partners highlighted for example that specific education and training are needed to support coping and communication skills to strengthen partner relationships after the onset of SCI to counteract negative effects on relationships caused by overprotective behaviours, asymmetrical dependency, loss of sexuality and intimacy, and difficulties in psychological adaptation . PromisiThis study contributed to current evidence in disability research by investigating structural and infrequently studied functional aspects of social relationship in relation to mental health in different countries across the world. We used state-of-the-art statistical methods and the adjusted analysis were based on DAGs, allowing for drawing causal inference. The measurement instrument to assess general mental health has been validated in different regions of the world and among persons with SCI  and the Various methodological limitations need to be discussed when interpreting our findings. Sampling bias might be an issue because only eight countries applied random sampling and 14 countries relied on convenience sampling. Although this may lead to limited generalizability to the total population of individuals with SCI in participating countries, our study does not focus on the epidemiological description of mental health, but rather on the detection of associations between social relationships and mental health. As we did not collect information on social relationships or mental health in non-responders, we cannot evaluate whether survey participation was independent of those constructs. Results of country-specific analysis need to be interpreted cautiously as small samples in some countries might limit the power to detect meaningful associations. In some countries, effect sizes were large but had very wide confidence intervals, indicating potential power issues. Further, it is not possible to assess whether the self-report nature of data lead to biased responses, since for example, information on mental health may be subject to social desirability bias. Also, it is important to mention that this study only investigated three specific aspects of functional relationships assessed with five items, and we therefore cannot make the claim to be representative for the full spectrum of functional aspects of social relationships. Moreover, reverse causation cannot be excluded as people with mental health problems might experience more difficulties in maintaining good quality relationships.Our findings highlight the importance of functional aspects of social relationships for mental health in people experiencing physical disability resulting from an SCI, as feelings of belongingness, satisfaction with relationships and few restrictions in social interactions were identified as important resources for mental health in different geographical settings. Future research to reduce the burden of mental health disorders could explore currently promising interventions to support persons with physical disabilities in establishing and maintaining good quality social relationships.


The opioid epidemic impacts all ages, yet few published medical education curricula exist to train physicians on how to care for opioid use disorder (OUD) in adolescents, a developmental stage where confidentiality protection is appropriate and contributes to quality health care. We developed a simulation-based educational intervention to increase addiction medicine and addiction psychiatry trainees’ confidence in managing adolescents with OUD.Trainees completed a confidence survey and viewed an educational video covering state-specific confidentiality laws pertinent to treating adolescents with OUD. One week later, trainees participated in a simulated encounter where they described the scope of confidentiality to a trained actor, used the Clinical Opiate Withdrawal Scale to assess symptoms of opioid withdrawal, and explained adolescent-specific OUD medication treatment options. Immediately afterward, trainees completed a self-reflection and satisfaction survey and participated in a debriefing session with a faculty member where they identified learning goals. One month later, they completed the confidence survey to quantify changes in confidence.n = 26) would recommend it to peers. In addition, learners identified future learning goals, including researching specific topics and seeking out additional opportunities to evaluate adolescents with OUD.Thirty-five fellows  completed the simulation-based educational intervention between 2016 and 2019. When asked to answer yes or no, 96% of participants described the exercise as effective and 100% (Based on our participants’ report, this simulation-based educational intervention is an effective teaching method for increasing trainee confidence in managing adolescents with OUD. The 2017 National Survey on Drug Use and Health estimated that over 70% of people who needed treatment for OUD did not receive it.5 This treatment gap may be partly due to a lack of skills and medical knowledge among treating physicians, particularly in the management of adolescents with OUD.6 Many factors, such as availability of skilled providers, accessibility of clinical sites, and source of payment, determine whether adolescents receive necessary, high-quality health care. Confidentiality is another key factor for this age group because concerns about privacy influence whether many minors will seek health care and openly communicate with health care professionals.7 A lack of knowledge of minor consent laws amongst medical trainees poses a barrier to offering high-quality health care for this population8 and speaks to the need to develop more training in this area. Medical training has traditionally lacked a rigorous approach to education about the prevention, identification, and treatment of OUD.9 Although curricula in peer-reviewed journals such as MedEdPORTAL cover medical education topics like opioid risk mitigation strategies,10 urine drug-screen interpretations,11 and overdose resuscitation,12 there are few published curricula targeting adolescent OUD. A recent assessment of progress and needs in addiction medicine training in North America noted few programs focusing on adolescent populations.13 The widespread implementation of trainee education on adolescent addiction treatment is further inhibited by limited access to faculty who are adolescent addiction specialists, a generally low volume of adolescent patients, and the low availability of adolescent treatment training sites in many communities. To overcome these limitations, it is necessary to create education modules on this topic that can be easily disseminated to trainees across different medical specialties.National rates of hospitalizations and deaths from opioid overdoses in adolescents have risen over the past decade.15 When simulation training is conducted under the right conditions and outcomes are measured by valid and reliable instruments, research has shown not only that graduate trainees can obtain desired skills in a controlled, simulated environment16 but also that these skills can transfer to the clinical setting.17The implementation of simulation-based scenarios could address this education gap in adolescent addiction training and prove a useful tool for disseminating information to physicians in training. When used in medical education, simulation improves medical knowledge and confidence in patient-centered conversations that involve complex interpersonal skills, including error disclosure and breaking bad news.18 Second, simulation with live actors allows trainees to practice nuanced interpersonal skills needed to provide effective counseling and referral to treatment for pediatric patients. Finally, the ability to debrief with expert faculty and reflect on one's performance facilitates learner-driven education and is a key piece of simulations. Our goal was to use dynamic interactions with a live actor to recreate the realistic challenges innate to discussing a sensitive topic with a pediatric patient and to help our trainees increase their confidence and self-efficacy in providing evidence-based treatment options for adolescent OUD.We developed a simulation-based educational intervention aimed at increasing addiction medicine and addiction psychiatry trainees’ confidence in the management of adolescents with OUD. The simulation focuses on three topics specific to adolescent care, including the understanding of unique aspects of confidentiality, the need to distinguish between mild and more severe forms of OUD, and the ability to communicate age-appropriate medication treatment options to a young patient. We chose to use simulation as our training modality for several reasons. First, most addiction trainees generally have limited opportunities to interact with a high volume of adolescent patients. Simulation scenarios with live actors are ideal for practicing high-stakes human interactions with patients with high acuity or uncommon conditions rarely encountered in clinical practice.We developed a simulation-based educational intervention  for the We created the simulation-based educational intervention in an interdisciplinary collaboration between addiction specialists, pediatricians, and medical simulation experts. Prior to implementing the simulation-based intervention, we pilot tested the simulation with an addiction medicine physician who had recently completed specialty training. The graduate provided feedback, which was incorporated into the final product. Once the curriculum had been developed, we incorporated it into addiction psychiatry and addiction medicine fellows’ first 6 months of subspecialty training. All trainees were X/Drug Addiction Treatment Act waivered; however, most participants had limited experience working with adolescents in clinical settings and less in working with adolescent substance use . All hadThe simulation experience lasted approximately 60 minutes, with time allotted for trainees to review their learner packet case materials , receivePrior to implementation, we created a video recording of the PowerPoint titled “Management of Adolescents With Opioid Use Disorder” . The vidStandardized patients (SPs), composed of both professional actors and volunteer students from other departments of the university, received the SP packet , which iFacilitators were trained addiction specialists, and one had additional expertise in working with adolescents. To offer this course elsewhere, we recommend that facilitators have some background knowledge of the management of OUD and be familiar with local state laws regarding adolescents. Included in The scenario took place in a simulated patient room resembling an outpatient evaluation room within a medical school–based immersive health care simulation center. The debriefing session was conducted in a separate confidential conference room. During the simulation, the SP used a cellular phone as a prop, and a chair was provided for the medical trainee. A facilitator stood in the control room next door behind a one-way mirror and offered one-way instructions through a handheld transceiver to the SP's earpiece. SPs received a packet  with theTo successfully use this case scenario, implementers will likely need at least one trained facilitator and an SP. In our situation, we had three trained facilitators in order to create efficiency and facilitate parallel, concurrent sessions for multiple learners. One facilitator oriented the trainees upon arrival and provided an exit interview. The other two facilitators were paired one to one with a trainee for the simulation/debrief, alternating their use of the simulation and debrief room. Learners participated in the simulation individually and were debriefed by the facilitators one-on-one. An SP who appeared young enough to resemble a teenager portrayed the adolescent patient for all the simulations within each given cohort. In addition, we had a simulation technician who helped set up equipment. Depending on a facility's available resources, the technician role could be filled by the facilitator.We invited addiction psychiatry and addiction medicine fellows to participate in the educational activity. Fellows were expected to attend the simulation and debrief as part of their addiction training curriculum; however, surveys were optional, and fellows could opt out of survey/data collection. One week prior to the simulation, trainees completed an electronic survey to measure their confidence in learning topics. Subsequently, learners participated in the presimulation educational didactic, which included (1) viewing a 10-minute PowerPoint video addressing learning objectives and (2) completing a baseline survey describing previous relevant clinical experience and basic demographic information. Trainees received a scheduled time and directions to the simulation center via email. On the day of the simulation program, a facilitator greeted trainees with the learner packet and answered initial questions before escorting them individually to the entrance of the simulated outpatient office. Once inside, the trainee interacted with the SP actor in a 20-minute simulated case encounter featuring a treatment-seeking adolescent with OUD in early signs of opioid withdrawal. Participants assessed the signs and symptoms of opioid withdrawal and explained treatment options and confidentiality to the adolescent. A facilitator stood in the control room next door behind a one-way mirror, offered one-way instructions through a handheld transceiver to the SP's earpiece, and completed the critical actions checklist . After t19 We followed the overall structure of the PEARLS (Promoting Excellence and Reflective Learning in Simulation) framework to develop our debriefing.19 First, the facilitator reviewed the learners’ self-debrief and evoked participants’ emotional reactions, revealing key issues important to the learners. This allowed a common agenda to develop. Participants then summarized the case, with the goal of highlighting their understanding of the learning objectives or lack thereof. Next, learner frames were evoked with the use of the advocacy/inquiry technique20 to probe for particular learner actions or challenges that facilitators had noted as critical learning points during the simulation. The facilitator then guided participants through critical reflection and delivered key learning points through focused teaching with the use of the debrief guide. Directions for future learning were developed in collaborative dialogue between learner and facilitator, and three learning goals were identified. In addition to the debrief guide, facilitators utilized a recent review on medication management for adolescents with OUD in preparation for the debrief  said they felt comfortable during the training, 96% (25) said they found the exercise effective, and 100% (26) said they would recommend the training to others.Between 2016 and 2019, a total of 35 fellows  participated in the educational activity. Seven of the participants reported having received some training on adolescents with substance use disorders prior to the exercise. Prior reported training consisted of elective clinical rotations, elective online courses, and attendance at conferences, among others . Of the •Allows for deliberate practice: “The simulation was a great way to practice communicating with an adolescent patient” (14).•Anxiety: “I felt anxious because: lack of experience with adolescents” (four), “lack of medical knowledge” (five), “being watched/evaluated” (two), “didn't know what to expect” (one).•Communicating with adolescents: “Talking to an adolescent is challenging” (six).•Realistic: “The case felt real and was reasonable” (six).At the conclusion of the simulation exercise, trainees completed a self-debrief that asked two questions: How did you feel about the case? Can you summarize what happened during the case? All participants were able to summarize the case scenario and the three main learning objectives. Recurrent themes describing feelings about the case included the following (the number of learners who described the theme is given in parentheses at the end of each item):•Continuing to practice communicating medical knowledge and establishing rapport with adolescents (15).•Identifying treatment options for adolescents in the community I practice/will practice (11).•Reading further on medications and behavioral interventions for adolescents with OUD (11).•Researching federal guidelines and policies surrounding methadone treatment for adolescents (eight).•Reviewing statutes of confidentiality in different states/countries (seven).•Engaging family in treatment for adolescents with OUD (five).Finally, after completing the debrief with a facilitator, learners identified future learning goals. Recurrent learning goals that emerged included the following :7 were known to have been published. The lack of opportunities during training to manage adolescents with OUD make our simulation-based curriculum ideal for this rare but critical health scenario. Our curriculum provides learners with an opportunity to demonstrate understanding of confidentiality laws as they pertain to substance use care in adolescents, assess for the presence of opioid withdrawal, and explain age-appropriate medication treatment options to adolescents with severe OUD.This step-by-step description of our activity demonstrates how to implement a simulation-based educational intervention on management of adolescents with OUD. At the time of writing, no peer-reviewed simulation-based curricula addressing the management of adolescents with OUD, a debilitating chronic condition that often begins during this vulnerable developmental stage in which concerns about privacy influence health care decisions and ability to openly communication with health care professionals,The exercise for subspecialty trainees was feasible, was well received, and increased confidence in all areas as evidenced by an increase in average mean scores in all domains. Most participants felt comfortable throughout, found the exercise effective, and stated that they would recommend the curriculum to others. Although some participants reported experiencing some form of anxiety , they also felt the simulation was a great tool to practice engaging with, developing rapport with, and communicating medical knowledge about OUD to an adolescent. Learning goals were identified by all participants, demonstrating the exercise's ability to inspire continued learning and inquiry.Although our training was conducted in an academic institution with a simulation center, it may be used by training programs without a simulation center, as there is no specific equipment required during the session. The use of a one-way mirror and handheld transceiver  is helpful but not necessary for successful execution. Developing rapport with an adolescent and effectively communicating medical knowledge and recommendations often require practice. The simulated case should be portrayed by a young adult to keep the experience as realistic as possible and mimic the discomfort that naturally occurs when counseling an adolescent. Training surrounding OUD and opioid withdrawal symptom manifestation may be necessary for young actors serving as SPs. In addition, postsimulation debriefs are encouraged to answer questions or respond to feelings from the SP that may arise during the interventions. For example, one of our actors shared a story of a friend with opioid addiction and the feelings of empathy for her friend that emerged during the exercise.24Even though this case was developed for addiction psychiatry and addiction medicine subspecialty trainees, it could potentially be adapted for application in other specialties, including general psychiatry, child and adolescent psychiatry, pediatrics, emergency medicine, and obstetrics and gynecology. The training could also be used with other professions, such as nursing, physician assistants, and clinical psychology. Tailoring the curriculum for nonaddiction trainees may require additional prework, such as material that recognizes addiction as a chronic medical condition with available lifesaving medications, as well as guidelines on how to diagnose OUD. The information provided to participants in the PowerPoint should be tailored to reflect state-specific confidentiality laws. Information about state-specific confidentiality laws can be found online through state-specific websites or at the Center for Adolescent Health & the Law.21 links in Limitations of our study include a small sample size of trainees at one training site. However, with most addiction medicine and addiction psychiatry fellowship programs graduating only one to two fellows per year, our sample size is comparatively large. Some participants did not complete the 1-month follow-up or completed it after the deadline. The evaluation did not allow us to determine whether the increase in confidence would translate into improved clinical competence or patient outcomes in real-world clinical settings. In addition, we were unable to discern what led to an increase in confidence scores: the prework, the simulation, or the combination of the two. Future studies should examine these results further by comparing confidence scores of individuals who complete the prework and individuals who do not. We were fortunate to have a pediatrician and psychiatrist, both with addiction expertise, evaluate and debrief participants. The expertise of the faculty may not be generalizable to other settings. However, we believe that nonaddiction experts could implement the curriculum by reviewing local state laws on confidentiality along with other material included in our curriculum (see prereading documentWe continue to use this educational activity with incoming addiction medicine and addiction psychiatry fellows at our institution. In response to some of the fellows’ identified learning goals, we have begun offering additional resources and learning material at the conclusion of the exercise, such as a detailed manual of local community resources. Future directions may include implementing the curriculum virtually and with other subspecialties, specialties, and health care professionals as well as expanding to national forums. In summary, we have found that a simulation-based educational intervention is an effective teaching method for the management of adolescents with OUD and that this curriculum can help advance the state of opioid education among health care professional trainees.OUD Simulation Case.docxDemographic Information Survey.docxConfidence Survey.docxCritical Actions Checklist.docxLearner Packet.docxLearner Satisfaction Survey.docxManagement of Adolescents With OUD.pptStandardized Patient Packet.docxDebriefing Guide.docxAll appendices are peer reviewed as integral parts of the Original Publication.


Biotransformation of host bile salts by gut microbes results in generation of secondary bile salt species that have biological and physicochemical properties that are distinct from the parent compounds. There is increased awareness that a bile salt–gut microbiome axis modulates various processes in the host, including innate and adaptive immunity, by interaction of microbial bile salt metabolites with host receptors. Omics and targeted approaches have vastly expanded the number and repertoire of secondary bile salt species. A new class of microbial bile salt metabolites was reported in 2020 and comprises bile salts that are conjugated by microbial enzymes. Amino acids other than those employed by host enzymes (glycine and taurine) are used as substrates in the formation of these microbial bile salt conjugates (MBSCs). Leucocholic acid, phenylalanocholic acid and tyrosocholic acid were the first MBSCs identified in mice and humans. The number of distinct MBSCs is now approaching 50, with variation both at the level of bile salt and amino acid employed for conjugation. Evidence is emerging that MBSC generation is a common feature of human gut bacteria, and initial links with disease states have been reported. In this review, we discuss this intriguing new class of secondary bile salts, with yet enigmatic function.  To communicate with the host, the microbiome converts diet- and host-derived molecules into metabolites with signaling function. These signaling molecules include bile salts, amphipathic molecules produced in the liver that are the active ingredient of the digestive fluid bile . Bile saN-amidated) with the amino acid glycine or taurine  nmol/mmol). The simultaneous presence in vast larger amounts of other receptor activating bile salts, combined with FXR/TGR5-binding affinities that are more or less similar for MBSCs and non-MBSC species, raises the question if MBSCs have a relevant contribution to host bile salt signaling or if their function relates more to gut ecology.Using a quantitative approach, we made a preliminary assessment on the presence of MBSCs in intestinal content of patients with acute intestinal failure . Figure. LC-MS aInherent to their detergent properties, bile salts have antibacterial activity, with physicochemical properties  an important determinant of bactericidal activity of individual species. Microbial metabolism of bile salts is widely viewed as a defensive mechanism that provides an advantage to adapted bacterial species. It is currently unresolved if microbial bile salt conjugation serves as an additional defensive strategy or has a more direct role in gut ecology. Common principles of microbial bile salt transformation are briefly discussed and viewed in light of MBSCs.Bacterial deconjugation of bile salts results in more hydrophobic molecules, which are in general more toxic, especially when accompanied by 7α-dehydroxylation. At the same time, deconjugated bile salts have lower solubility in the fecal water phase, and precipitation, as well as enhanced binding to fibrous dietary matter , rendersClostridiales [bai proteins and may play a role in niche restriction by forming hydrophobic secondary bile salts. This is exemplified by Clostridium scindens, which inhibits germination of the pathogen Clostridium difficile by producing secondary bile salts. Reconjugation may protect capable species from the growth-repressive effects of ‘free’ bile salts [Microbial BSHs catalyze the deconjugation of bile salts. BSH activity is widespread in gut bacteria, especially in Gram-positive bacteria, that due to their lack of an outer membrane are more sensitive to detergent effects of bile salts . Bacteriridiales . 7α-dehyle salts .Salmonella enterica or Escherichia coli is expression of efflux pumps that maintain low levels of bile salts inside bacteria [Generation of more hydrophilic species is a parallel strategy for microbial detoxification of bile salts. Reaction types include dehydrogenation/oxidation and epimerization of hydroxyl groups in the steroid nucleus. Site-specific enzymes that catalyze these conversions  are widespread in gut bacteria ,45. Oxidbacteria ,49. It iFunction of MBSCs as a niche factor was explored in the original publication . AdditioClostridium bolteae strains capable of synthesizing them. Initial explorations indicated that MBSCs were able to activate the bile salt receptor Fxr in the liver when administered to mice. Meanwhile, the number of bacterial strains capable of MBSC formation in vitro, as well as the diversity of MBSCs, has greatly expanded. Insight into the functions of MBSCs is, unsurprisingly, still lagging behind. Of particular interest would be to learn if MBSCs are absorbed by the host in intact form, in amounts that would make a relevant contribution to host signaling on top of abundant receptor-modulating bile salts in the circulation.The pioneering study of Quinn et al. that was published in March 2020 resulted in the identification of three MBSCs and two This is the more relevant, as MBSCs appear prone to degradation by bacterial and host enzymes that are also present in the gut lumen. Disease associations have been uncovered for fecal MBSCs and Crohn’s disease and cystic fibrosis and overnutrition-related disorders. Analysis of samples of the above patient groups may provide the first clues to a possible impact of MBSCs on the host and/or the disease process. If the consequence of an altered gut microbial community structure, MBSCs may have potential as a biomarker for gut dysbiosis. Conversely, MBSCs may engage in intermicrobial communication and have a function in gut ecology. Much is to be learned about MBSCs, and the future will tell if, like their equivalent in pop music, these new kids on the block are here to stay.


Oligodendrocytes are multifunctional central nervous system (CNS) glia that are essential for neural function in gnathostomes. The evolutionary origins and specializations of the oligodendrocyte cell type are among the many remaining mysteries in glial biology and neuroscience. The role of oligodendrocytes as CNS myelinating glia is well established, but recent studies demonstrate that oligodendrocytes also participate in several myelin-independent aspects of CNS development, function, and maintenance. Furthermore, many recent studies have collectively advanced our understanding of myelin plasticity, and it is now clear that experience-dependent adaptations to myelination are an additional form of neural plasticity. These observations beg the questions of when and for which functions the ancestral oligodendrocyte cell type emerged, when primitive oligodendrocytes evolved new functionalities, and the genetic changes responsible for these evolutionary innovations. Here, I review recent findings and propose working models addressing the origins and evolution of the oligodendrocyte cell type and adaptive myelination. The core gene regulatory network (GRN) specifying the oligodendrocyte cell type is also reviewed as a means to probe the existence of oligodendrocytes in basal vertebrates and chordate invertebrates. Vertebrate central nervous systems (CNSs) possess several distinct glial cell types that are essential for CNS functions. Oligodendrocytes are most frequently associated with myelination, but also perform other functions that are less well characterized. For example, oligodendrocyte lineage cells provide metabolic support to axons, influence axonal and dendritic growth, form synapses with neurons, regulate inflammation and angiogenesis, produce extracellular matrix to form perineuronal nets, and influence blood-brain barrier function . The evoIf vertebrate myelin evolved in ancestral hinge-jawed fish, did oligodendrocytes evolve coincidentally, or did the cell type emerge prior to the evolution of the myelinating phenotype? To date, an oligodendrocyte-like cell has not been characterized in organisms that lack myelin, but the CNS glia of invertebrate chordates and jawless vertebrates are incompletely characterized. Rather than making assumptions based on the absence of evidence, it will be beneficial to further expand initial characterizations of glia and oligodendrocyte-associated gene expression in invertebrate chordates and agnathans to either support or rule out their presence. Unlike myelin, which is structurally unmistakable in electron micrographs, identifying primitive oligodendrocytes is less straightforward. These cells may have been multifunctional and performed functions that were not retained by the gnathostome oligodendrocytes. Alternatively, primitive oligodendrocyte functions could be similar to the non-myelinating functions of gnathostome OPCs and oligodendrocytes. In comparison to myelination, these non-myelinating functions are less well characterized and do not necessitate specialized structures that would be morphologically identified as easily as myelin sheaths. In addition, the upstream transcriptional regulators and effector genes mediating oligodendrocytes’ non-myelinating functions are less well understood. In evolutionary studies, the core regulatory factors expressed by a cell type may be a more accurate indicators of cell identity than morphology and function . Therefo+ cell populations are incompletely characterized, due to the master regulatory roles of SoxE family transcription factors in gnathostome oligodendrocyte development, these important findings raise the possibility that chordates and agnathans possess an oligodendrocyte-like cell type that lacks the ability to myelinate axons. This elevates the spinal cord as a CNS region of interest in the search for ancestral oligodendrocytes.Relatively few studies have investigated glia and potential oligodendrocyte homologous cell types in living chordates or agnathans such as amphioxus, tunicates, hagfish and lamprey. However, some important studies and discoveries have been made, which together raise the possibility that the oligodendrocyte cell type may have evolved prior to the myelinating phenotype. Ultrastructural characterization of the amphioxus CNS revealed the presence of cells termed axial glia . These cThe purpose of this review is to evaluate evidence and potential models for the evolution of the oligodendrocyte cell type as well as the adaptive myelination phenotype. Taking into consideration evidence from both aquatic and terrestrial vertebrates, a working definition for the oligodendrocyte cell type based on core GRNs is proposed. I also propose models for how co-option, module integration, and acquisition of novel genes can explain the evolution of the adaptive myelination phenotype. Finally, the importance of increasing diversity of research organisms used to study oligodendrocytes and myelin is discussed as an essential means to understand the evolutionary mechanisms responsible for this novel cell type and its myelinating and non-myelinating functions.Cell types have classically been defined based on structural and functional properties, but the availability of genetic profiling techniques such as single-cell RNA sequencing provide new means to investigate and define cell identities. When cell morphology or function are used, it is important to recognize that cells are dynamic systems that may show a range of phenotypes depending on the context. For example, many cell types change cellular state over time or in response to environmental stimuli . Due to Oligodendrocytes provide another example of the issues and challenges associated with defining cell types based on morphologies and functions. The oligodendrocyte cell type was discovered by What are the defining properties of oligodendrocytes? This is challenging from a morphological standpoint because oligodendrocyte lineage cells may exhibit a simple bipolar morphology, may develop a complex and branchy morphology with dynamic process extensions, or may possess myelin sheaths . UltrastDefining cell types based on molecular signatures offers an additional and complementary approach. In contrast to cellular structure and phenotypes, which change depending on the various states that cells encounter and can blur when comparing homologous cell types, the transcription factors expressed by a cell type tend to show strong evolutionary conservation  domain is a feature common of all gnathostomes observed to date. The pMN domain is one of several distinct progenitor cell populations that are patterned by opposing Hedgehog and BMP morphogen gradients along the neural tube dorsal-ventral axis  and agnathans (lamprey), it is unknown if these cells produce glia such as oligodendrocytes. In this model, we consider a scenario where the Olig-expressing pMN progenitors first arose as a neuron-producing domain, and that these progenitors subsequently evolved the ability to specify oligodendroglia  is a direct downstream target of Olig2. Induction of Sox10 expression is facilitated by an enhancer that is conserved between birds and mammals  and effectors associated with myelination  (In addition to Olig-dependent regulation of SoxE factors, in mammals, Olig1 and Olig2 regulate the expression many downstream genes involved in oligodendrocyte differentiation. It is likely that some novel Olig target genes other than nd Mobp] . InvestiOne mechanism thought to drive the evolution of novel cell types is the duplication of regulatory identities followed by divergence, specialization, and mutual repression of the alternative identity. In this way, multi-functional ancestral cell types diverge into two sister cell types. These sister cell types may lose features of the ancestral cells, may become more specialized, and may also co-opt new regulatory modules to take on new form and function. Here, an additional model is discussed whereby pMN-derived multifunctional glia existed prior to the origins of oligodendrocytes. Genetic changes occurred that drove formation of two distinct core regulatory networks, resulting in two sister cell types, one of which was the ancestral oligodendrocyte . A key dThe functions of the ancestral pMN-derived glial cell type cannot be addressed with current knowledge. This cell type may have been multifunctional, or it could more closely resemble the properties of a glial cell type found in extant vertebrates. If for example a multifunctional glia existed in an ancestral chordate prior to the divergence of the vertebrate lineage, we would expect it could be retained in living chordates such as amphioxus. This is possible and amphioxus possess glia including those with OPC-like morphologies , but the+ multifunctional glial origin. This model is supported at the core GRN level, because the regulatory factors expressed by each cell type overlap. It is also proposed that sister cell types evolve ways to mutually repress the alternative fate. Although how this occurs is not yet clear, oligodendrocytes repress expression of Foxd3 and Wif1 (One sister cell type candidate are motor glia. Due to the common origins and anatomical position of motor neurons and oligodendrocytes, and Wif1 , and it A second possibility is that rather than MEP glia, vertebrate astrocytes and oligodendrocytes are sister cell types. In this scenario, the ancestral multifunctional glia would have performed astrocytic and non-myelinating oligodendrocyte functions. At some point, the core GRN specifying this cell type diverged into primitive astrocytes and oligodendrocytes. This seems unlikely because we would expect to see some similarity in the core GRN for astrocytes and oligodendrocytes, but instead there is remarkably little similarity . HoweverIn order to support or reject the models described above it will be important for future studies to characterize CNS glia in invertebrate chordates and agnathans. These studies may reveal primitive oligodendrocytes or multi-functional glia in these species, or alternatively, may instead support the notion that the oligodendrocyte cell type evolved concurrently with the myelinating phenotype. Evidence that the pMN domain and oligodendrocyte-like CNS glia are present in in all chordates would most strongly support the “novel progenitor zone” or “novel pMN derivatives” models. Alternatively, evidence that neither oligodendrocyte-like cells or the myelinating phenotype existed in chordates or agnathans would support the idea that the cell type and myelination arose concurrently. This would be most consistent with the “novel pMN derivatives” or “sister cell type” models. A deep homology event is another possibility not considered in the models described above. For example, the oligodendrocyte cell type may have evolved through convergence to other pre-existing cell types, acquiring a small set of transcriptional regulators such as Olig2 and Sox10, rather than by co-opting a larger transcriptional network or through the divergence of sister cell types. Additional studies that investigate the genetic relationships between CNS glia across multiple chordate and basal vertebrate species will distinguish between these possibilities.Oligodendrocytes are multifunctional cells that can exist in multiple cellular states. To transition to the myelinating state necessitates changes in gene expression resulting in the production and localization of gene products needed to form myelin sheaths. Myelin associated gene expression does not define the oligodendrocyte cell type identity or core GRN. Instead, the GRN for myelination is an accessory module specific to one of several cellular phenotypes exhibited by oligodendrocytes. Cell-type specific phenotypic modules such as myelination have also been referred to as apomeres  and the Myelin was once thought of a fixed and unmodifiable insulation, but the discovery that myelination is adaptable to external experiences increases the molecular complexity and repertoire of gene products involved (reviewed by In addition to the obvious resemblance to other ensheathing cells, oligodendrocytes display functional and gene expression features that resemble the post-synaptic compartments of neuronal dendrites . Like neConsidering the similarities described above, it is logical to propose that the oligodendrocyte apomere for myelination evolved by integrating gene expression and protein targeting modules associated with ensheathing glia and neuronal dendrites in combination with the acquisition of novel myelin-associated genes that are unique to gnathostomes . Neurona2+ signaling (2+ signaling in activity-dependent dendritic spine remodeling. Taken together, there are many similarities when examining how dendritic and oligodendrocyte process extensions respond to axon derived cues.Dendritic and myelinating processes are both highly specialized and comprise sets of proteins that are not expressed by most cell types in the vertebrate body plan. Despite this, the overlap in proteins typically designated as synaptic proteins that are present in both oligodendrocyte and dendritic processes is extensive. These have been nicely reviewed by ignaling , which i2+-dependent transcription factors by oligodendrocytes could orchestrate synaptic gene expression in oligodendrocytes (The transcriptional regulation of neuronal synapse-associated genes in oligodendrocytes is not well understood, but is likely to hold the key to understanding how oligodendrocytes evolved the ability to recognize axons and adapt their behavior to changes in circuit activity. For example, mammalian oligodendrocytes express factors with known roles in synaptic gene expression in neurons such as CREB and Nfat-family factors . This sudrocytes . An alteAs previously described, this co-opted neuronal module of the myelinating apomere may account for several mechanistic features of myelination, but alone, this module is insufficient to drive myelin sheath formation. Further integration with other gene products mediating axon ensheathment and multilamellar, compact myelin sheath formation was necessary for the evolution of myelination. It is unknown if ancestral chordate or agnathan CNS glia preceding the origins of myelination possessed ensheathing capabilities. Recent ultrastructural studies examining lamprey and amphioxus CNS glia identified axon-contacting glial processes, and it is possible these represent ancestral ensheathing glia from which the oligodendrocyte ensheathment module was derived or co-opted . Alternambp gene correlates with the emergence of myelination in gnathostomes (reviewed by mbp is an example of a novel gene, it is not surprising from an evo-devo perspective that most myelin effectors are not novel genes, and that gene re-purposing or neofunctionalization played a major role in the evolution of vertebrate myelination (An additional and essential element of the myelinating apomere is the recruitment genes involved in forming compacted myelin sheaths. Re-purposing of existing genes through co-option cannot fully account for the myelinating phenotype because some genes necessary for myelination are not present in invertebrate chordate or jawless vertebrate genomes. A classic and well described example is Mbp, which is necessary for myelin formation and maintenance and is one of the most abundant proteins in myelin. The appearance of the lination . This prlination .Notably, the apomere integration model proposed above assumes that vertebrate myelination has always been adaptive, and that there is no such thing as non-adaptive myelination. Substantial evidence for activity-dependent myelination in both mammals and teleost fish suggests that myelination was adaptive at least 380 million years ago in ancestors of modern bony fish. Because adaptive myelination has never been investigated in cartilaginous fish, it cannot be ruled out that the adaptive feature of myelination is an innovation that emerged during the relatively short evolutionary period between the divergence of cartilaginous and bony fish lineages. Future studies investigating adaptive myelination in cartilaginous fish such as sharks or skates would provide further insight into this question.The origins and evolution of the oligodendrocyte cell type and myelinating phenotype are among many unsolved mysteries in neuroscience. Few studies have directly addressed the origins of the oligodendrocyte cell type, but myelin evolution has been studied for several decades. Many discoveries have been made using rodent models along with some studies of myelin ultrastructure and biochemistry in lamprey, hagfish, shark, and ray CNSs . More reOne important area for future investigation is to determine which extant species have oligodendroglia, and which lack a homologous cell type. In addition to investigating expression of core regulatory factors described above, single-cell RNA-Seq profiling and comparisons between species are likely to be informative, but complex. Currently, it can be challenging to identify and classify distinct cell types within a single species. Investigations comparing species separated by tens or hundreds of millions of years of independent evolution will be difficult but are important endeavors. Important foundational work has been performed using amphioxus and lamprey. Both species offer expanding genomic resources, animal breeding, embryology methods, and tools for gene manipulation and reporter expression. For instance, seminal studies have identified CNS Olig and SoxE populations in amphioxus and lamprey that are yet uncharacterized . It willA second important area for investigation is to more fully characterize how the earliest gnathostome oligodendrocytes were specified and operated using comparative studies in extant species. It is not possible to understand how these cells arose without establishing the genes and core network that are involved and required. Fossil evidence has been used to propose that myelinating oligodendrocytes evolved prior to the divergence of placoderms, which of course cannot be confirmed or used to investigate the molecular evolution in living organisms . Based oIt is unlikely that a cartilaginous fish model system will emerge that comes close to the high throughput and rapid generational cycles of bony fish models such as zebrafish. Therefore, much of our understanding of the successful formula for oligodendrocyte and myelin evolution will need to partially rely on bony fish. Zebrafish are widely recognized as a powerful model organism, but like any species, present some drawbacks. For instance, zebrafish and other teleosts are small and rapidly growing animals and also have a duplicated genome. Use of additional bony fish models as well as other non-mammalian vertebrates such as Xenopus is important to overcome these drawbacks and avoid over-reliance on a single model . Rather The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We test this possibility using the odds of a live-born twin among male births in Norway as an indicator of the depth of selection in birth cohorts.The suspicion that a population stressor as profound as the COVID-19 pandemic would increase preterm birth among cohorts in gestation at its outset has not been supported by data collected in 2020. An evolutionary perspective on this circumstance suggests that natural selection We apply Box–Jenkins methods to 50 pre-pandemic months to estimate counterfactuals for the nine birth cohorts in gestation in March 2020 when the first deaths attributable to SARS-CoV-2 infection occurred in Norway. We use Alwan and Roberts outlier detection methods to discover any sequence of outlying values in the odds of a live-born twin among male births in exposed birth cohorts. We find a downward level shift of 27% in the monthly odds of a twin among male births beginning in May and persisting through the remainder of 2020.in utero, birth cohorts exposed in utero to the onset of the COVID-19 pandemic yielded fewer male twins than expected.Consistent with evolutionary theory and selection Our finding of fewer than expected male twin births during the onset of the COVID-19 pandemic provides more evidence that evolution continues to affect the characteristics and health of contemporary populations. Constants and autoregressive parameters all exceeded twice their standard errors.In which [a priori—from the dependent variable before measuring its association with the ‘interruption’ . Our test, therefore, ‘controls’ for autoregression at 2 and 7 months for males and females respectively even though we did not predict this autocorrelation a priori.As noted above, to satisfy the assumptions of statistical estimation of association, time-series modeling removes all forms of autocorrelation—whether or not hypothesized in utero in Norway. If so, the unexpectedly low rates of preterm birth observed in 2020 may reflect, at least in part, spontaneous abortion of fetuses that in more benign times would have survived to live birth later in pregnancy but before 36 complete weeks of gestation.We find evidence that, consistent with theory, the onset of the COVID-19 pandemic and the steps taken to avert its worst effects, may have deepened selection in utero [Norway experienced relatively low rates of SARS-CoV-2 infection and excess mortality during our test period, making it a good setting in which to estimate the association between social processes, as opposed to infection, and twin births. The pandemic could have engendered stress not only by threatening and causing infection, but also by imposing demands on individuals and households (e.g. caring for children out of school or ill relatives) while also disallowing access to social resources (e.g. supportive co-workers), and constraining access to settings  that Norwegians would otherwise rely on for effective restoration. We, however, have no measurements of access to social and other coping resources in Norway during the pandemic. We cannot, therefore, estimate whether an increase in infection or a decrease in protective or restorative resources better ‘fits’ the drop in twin births that we found. We note, though, that previous studies with Norwegian and Swedish data have found indications of higher rates of spontaneous abortion under stressful circumstances that do not involve infection , 43–45. in utero . The panin utero .2 dipped nearly 10 μg/m3 below pre-COVID-19 levels [We further note that pandemic management strategies may have reduced the dose of a least one ambient stressor, traffic-related air pollution (e.g. nitrogen dioxide) , thought9 levels . Our finin utero appeared most susceptible to the COVID-19 pandemic. Data on gestational age of live births and spontaneous losses would allow the creation of a time series arrayed by estimated month of conception (rather than by month of birth). Such series would allow identifying which conception cohorts contributed most to our birth cohort findings. That information would, in turn, suggest the gestational age at which stressors on the population trigger selection in utero against male twins.Limitations of our tests include that we cannot know the gestational age, or ages, at which twins Pandemics, by axiom, affect most human populations. That fact, and the lack of twin birth data from other societies, precludes our using a comparison population design to control for generally occurring confounders . The perInterventions in Norway intended to increase social distancing early in the pandemic included suspending medically assisted reproduction (MAR) services . Twins, in utero. We and others have argued that this literature leads to the prediction of fewer than expected twins among male births in stressed populations. Other literature may also lead to the prediction and suggest other lines of further research. Work describing competition between twins for resources in utero [We were led to our hypothesis by theoretical and empirical literature arguing that natural selection conserved biological mechanisms that manifest in reproductive suppression, brood reduction, offspring sex selection and selection in utero  could, fa priori on any dimension of dose. If this were possible, future research could test the intuitively appealing argument that differences in the strength of association reflects not only differences in the characteristics of the populations tested, but also in the dose of stress they likely suffered.Although multiple studies have supported the argument that twins will appear less frequently than expected among males born to stressed populations, the stressors tested vary widely in their nature as do the reported effect sizes. No work has yet attempted to array the stressors in utero shapes the characteristics of human birth cohorts and that collective as well as individual choices likely affect the targets and depth of that selection [in utero.We cannot claim that our findings have clear clinical implications although they suggest increasing, during stressful times, public health programming focused on women of reproductive age. Our findings do, however, have implications for our understanding of how evolution affects the nature and experience of contemporary populations. They call attention to the facts that selection election . They, mConflict of interest: None declared.


In the era of precision medicine, radiation medicine is currently focused on the precise delivery of highly conformal radiation treatments. However, the tremendous developments in targeted therapy are yet to fulfill their full promise and arguably have the potential to dramatically enhance the radiation therapeutic ratio. The increased ability to molecularly profile tumors both at diagnosis and at relapse and the co-incident progress in the field of radiogenomics could potentially pave the way for a more personalized approach to radiation treatment in contrast to the current ‘‘one size fits all’’ paradigm. Few clinical trials to date have shown an improved clinical outcome when combining targeted agents with radiation therapy, however, most have failed to show benefit, which is arguably due to limited preclinical data. Several key molecular pathways could theoretically enhance therapeutic effect of radiation when rationally targeted either by directly enhancing tumor cell kill or indirectly through the abscopal effect of radiation when combined with novel immunotherapies. The timing of combining molecular targeted therapy with radiation is also important to determine and could greatly affect the outcome depending on which pathway is being inhibited. A plethora of factors are involved in the development and progression of cancer in individuals such as family history, age, sex, primary site of origin and driver mutations; thus, treatment depends upon the goal of therapy - curative or palliative. Treatment for cancer involves multiple approaches including surgery, chemotherapy, immunotherapy, small molecules that target certain cancer signaling pathways, and radiation depending on cancer type or status. The use of multiple treatments concurrently is referred to as multi-modality treatment. Radiation therapy plays a crucial role in the management of cancer. Also known as radiotherapy (RT), it is a method of impeding cancer cell division by using high-energy ionizing radiation to induce DNA damage and disrupt cell cycle progression. In the treatment of cancer, RT can be given alone or coupled with chemotherapy or surgery and is aimed at reducing local tumor burden. The primary advantage, however, that RT confers over chemotherapy is the ability to precisely target the tumor and reduce systemic side effects. Epidemiological studies have reported that almost 54% of breast cancer survivors were treated with radiation therapy in 2016 and this is projected to become 60% by 2030 , which includes Stereotactic Body Radiation therapy (SBRT) and Stereotactic Radiosurgery (SRS). EBRT most typically uses a linear accelerator to deliver radiation directly into the cancer site in the form of photons. Depending on the location of the tumor, this radiation can be of high or low energy. For instance, high energy EBRT is used in the treatment of head and neck cancer, breast, lung, and eye cancer –5 while Conventional fractionated EBRT was traditionally based off the classical “four R’s” of radiation biology: reassortment, repair, reoxygenation, and repopulation , to whicAdditionally, SBRT or SRS is used to deliver very high doses of radiation to the primary sites or metastatic sites in few treatments –5, with Ionizing radiation introduces energy into molecular structures which then releases electrons creating ions that are capable of breaking covalent bonds. The breakdown of these covalent bonds within DNA produces DNA breaks, including double-stranded breaks. Radiation also leads to the generation of reactive oxygen species (ROS) which oxidize lipids and proteins and are capable of damaging DNA in many ways, including single-strand breaks. This damage leads to cell death and failure of mitosis.Consequently, highly proliferating cells are most susceptible to damage due to radiation. DNA damage is not an uncommon phenomenon, with as many as 50,000 lesions, or instances of DNA damage, in each cell, every day. Cellular mechanisms of DNA repair are able to fix this continuous damage and maintain functional DNA. Endogenously induced lesions are generally isolated and more evenly distributed throughout DNA. Damage resulting from radiation is far less dispersed. When two or more lesions are found within two helical turns, this is referred to as a clustered damage site, and these are far more difficult to repair than isolated lesions , 22. Thevia several mechanisms which enhance their DNA repair capacity or suppress the functions of tumor suppressors , DCISionRT® is a multigene assay  positive oropharyngeal cancers who are thought to have improved survival outcomes due to impaired DNA repair –95. In tThe equally important aspect of radiation therapy, which is crucial for an optimal therapeutic ratio, is better understanding and prediction of normal tissue toxicity, particularly late side effects, which are usually irreversible and can severely impact quality of life. While demographic and clinical factors are well-recognized culprits of late tissue toxicity, the evolving field of radiogenomics proposed genetic factors as key players as well. Kerns et al. proposed two arching goals for the field: first, identifying key molecular pathways that can predict radiation-induced normal tissue toxicity and second, developing an assay to identify the patients who are more likely to develop late tissue toxicities and therefore require tailored treatment . SeveralDespite the efforts outlined above, the radiation oncology field significantly lags behind in designing clinical trials that are poised to prospectively test whether specific combinations of radiation and targeted therapy can particularly benefit a genomically distinct patient population. To that end, several collaborative efforts aimed to outline guidelines to usher the field toward optimizing the clinical development of novel drug-radiotherapy combinations. Two key points were proposed: 1) reconsidering novel endpoints in clinical trial design such as local control, organ preservation, and patient reported outcomes, and 2) prioritizing the development of promising therapeutics that target relevant pathways to radiation such as DNA repair inhibitors and immunotherapies –106.Traditional radiosensitizing agents (such as cisplatin and 5-fluorouracil) typically exert their effect by augmenting DNA damage . As largPreclinical evidence suggests that DDR inhibitors can act as potent radiosensitizers and potentially have greater cytotoxic effects in cancer cells compared to normal cells Figure 2In the era of immunotherapy, modulation of the host and the tumor microenvironment holds a lot of promise when combined with radiation as demonstrated in a plethora of eloquent preclinical studies. Radiation and immunotherapy agents are thought to interact through five distinct mechanisms based of the modified Steel hypothesis : (1) spaThus far, the failure to predict treatment efficacy using genetic variables represents one of the most significant obstacles to the personalization of radiation-based treatment regimens. The potential success of radiosensitizing-targeted therapy is contingent upon our better understanding of radiogenomics, which pertain to defining biomarkers of response and genetic determinants of late tissue toxicity , 131. MoFor a small molecule to be maximally effective as radiosensitizer, it must be highly specific and directly toxic to the tumor. Tumor cells depend more heavily on certain signaling pathways over normal tissues, therefore combination of RT with small molecule inhibitors of these pathways offers an alternative strategy to chemoradiation that is potentially less toxic to surrounding healthy tissues. A general limitation to this is the lack of preclinical models that mimic the human cancer to a molecular level which provides information regarding predictive biomarkers that differentiate between radioresistance and radiosensitivity.in vitro models as well as in vivo models with appropriate genetic manipulations to capture the aforementioned characteristics in response to RT. Methods include genetic knockdown, knock-in, activation, tissue-specific expression, inducible expression, and sequential expression in traditional cell culture, 3-dimensional (3D), organoid, and xenograft models. My laboratory has focused on generating 3D mono- and co-cultures using various cancers such as pancreatic, colon, and bladder  possesses multiple functions that could affect the cellular response to RT Figure 2In addition to DNA repair activity, APE1/Ref-1 also plays an important role in signaling within the tumor and TME through the transcription factors (TFs) it regulates, and many of these TFs also play a role in inflammation . Functioin vitro and in vivo  inhibitors have widely been studied for combination radiotherapy in several different cancers which enhance anti-tumor effects  in vivo –188.via several mechanisms, including activating proteins or pathways involved in repairing radiation-induced DNA damage  are naturally radioresistant, and data suggests that radiation can induce their pro-tumorigenic capabilities. However, the concept of combining RT with CAF targeting has not been investigated to date . AlternaFinally, owing to rapid advances in nanotechnology, nanomaterials have attracted particular attention to enhance the anticancer efficacy of radiotherapy , 223. NaEven with the multitude of preclinical studies looking at combining RT with targeted therapy, chemotherapy, or immunotherapy, there are still very few examples of combinations that have translated into success clinically. We will now highlight some examples as well as future directions Figure 2A large body of preclinical evidence exists to support novel radiation-targeted therapy combinations. However, to date the EGFR inhibitor cetuximab remains to be the only molecular targeted agent approved by the U.S. Food and Drug Administration (FDA) for use with radiation therapy in head and neck cancer . InteresAlthough cetuximab was relatively successful as a radiosensitizer in the setting of head and neck cancer, it failed to show promising results in other cancers where EGFR signaling is relevant Figure 2Another targeted radiosensitizer that has been relatively successful in the clinical setting is nimorazole. Nimorazole is a targeted radiosensitizer which selectively targets hypoxic tumor cells and has been shown in a phase III trial to significantly improve locoregional control by 16% in patients with cancer of the supraglottic larynx and pharynx when combined with radiation compared to radiation alone . Howeverin vitro screen of 32 cell lines that represented lung, colorectal, head and neck, and genitourinary cell lines and identified Midostaurin as a potential radiosensitizer for KRas mutant cancers   . Other cyear OS) . In thatyear OS) . This seyear OS) . ATM, ATyear OS) .Nanotechnology offers a new area of exciting research where nanoparticles can be used for targeted radiotherapy, either as sensitizers of external beams or as delivery vehicles for therapeutic radionuclides . In a phThe PACIFIC trial has revolutionized the management and therefore the outcomes of patients with locally advanced NSCLC. It has also set unprecedented clinical evidence supporting the interplay of chemoradiation and immunotherapy , 281. ImIn the metastatic setting, several prospective trials have been conducted to test the abscopal effect of radiation, which stems from many anecdotal reports and arguably stimulated much of the hype regarding the combination of radiation and immunotherapy . The absAs outlined above there are many clinical trials testing different radiosensitization paradigms. That is not meant to be a comprehensive list but rather to paint a picture for the diverse nature of signaling mechanisms that could potentially be targeted to improve the therapeutic ratio of radiation. Importantly, while there are examples of successful radiation-targeted therapy combination in clinic, failures certainly outweigh those few successes. Therefore, a lot remains to be done in to decrease attrition rates of novel radiosensitizers in the clinic.Oncology drug development has witnessed a significant growth over the last decade that was coupled with improved cancer outcomes and unprecedented drop in cancer related death rates . HoweverME and NC contributed equally to the writing and planning of the content of the review. RR provided clinical input and expertise to the review and MF edited and directed the writing and content within the review. All authors contributed to the article and approved the submitted version.MF was supported by grants from the National Institute of Health and National Cancer Institute R01CA167291, R01CA211098, R01 CA254110. NC and MF were also supported by NIH/NCI grant U01HL143403. MF was additionally supported by the Riley Children’s Foundation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coronary artery thrombosis is the major risk associated with Kawasaki disease (KD). Long-term management of KD patients with persistent aneurysms requires a thrombotic risk assessment and clinical decisions regarding the administration of anticoagulation therapy. Computational fluid dynamics has demonstrated that abnormal KD coronary artery hemodynamics can be associated with thrombosis. However, the underlying mechanisms of clot formation are not yet fully understood. Here we present a new model incorporating data from patient-specific simulated velocity fields to track platelet activation and accumulation. We use a system of Reaction-Advection-Diffusion equations solved with a stabilized finite element method to describe the evolution of non-activated platelets and activated platelet concentrations [AP], local concentrations of adenosine diphosphate (ADP) and poly-phosphate (PolyP). The activation of platelets is modeled as a function of shear-rate exposure and local concentration of agonists. We compared the distribution of activated platelets in a healthy coronary case and six cases with coronary artery aneurysms caused by KD, including three with confirmed thrombosis. Results show spatial correlation between regions of higher concentration of activated platelets and the reported location of the clot, suggesting predictive capabilities of this model towards identifying regions at high risk for thrombosis. Also, the concentration levels of ADP and PolyP in cases with confirmed thrombosis are higher than the reported critical values associated with platelet aggregation (ADP) and activation of the intrinsic coagulation pathway (PolyP). These findings suggest the potential initiation of a coagulation pathway even in the absence of an extrinsic factor. Finally, computational simulations show that in regions of flow stagnation, biochemical activation, as a result of local agonist concentration, is dominant. Identifying the leading factors to a pro-coagulant environment in each case—mechanical or biochemical—could help define improved strategies for thrombosis prevention tailored for each patient. Computational studies aiming to model thrombosis often rely on an arterial wall injury. Collagen and other extracellular matrix components are exposed to the bloodstream, which facilitates platelet adhesion to the wall and subsequent clot formation. However, these models are not adequate to explain thrombosis in other settings where even in the absence of a focal lesion, clots may still form under certain flow conditions. Coronary artery aneurysm thrombosis following KD is an example of the need to understand the mechanisms of thrombus initiation in the absence of an extrinsic factor. This study provides a new framework to investigate thrombus initiation in KD from a patient-specific perspective, which integrates fluid mechanics and biochemistry and which could help quantify the pro-coagulant environment induced by the aneurysm and become a predictive tool. The work presented here has broad relevance to other clinical situations where flow stagnation and transport are driving factors in thrombus formation. Coronary artery thrombosis is the major risk associated with Kawasaki disease (KD) . Long-teThe coagulation cascade has been widely studied. In particular, in the setting of hemostasis. The two coagulation pathways, the intrinsic or contact pathway and the extrinsic pathway initiated by tissue factor (TF), converge to the common pathway leading to activation of factor X and generation of thrombin, fibrin, and ultimately a stable clot . PlateleComputational studies aiming to model arterial thrombosis often rely on an arterial wall injury that exposes collagen, vWF, and other sub-endothelial components to the bloodstream, which facilitates platelet adhesion to the wall even in a non-activated state , and subTypically thrombosis models have been coupled with the flow, widely using a 2D approach, relying on idealized flow waveforms , 34–38 aOne limitation of current thrombosis models is their reliance on assumptions about the thrombotic potential of the wall, for which there is very limited quantitative data. These models require that initial extrinsic factor, TF, or assumed collagen or vWF concentration is prescribed as the initiator for the coagulation cascade. Although these assumptions hold in the case of trauma or athero-thrombosis, there are other scenarios where no data on the thrombogenic potential of the arterial wall is available. Therefore, these models fail to accurately represent the process of clot formation in such cases. Coronary artery aneurysm thrombosis following KD is an example of the need to understand the mechanisms of thrombus initiation in the absence of an extrinsic factor. No obvious wall injury, which would enable a local release of TF, has been reported concerning thrombosis in KD aneurysms. Therefore, we hypothesized that the intrinsic or contact coagulation pathway may be responsible for thrombus formation in this setting. Our previous studies related to KD aneurysms, have related thrombosis risk with abnormal hemodynamics, suggesting that hemodynamics are likely the primary driver. We present a new model that incorporates information from patient-specific simulated velocity fields to track platelet activation and accumulation in patient-specific coronary artery aneurysms caused by KD. Increased concentration of activated platelets and agonists induces a pro-coagulant environment that may drive thrombus initiation and growth. This hypothesis is based on recent studies that have suggested that critical concentration of PolyP, released from platelets upon activation, can activate factor XII and initiate the intrinsic coagulation cascade –10 and dThis study was approved by the Institutional Review Board at Stanford University and The Hospital for Sick Children, University of Toronto. Written subject consent or assent and parental written consent were obtained as appropriate for the imaging and simulation studies.We considered six KD patients with aneurysms and one control patient with normal coronary arteries. The diseased group was drawn from our previous study on patient-specific hemodynamics and risk of thrombosis in KD . This grE is the rate of strain tensorTo quantify cumulative shear rate exposure in the 3D domain we used a Lagrangian approach, computing shear rate exposure along path lines calculated from a patient-specific velocity field. Shear rate is defined as:Cumulative shear rate was defined as a linear function of shear rate exposure timeTo initiate the calculation, massless particles representing platelets (Stokes number ≪ 1) were released at the most proximal section of the coronary artery model (ostium) and their trajectories were time-integrated using a fourth order Runge-Kutta scheme based on the velocity field obtained from a patient-specific blood flow simulation . We obtaTo describe platelet activation related to high cumulative shear rate, we used an activation function, based on the cumulative shear rate levels observed in the healthy coronary model, and reported literature values for platelet activation indices found in regions prone to thrombosis , 25. TheWe chose four time points equally distributed along the cardiac cycle for particles release to compute path lines . Although we observed differences in cumulative shear rate distribution when comparing results from particles released at the beginning of the cardiac cycle to other time points, we did not observe substantial differences when increasing the number of time points up to ten.RP] and activated platelets [AP], and local concentrations of Adenosine Diphosphate [ADP] and PolyPhosphate [PolyP]. To perform this simulation, sub-models of the right and left coronary branches were considered independently. Activation of platelets is modeled considering two different contributions to the reaction term: mechanical activation, a function of cumulative shear rate, and biochemical activation, a function of the local concentration of activated platelets and agonists such as ADP. We define the advection-diffusion operator as:Ai] represents the concentration of the ith chemical specie considered and u is the patient-specific pre-computed fluid velocity. The velocity fields were computed using Simvascular rel and [ADP]rel account for ADP and PolyP released from dense granules upon platelet activation.Polyphosphate (PolyP):The definition of the RAD equation, for each of the chemical species considered in our model is presented below:11 platelets/l) with 5% of activated platelets circulating in the bloodstream content = 3 mol/1017plt and [PolyP]content = 0.743 mol/1017plt. Assuming at least 75% percent of the platelets dense granules content is released, we can estimate the corresponding concentrations of released [ADP]rel and [PolyP]rel:One of the primary mediators of biochemical platelet activation is the release of chemical agonists from platelet dense granules upon activation. We assumed the following values for the contents of platelet dense granules based on the literature : [ADP]coAP] and [RP] we included an activation function that limits the activity of this reaction based on a minimum ADP concentration, [ADP] > 0.2 μM, which is reported in the literature as the threshold value for reversible changes in platelets [ADP] = 0.2 μM (reversible activation) and [ADP] = 1 μM (irreversible activation).Although reaction constant rates are given in terms of [latelets . The actAP] in a normal coronary tree, and low and high thrombosis risk cases, and quantified the concentration of resting and activated platelets within the aneurysms. In addition, we quantified the concentration of agonists such as ADP and PolyP to determine if their concentration reached the critical values associated with irreversible changes in platelets and platelet aggregation in the case of ADP, and activation of factor XII (intrinsic coagulation pathway) in the case of PolyP. Critical values were set to [ADP]crit = 1μM [PolyP]crit = 2μgr / ml according to experimental values reported in the literature  ≥ 10nM.Volume fraction of the domain where the concentration of activated platelets is greater than or equal to a specified level of platelet activation. We define ADP] and [PolyP] are above the specified critical thresholds.Volume fraction of the aneurysm where  spatial distribution between coronary arteries with aneurysms and the control case showed that high [AP] correlates with regions of confirmed thrombosis , at the specific locations where thrombosis was reported, which implies full platelet activation. The volume fraction of the aneurysm where [AP] > 10 nM, representing full platelet activation, ranged from 10–100% for the high thrombotic risk patients. On the low-risk cases, this volume fraction was smaller, ranging from 0–10% . On the other hand, we observed low correlation of AP10 (volume fraction) with low wall shear stress exposure, AWSS1 .We observed a qualitative correlation between areas of low shear stress and high residence time with regions of increased pro-coagulant activity, measured by  and [PolyP] in the coronary models analyzed was negligible. We observed that although higher cumulative shear rate correlated with thrombus location to some extent (SRcum > 250) and our results did not differ substantially. Completely removing the mechanical contribution to platelet activation also did not substantially change the result. This was expected since the dominant contribution in the cases analyzed here was due to agonist-induced platelet activation.Our simulation results show that the contribution of shear rate to platelet activation is almost negligible for the cases considered in this study. However, calculated cumulative shear rate distributions suggest that stenosis proximal to the aneurysm may substantially increase the contribution of shear rate to platelet activation. The biochemical activation term is dominant in areas of high flow stagnation, where the accumulation of circulating pro-coagulants results in concentrations that may be sufficient to induce platelet aggregation or initiate the intrinsic coagulation cascade via factor XII activation . In the e extent , such vae extent . To assee extent , we also2. According to experimental assessments of thrombus deformation and growth at different shear rates  distribution from patient-specific velocity fields obtained from rigid wall or fluid-solid interaction simulations. However, we observed that as the flow becomes more complex, especially in the presence of large flow stagnation areas, wall deformation may have a more significant effect on near-wall transport. This should be considered in future studies, in particular, if large wall deformations are expected, such as in aortic dissection.In our computational experiments, we did not find substantial differences between  > [PolyP]crit). Empirical PolyP biochemical data including activation rate constant for factor XII activation needs to be incorporated to achieve a patient-specific intrinsic coagulation cascade model from patient-specific anatomy to clot formation. In addition, future studies aiming to simulate thrombus growth and concentrated on the microscale region near the wall would need to consider a more precise diffusivity value for PolyP, which is specific for PolyP chains released upon platelet activation.Our model simulations did not include the full intrinsic coagulation cascade, but the estimation of the critical conditions that may trigger the intrinsic coagulation pathway ([This computational modeling study illustrates possible mechanisms for thrombus initiation under low shear conditions, such as those induced by aneurysms. In the absence of extremely high shear rates, prolonged exposure to moderate shear rates has the potential to activate platelets. In the normal coronary case we observe that even if platelets are activated due to high cumulative shear rates, the activated platelets and chemical agonists released due to platelet activation are typically washed out of the domain after one cardiac cycle. That is, platelets do not accumulate anywhere in the coronary model. On the contrary, in the case of aneurysms, we observe a significant accumulation of both activated platelets and chemical agonists due to flow stagnation. Furthermore, the local concentration of agonists in some aneurysms reaches levels comparable to those reported as critical thresholds to induce platelet activation, amplifying the effect of shear rate activation and creating an environment prone to thrombus initiation, potentially though the intrinsic coagulation pathway. Current guidelines rely only on the diameter of the aneurysm as the criterion for initiating systemic anticoagulation. Our results suggest that it is precisely for those patients, such as patient P2, who are near the cutoff Z-score value that alternative metrics, based on hemodynamic and biochemical simulations, such as pro-coagulant environment volume quantification, can add value to thrombotic risk prediction. In addition, we showed that coronary artery aneurysm thrombosis may depend to a great degree on platelet activation, suggesting some type of antiplatelet therapy as the most efficient antithrombotic therapy.This work provides a new framework to investigate thrombus initiation from a patient-specific perspective. The model presented here could help identify regions at higher risk of thrombosis based on patient-specific hemodynamics, even in the absence of a focal lesion, as well as the best strategies for thrombosis prevention by analyzing the most significant contributions to platelet activation in each case. Results presented here would motivate prospective studies of larger patient cohorts. Future work should include information on patient-specific coagulation profiles to increase patient-specificity and clinical relevance. This approach could also enable the addition of anticoagulation drugs into the model to evaluate their efficiency and dose management, and design treatment strategies tailored for each patient.