Copy number variants (CNVs), comprising gene amplifications and deletions, are a pervasive class of heritable variation. CNVs play a key role in rapid adaptation in both natural, and experimental, evolution. However, despite the advent of new DNA sequencing technologies, detection and quantification of CNVs in heterogeneous populations has remained challenging. Here, we summarize recent advances in the use of CNV reporters that provide a facile means of quantifying de novo CNVs at a specific locus in the genome, and nanopore sequencing, for resolving the often complex structures of CNVs. We provide guidance for the engineering and analysis of CNV reporters and practical guidelines for single-cell analysis of CNVs using flow cytometry. We summarize recent advances in nanopore sequencing, discuss the utility of this technology, and provide guidance for the bioinformatic analysis of these data to define the molecular structure of CNVs. The combination of reporter systems for tracking and isolating CNV lineages and long-read DNA sequencing for characterizing CNV structures enables unprecedented resolution of the mechanisms by which CNVs are generated and their evolutionary dynamics. Escherichia coli limited for lactose  have been known to play a central role in rapid adaptive evolution. Foundational studies in nutrient-limited chemostats discovered that amplifications of genes encoding transporters of the limiting nutrient were repeatedly selected in different bacteria including  Hansche . CNVs ha Hansche , and sel Hansche . However Hansche . As CNVs Hansche , the stuIn this paper we present two approaches that we have employed to study CNVs in the context of experimental evolution in chemostats. The first method entails the use of a CNV reporter, which we developed to study CNVs at a specific locus of interest. This straightforward method relies on the use of a phenotypic reporter of gene amplifications and deletion at a locus in single cells. Although conceptually simple, this system provides unparalleled resolution of CNV dynamics and practical utility in the identification and isolation of CNV-containing lineages. The power of the CNV reporter system is complemented by the use of long-read DNA sequencing using Oxford Nanopore Technology (ONT) to characterize the complex novel structures that can arise in genomes during experimental evolution. Although the motivation for the development of these methods has been understanding CNVs in the context of experimental evolution in chemostats, this combination of tools has general utility for a variety of approaches to experimental evolution as well as potential applications in other experimental systems.One of the major limitations in studying CNVs in evolving populations is the challenge of identifying alleles at low frequencies in heterogeneous populations. Typical methods to detect CNVs include DNA sequencing, quantitative PCR, Southern blotting, and DNA microarrays. However, these molecular methods are best suited to the analysis of clonal samples and are unreliable for detecting de novo CNVs in heterogeneous populations. Although in principle it is possible to sample populations, isolate clones, and analyze CNVs using these methods, this approach is not practical in terms of cost, effort, and time. By comparison, the estimation of single-nucleotide variants (SNVs) allele frequencies from Illumina short-read sequencing data are routine and widely used to study SNV dynamics in experimental evolution. The inherent limitations of existing approaches for accurately identifying CNVs have hindered studies of the evolutionary dynamics of CNVs.To overcome these challenges, we developed a CNV reporter that enables efficient and real-time tracking of CNV dynamics in evolving populations with single-cell resolution  GAP1 amplification alleles are selected in glutamine and glutamate-limited chemostats, (ii) GAP1 deletion alleles are selected in urea- and allantoin-limited chemostats  green fluorescent protein (GFP) variant mCitrine  and loci (PUT4 and MEP2) and observed similar utility as a CNV reporter (unpublished). In constructing different CNV reporters we have placed the construct ~ 1 kb upstream or downstream of the coding sequence. In principle, the CNV reporter should be effective at any locus of interest.To construct the reporter, we integrated a constitutively expressed (using the HO locus), (ii) a two-copy control containing two copies of the CNV reporter at two neutral loci (we use HO and the dubious ORF YLR123C), and (iii) a zero-copy control that lacks the CNV reporter. As protein fluorescence is dependent on growth conditions and flow cytometry measurements can be impacted by several experimental variables, the three control strains are propagated in the same selection conditions as the experimental lineages and analyzed in parallel with experimental samples. These control strains serve two functions: (1) they are used to define flow cytometry gates for CNV detection and (2) they provide a measure of CNV reporter stability during the experiment.Detecting CNVs using the reporter systems requires the use of three important control strains: (i) a one-copy control containing a single copy of the CNV reporter at a neutral locus  and measure the fluorescence of a sample from the population. Due to the large population sizes used in our experiments, we routinely measure 10,000–100,000 cells per sample to minimize sampling bias. We analyze cells on the flow cytometer immediately after sample collection, providing real-time tracking of CNVs. In addition, every 16–20 generations, we freeze samples down as cell pellets for subsequent analysis. By the end of a long-term experimental evolution, we typically have ~ 25 timepoints across ~ 250 generations. This timescale is sufficient for observing de novo CNVs under conditions of strong selection , resulting in a protein concentration estimation. By engineering strains with different copy numbers of the reporter we have found that the concentration of fluorescent protein is proportional to the ploidy normalized copy number of the fluorescent protein gene, i.e., one-copy in a haploid results in a signal equivalent to two copies in a diploid, and two copies in a haploid results in a signal similar to four copies in a diploid .We have used a variety of flow cytometers and fluorescent-activated cell sorting (FACS) machines to analyze CNV dynamics using the CNV reporter. Although each machine typically has some sort of analysis software, our standard practice is to export results from these machines as .fcs files and undertake our own analysis using R. This provides greater flexibility and ensures reproducible computational analysis. We use the R package  Hammill . We brieWe use gating to define cells and subpopulations of interest within the total flow cytometry sample. Although we have tried clustering algorithms to automatically draw gates we have found manual gating to be more accurate and straightforward. Before gating, we transform the data in such a way that helps further distinguish subpopulations. We use a combination of logarithmic transformations for forward scatter (FSC) and side scatter (SSC) values and a logicle transformation for fluorescent values Fig. B before The set of drawn gates defines the gating template which is then applied to sample data. Because of day-to-day variation in machine sensitivity, we have found that in some cases a universal gating template is not appropriate for a months-long evolution experiment. In this case, a gating template can be constructed for every timepoint using the corresponding measurement for the control populations at the same time point. Importantly, to assess the gates, we use a threshold of > 85% of control cells across all time points lying within the corresponding control gate. Gates are manually redrawn until it passes this assessment. The multiple attempts at drawing gates that fulfill these criteria is known in our lab as the “art of gating.” Once gates have been defined, we obtain the proportion of cells within each gate per time point and graph the proportion of each population with CNVs over time. These data are then used to quantify CNV dynamics as described below.up, the generation at which CNVs first appear, for each of the evolved populations. Previously, to calculate Tup, we first defined a false positive rate of CNVs by calculating the proportion of one-copy controls appearing in the CNV gate across all generations  for three consecutive generations. Our lab currently uses this second approach for calculating Tup.Alternatively, Tup, the percent increase in CNVs per generation for each evolved population. To do this, we take the natural log of the proportion of population containing CNVs divided by the proportion of population without CNVs for each time point. We plot these values across time and perform linear regression during the initial population expansion of CNVs. The slope of the linear fit is Sup.Next, we calculate Sdown, the percent of CNV decrease, using the same approach as in Sup, except the resulting slope is negative. If observed, we calculate Tdown, which is the generation CNVs go below the same threshold used for defining Tup   normalized fluorescent signal for additional analyses. For example, density plots of the normalized fluorescence are useful to examine as is the median normalized fluorescence for each sample. It is important to note that the median fluorescence convolutes CNV population frequency and variable copy number within cells and thus must be interpreted with caution. However, we have found that this metric provides a useful summary of population dynamics for comparison among replicate populations.The ability to detect individual cells with CNVs enables the isolation of CNV-containing lineages for subsequent analysis. We have successfully used FACS to isolate CNV-containing lineages by first sorting the subpopulation of cells with increased fluorescence indicating increased copy number, and then isolating clones through single colony purification. It is our standard practice to then use flow cytometry to confirm that the clonal isolate exhibits homogeneous fluorescence consistent with a CNV. Isolated CNV lineages can then be studied using fitness and phenotypic assays, and genome characterization by methods such as long-read nanopore sequencing, as described below.HO locus in our strains using a single construct that combines both parts of the landing pad.The combination of a CNV reporter and lineage-tracking barcode enables additional levels of resolution on CNV-mediated adaptive evolution. Barcode sequencing allows the tracking of evolving lineages within populations by engineering unique genomic barcodes at a neutral locus in the background of a CNV reporter strain. Consideration of barcode length and estimated population size are important factors to consider when doing so   and Nanopore (Oxford Nanopore Technologies) sequencing  they can produce short, novel sequences, which are identifiable using short-read data Fig. . HoweverA difficulty in any evolution experiment is to meaningfully capture the diversity present within an actively evolving population. While sequencing the entire population in bulk is possible, important information  and when the colonies are large enough (~ 0.1 cm) inoculate overnight cultures  and harvest cells 12–24 h later.In our lab’s experience using Finally, caution should be exercised when drawing inferences from the clones selected about the state of the evolved population. While it is more likely that the clones recovered from a population represent the more abundant lineages within that population, it is not necessarily the case. Estimating population variant frequencies is difficult to do using NGS data because of the variability in site coverage and the difficulty in extrapolating from proportion of reads with variants to proportion of population with variants , aligners, and variant callers can generate significantly different sets of candidate CNVs  and deep-neural network (DNN) platforms a trend has begun to emerge that seeks to extend beyond sequence level information to deeper biological signals that are present within the data but which are subtle, complex, or poorly understood (Wan et al. CNVs are an important class of genetic variation with important roles in adaptive evolution. Experimental evolution is well-suited to studying the role of CNVs and addressing fundamental questions about CNV diversity and dynamics, molecular processes that promote or inhibit the formation of CNVs, and the consequences of CNVs for the organism. Here we have summarized two tools that are critical for the efficient and accurate study of CNVs. The combination of CNV reporters and long-read sequencing enables unprecedented resolution of CNV-mediated adaptation and opens the door to a range of newly addressable questions.


Amyloidosis is a prevalent age-associated pathology caused by the accumulation of fibrous, insoluble protein fibrils in tissues. The most common human amyloid is aortic medial amyloid (AMA), caused by aggregation of a 50-amino acid peptide called medin, which is cleaved by an unknown mechanism from its parent protein, milk fat globulin EGF-factor 8 (MFGE8). Medin is present in the vessel wall of 97% of Caucasians aged over 50-years, yet despite its prevalence in the ageing population there is a very limited understanding of the mechanisms driving AMA. The novel data presented in the paper by Whitehead et al. provides evidence that vascular smooth muscle cell (VSMC)-derived small extracellular vesicles (sEVs) are key mediators of medin accumulation in the vessel wall . In addiIn vitro studies, using human VSMCs, demonstrated that the ECM synthesised by senescent cells contained medin in a fibril-like form. This was in contrast to the small, round aggregates found in the ECM from healthy, proliferative VSMCs, suggesting senescent cells create an environment permissive for medin aggregation. Further mechanistic studies found that sEVs secreted from VSMCs carried medin as a cargo and were responsible for medin release and also its aggregation in the ECM. Importantly, senescent VSMCs showed enhanced sEV secretion and senescent sEVs could accelerate medin aggregation compared with sEVs from proliferative VSMCs. The authors hypothesized that this was due to the formation of fibril-like amyloid seeds which were observed exclusively on the surface of senescent sEVs using super-resolution microscopy. To evaluate what factors might mediate these changes, proteomic analysis was performed on both sEVs and the ECM from senescent VSMCs. This demonstrated significant changes in sEV cargo-loading with senescence and also enrichment of the senescent ECM with the proteoglycan, HSPG2. Importantly, HSPG2 was shown to have a strong affinity for binding sEVs and sEVs from senescent VSMCs showed enhanced binding to HSPG2. The authors concluded that the changes in sEVs and the ECM during VSMC senescence synergise to trigger medin accumulation and aggregation into fibril structures in the aged ECM.Despite several forms of amyloidosis, including AMA and Alzheimer’s disease (AD), being frequently associated with ageing, there has been limited research to date on the effect of cellular ‘ageing’, termed senescence, on amyloidosis. Senescence is considered a stable cell cycle arrest provoked by various stresses associated with ageing, including persistent DNA damage and telomere shortening . SenesceHowever, there are a number of questions that remain unanswered. The physical role of sEVs on medin aggregation was not fully investigated and it would be interesting to understand what changes in senescence-associated sEVs trigger accelerated medin aggregation. This could be related to cargo-loading, although no clear candidates were identified by proteomics. Alternatively, the lipid composition of vesicles may be involved. Recent research has shown there are changes in lipid membrane composition in sEVs derived from senescent cells and lipid membranes have been shown to be important in amyloid fibrillation.in vitro, highlighting this pathway as another potential target for intervention in the vasculature.Interestingly, this study highlighted the many mechanistic parallels between vascular and brain amyloidosis. For example, it is well established that amyloid-β (Aβ), the amyloid associated with AD, can be secreted by small sEVs in the brain, which initiates its build-up in the extracellular space . SeveralThe pathological effects of medin accumulation in the vasculature, as well as the forms of medin responsible for inducing damage, remain poorly understood. Studies have shown that small medin aggregates can induce endothelial cell dysfunction and inflammation while accumulation of fibrillar amyloid species can contribute to weakening and stiffening of the vessel wall . These e


Episcopic imaging using techniques such as High Resolution Episcopic Microscopy (HREM) and its variants, allows biological samples to be visualized in three dimensions over a large field of view. Quantitative analysis of episcopic image data is undertaken using a range of methods. In this systematic review, we look at trends in quantitative analysis of episcopic images and discuss avenues for further research. Papers published between 2011 and 2022 were analyzed for details about quantitative analysis approaches, methods of image annotation and choice of image processing software. It is shown that quantitative processing is becoming more common in episcopic microscopy and that manual annotation is the predominant method of image analysis. Our meta-analysis highlights where tools and methods require further development in this field, and we discuss what this means for the future of quantitative episcopic imaging, as well as how annotation and quantification may be automated and standardized across the field.  As such there are of interest to many fields of biological imaging.Episcopic microscopy refers to imaging techniques that utilize serial sectioning and block-face imaging to create high-resolution large-volume three-dimensional (3D) datasets. High Resolution Episcopic Microscopy (HREM) and its associated techniques  and multi-fluorescent HREM (MF-HREM) A), requiEach of the episcopic techniques above have established experimental protocols across a range of biological samples; and, in the case of optical HREM and EFIC, have been used to test biological and medical hypotheses for over a decade . AlongsiIn this review we outline the goals and methods of quantitative 3D image processing and present results from a systematic review into the current practices for image processing approaches in the episcopic imaging literature. Additionally, we highlight obstacles preventing wider use of quantitative image analysis. Finally, we propose avenues for further research and suggest what future developments we may expect.HREM was originally developed as a high-throughput platform for phenotyping transgenic mouse models ,3,4 and Episcopic Fluorescence Image Capture (EFIC) is a variant of HREM in which samples are embedded in non-fluorescent resin and contrast is provided solely by the native autofluorescence of the tissue . More reVariants of episcopic imaging have been developed that utilise cryosectioning to circumvent some of the difficulties of standard HREM sample preparation ,8. RatheApplications of HREM and its variants have included large-scale embryo phenotyping to build up an atlas of embryonic-lethal gene knockouts `Deciphering the Mechanisms of Developmental Disorders (DMDD)’ , assessiQuantitative image analysis is required biological hypotheses to be rigorously tested and results to be reported with confidence. Moreover, quantification can be used to extract more information from image data and, when automated, facilitate high throughput analysis. Well established quantification pipelines allow researchers to standardise their image analysis, and the objectivity of quantitative analysis makes for more robust interpretations of data. Finally, quantised results can be used for further experiments such as in-silico modelling or training of a machine learning model. However, performing quantitative analysis on the large 3D data sets typically produced by episcopic imaging is a non-trivial task that requires specialised approaches depending on the data type and the aims of the analysis. There are a few key methods for quantification that are relevant to episcopic imaging data analysis; as the pixel intensities of episcopic images do not correlate with biological properties (unlike techniques such as Bioluminescence Microscopy), analysis is largely dependent on image annotation. Annotation can take the form of object detection or segmentation. Object detection usually takes the form of a bounding box or coordinates to identify the feature of interest, while segmentation labels every pixel within the feature as belonging to that feature  . Object Annotation can be performed manually, by tracing or highlighting objects, or by computational means. Widely used methods include pixel intensity-thresholding, filtering  and, moLiterature searches were performed in Google Scholar using the term “high resolution episcopic microscopy” OR “episcopic fluorescence image capture” (case ignored) with a date range of January 2011 to December 2022. 463 matches were initially returned. Based on the title, abstract and methods for these results, review articles were excluded alongside articles which did not use HREM, EFIC or a related episcopic technique. This left 167 relevant articles. YearContrast typeStructure under investigationModel organismWas annotation performed?If yes: was this segmentation, object detection, or both?Was quantitative data presented?Method of annotation /quantification—organized into categories determined during analysisSoftware usedWas the raw image data deposited in an online repository?The type of publication Subsequent exclusion criteria removed articles which did not use biological samples or were protocol descriptions only, without new experimental data or new image processing methods. Conference abstracts without enough detail to extract meta-analysis data were also excluded. From the remaining 123 articles, ten features were extracted: Since 2011, episcopic microscopy data has featured in 123 publications, including 115 journal papers, three conference papers, two correspondences, two doctoral theses and one case report. A table summarizing all these publications can be found in the The most commonly imaged species was mouse (85 papers), followed by human tissue (20 papers). There has been a strong focus on visualizing mouse embryos, in particular for assessing cardiac morphology (represented in 55 papers).Over these past ten years, the number of papers published has averaged ten per year, with an increase in the last two years to 17 in 2021 and 16 in 2022. Of these papers, the proportion which provide quantitative image data has fluctuated over time B. In totA quarter of the papers reviewed used episcopic imaging for visualization without annotation; often this data was presented alongside other experimental techniques such as in-vivo imaging, immunohistochemistry and genetic sequencing. For example, Dupays et al.  used HREScoring/counting of abnormalities (as defined in an atlas)Object counting (no agreed ontology needed)Registration basedHand 2D measurements of anatomic features after reslicing data3D morphological analysis, for example: volumes, networksFractal analysis of heart (standardized method)Other Through the analysis of the last decade of HREM papers, we have identified seven broad categories of quantitative analysis being performed on episcopic image data. We define these categories as: 3D morphological measurements were the most commonly applied method of quantification, occurring in 25 papers and becoming more prevalent over time A,B. ThisScoring or counting of abnormalities was the second most widely employed method, appearing in 19 papers, and notably increased in use from 2016 onwards. This is potentially associated with the publication a couple of years earlier of the Deciphering Mechanisms of Developmental Disorders (DMDD) database: an open-source archive of HREM data cataloguing phenotypical changes associated with different embryonic lethal gene knockouts . As partSeven papers included fractal analysis of the heart. This method produces a quantitative measure of complexity or surface roughness  and is wieldy used in cardiology . Captur Other quantification methods began appearing in papers from 2017 onwards. Analysis of cell orientation was performed in three papers ,23. CarcManual annotation was the most common method for both object detection and segmentation. Overall, 76% of papers presenting quantitative image analysis relied, at least in part, on manual annotation  B. AutomaA wide range of software was used to facilitate episcopic image analysis. Of the 20 software packages listed across all the reviewed papers, the most widely used were Amira (Thermofisher) (48 papers), OsiriX (Pixmeo) (24 papers) and Fiji/ ImageJ (18 papers) A. Amira In three papers, deconvolution algorithms were used to overcome the image blurring caused by out-of-plane light from below the sample surface ,26,30. TAsymmetric blurring is one of the reasons that automated methods might struggle to correctly annotate episcopic images. Additionally, where fluorescent labels are used, the fact that samples are whole-mount stained means they are likely to suffer from heterogenous levels of staining (due to poor penetration of the staining molecule into the center of the sample). The different forms of episcopic imaging also require different tools for annotation. This could explain the high proportion of papers relying on manual annotation, which represents a significant bottleneck for episcopic image analysis. This result highlights the need for new automated methods of annotation designed specifically for episcopic image data. One area of research that could address this need is future is machine learning-based image annotation.Publication of episcopic imaging data has increased over the last decade, demonstrating that episcopic microscopy continues to be a valuable tool for analyzing biological samples. Quantitative analysis of this data has also become more prevalent, with an increasing range of methodologies being used to extract quantitative data from episcopic images. However, the proportion of papers providing quantitative image analysis is still fairly low, averaging 54%, possibly due in part to the difficulties associated with image annotation: episcopic images are mostly annotated by hand, which can be extremely labor intensive. While some areas of analysis follow standardized methods  much of the quantification being performed is not standardized across research groups. 20 different software packages are used for analysis, most of which are commercially licensed and potentially expensive, while 22 papers did not list the software used. This makes reproducing and comparing results challenging, especially as different software packages provide different tools for visualization  which can influence the manual segmentation produced. For quantitative episcopic imaging to continue to grow as a technique, future work must focus on automated image annotation and standardized pipelines for quantitative analysis. Given recent trends, it is likely we will see continue to see more use of complex or multi-stage methodologies, for example segmentations that feed in to computer simulations. In these cases, a reproducible analysis pipeline is even more crucial to provide confidence in the analysis results. Making data easily available, using open source software, and clearly defining the analysis pipeline used in a published study are all ways researchers can contribute to more standardized and reproducible quantification methodologies. One exciting avenue for further research is machine learning-based image annotation. Episcopic image data is a prime candidate for machine learning (ML) approaches due to the availability of data in online repositories that could be used for training. The development of new tools, such as ML models, for episcopic image analysis will expand the use of episcopic imaging and create further possibilities for new applications.


Sarcopenia, the age-related decline in muscle mass and function, is a significant cause of disability and frailty in older adults and leads to poor quality of life and enormous health care costs. The pathogenesis of these changes is multifactorial and the related etiologies are not fully understood. A more complete understanding of the mechanisms driving these sarcopenic changes is required to identify both appropriate biomarkers and novel interventions to preserve and restore muscle mass and function in older adults and to improve healthspan and lifespan in older adults.Growing evidence suggests that immunoglobulin G (IgG) mediated autoimmune process may be a critical, but under-recognized, contributor to sarcopenia. Autoimmunity increases with age  and two in vivo, or treatment of isolated IgG on cultured skeletal muscle cell lines in vitro. Whether pathogenic or not, the newly discovered aging related IgG deposition/infiltration in skeletal muscle, at least as a marker of increased muscle fiber membrane permeability, could potentially be used as a novel biomarker to predict sarcopenia development and/or individual responses to clinical interventions. Similarly, anti-cTnT autoantibodies and any other skeletal muscle reactive autoantibodies to be discovered in blood of older adults may also serve as novel biomarkers for sarcopenia diagnosis and development prediction. Autoimmunomic signatures of aging therefore could be of great potential to identify novel sarcopenia related circulating biomarkers in older adults, as was found in age-related neurodegenerative diseases [Although detection of autoantibody is essential for diagnosis of any autoimmune diseases, a direct causal relationship has been demonstrated for several, but not all, autoimmune diseases . As for diseases .Based on IgG infiltration in skeletal muscle and/or skeletal muscle reactive autoantibodies in blood, a possible new subtype of sarcopenia could be established, which could be originated from age-dependent plasma membrane senescence with or without subsequent autoimmune responses. Genetic and cellular/molecular mechanism underlying this age-related membrane integrity impairment and autoimmunity in skeletal muscle will need to be explored for better developing new preventive and therapeutic strategies on sarcopenia prevention and intervention. Therefore, epidemiology study will be the next step to identify skeletal muscle reactive autoantibodies and determine their prevalence and association with sarcopenia in older adults. Together with basic research studies in aging mouse models and transgenic mouse models, a more in-depth understanding of mechanisms, risk factors, and clinical implications of autoimmunity in sarcopenia pathogenesis and development will guarantee rich practical rewards.


While misuse of psychoactive substances can be traced back to pre-historic times, their effects on invisible target—such as the developing fetus—only gained recognition within the last century –4. InitiThe mechanisms by which prenatal exposure to psychoactive substances alters developmental trajectories are variable, from epigenetic alterations to fine-tuning of blood supply to the brain , 6. In sThe current Special Issue aims at describing recent advances in our understanding of how maternal and paternal substance abuse affects early development of progeny. Contributions into this Special Issue span from methodological advancements, into reviews of current standing in the field, original research report, and novel diagnostic tools. Specifically, Mbolle et al. take the reader on a journey into modern technology for high-resolution imaging of prenatal development. While this task is certainly streamlined in obstetrics clinics, major challenges remain at the bench, as current resolution capabilities are often below the thresholds needed for accurate visualization of fetal structures within small rodent species such as mice which are used for mechanistic studies. Development of deep-tissue high-resolution imaging and accompanying computational reconstruction technology will serve many researchers who are trying to elucidate the underpinnings of prenatal drug exposure effects. Before such methodology could advance the field, a thorough analysis of existing knowledge is needed, so that gaps could be identified and pursued with precision targeting. To address this task, Mulligan and Hamre offer an in-depth review of current standing on the influence of prenatal cannabinoid exposure on early development and beyond. The task is not trivial, as there is a clear difference of opinion between medical professionals and societal attitudes. The authors conclude that while there is evidence that prenatal cannabis exposure might alter developmental trajectories, the cause-effect relationships are not proven and, in most cases, require additional studies.Strong focus in this Special Issue is maintained on vascular and cerebrovascular consequences of prenatal alcohol exposure. Momin et al. review vascular contributors to the neurobiological effects of prenatal alcohol exposure. This systematic review of the literature leads to a conclusion that although the brain has been traditionally considered as a main target of prenatal alcohol exposure, data from human samples and bench studies document that developing vasculature is equally sensitive to an early alcohol hit. Saha and Mayhan concur with this conclusion and expand upon it by presenting a review of mechanisms that underly cerebral vascular damage by prenatal alcohol exposure and which enable lifelong consequences of this adverse developmental event. Both reviews highlight the fact that our approach to treatment of adverse developmental outcomes should involve a multi-organ strategy.in press) offer an early, time-efficient screening tool which could assist in efforts to diagnose fetal alcohol spectrum disorders in large cohorts of children. Wide clinical utility of this newly offered tool remains to be documented.Waite and Burd conclude the chapter of alcohol prenatal effects with a review of literature on common developmental trajectories and clinical identification of children with fetal alcohol spectrum disorders. They describe current challenges in the diagnosis of fetal alcohol spectrum disorders as opposed to prevalent behavioral disorders. Finally, Fleming et al (Following current trends of increased opioid use, misuse, and opioid-related deaths, Madurai et al. describe alterations in the peripheral inflammatory and central immune landscapes following prenatal exposure of rats to methadone. The authors conclude that such alterations may underly long-term consequences of developmental brain injury including cognitive and attention deficits. However, identification of altered patterns may also serve as a biomarker and a therapeutic target. Translation of these findings from bench to bedside remains a goal for near future.


Person-centered communication is foundational to cancer care. In pilot research, a questionnaire eliciting patients' illness and treatment understanding (ITU) and core health-related values (HRV) through the electronic patient portal demonstrated feasibility, acceptability, and efficacy. The aim of this study was to elicit stakeholder feedback to refine the design of the portal-based intervention, remain end-user centered, and optimize future system-wide integration.Between April and June 2023, we purposively sampled patients and clinicians from a previous pilot study to participate in a 20–30-minute semistructured interview about their opinions of and experiences with the portal questionnaire on ITU and HRV. An interdisciplinary coding team used a two-phase rapid analysis to identify themes, subthemes, and illustrative participant quotations.n = 26). Colorectal cancer was the commonest malignancy (64%) among patients. Clinicians were mostly physicians (50%), nurse practitioners (33%), and registered nurses (17%), with two-thirds having >15 years of experience in their specialty. Analysis generated four themes: (1) clinical utility of questionnaire, (2) barriers to questionnaire implementation, (3) considerations and strategies for modifying the questionnaire, and (4) considerations and strategies for questionnaire implementation. Themes captured key information about incorporating this questionnaire into clinical practice.Fourteen patients (mean age = 68 years) and 12 clinicians participated (total Patients with cancer and their clinicians found a portal-based ITU and HRV questionnaire clinically useful to improve multiple aspects of person-centered communication. Participant recommendations about questionnaire timing and sharing of questionnaire responses with the clinical team will inform future questionnaire implementation and scaling in clinical settings. Although person-centered communication is known to positively affect psychological wellbeing5 and increase documentation of patients' care goals,6 research to date has primarily focused on such communication for patients nearing death,7–10 and on specific end-of-life treatment preferences.11–13 Furthermore, person-centered communication is fundamental to effective advance care planning (ACP) to promote goal concordance in cancer and other serious illnesses.14–17Person-centered communication is a two-way process ensuring that patients can adequately understand their illness and treatment options and clinicians know patients as individuals.4 potentially leading to adverse outcomes and missed opportunities for shared decision making. However, as health systems continue to leverage technology—specifically, patient portals—to improve communication and cancer-related outcomes,18–20 there is a pressing need to consider patient and clinician perspectives about the use of portals to improve person-centered communication, both to enhance the clinical utility of portal-focused innovations and to balance factors such as treatment benefits and burdens, survival time, and quality of life.16Oncologic therapies that may improve survival and/or quality of life entail progressively intricate choices and less certain outcomes that require effective communication. Yet several real-world clinical factors  create barriers to person-centered communication,Patient portals to Elicit essential patient-Reported elements of communication Supporting person-centered ONcologic care)22 in a sample of gastrointestinal (GI) oncology clinics within our comprehensive cancer center to elicit information about personhood, ITU, and HRV .24 This pilot work provided evidence of feasibility and acceptability, while also showing an association between the PERSON approach and higher patient ratings of clinicians' communication quality.22 In the present study, we explored the perspectives of patients and clinicians who participated in the pilot study for additional feedback about workflow considerations, processes, and portal use related to PERSON. Our aim to identify barriers and facilitators to effective portal-based elicitation of ITU and HRV will assist in refining the design of our questionnaire, ensuring it remains end-user centered, and optimizing future system-wide PERSON implementation.We have pilot tested a portal-enabled approach called “PERSON”  through the patient portalA qualitative methods specialist (QMS: J.G.) conducted interviews with patients and clinicians in English through Zoom  or by telephone using a written interview guide . Partici26 Rapid analysis (RA) is a qualitative approach that quickly produces information to inform ongoing implementation efforts, and has been shown to generate results consistent with in-depth text analysis.26 RA was conducted in two phases: a “vertical” phase and a “horizontal” phase.28 In the vertical phase, the QMS reviewed the individual interview summaries, and significant statements were identified based on a priori constructs from the interview guide as well as emergent ideas from the interviews. These statements were sorted into categories relating to importance of the information being collected, experience with the questionnaire, perceptions of usefulness and clinical utility, clinical workflow implications, and institutional support. In the horizontal phase, members of the interdisciplinary analytic team  reviewed the statements grouped within each category, across participants, to identify primary themes.Following the interview, the QMS drafted a detailed summary of participants' responses using the audio recording to include verbatim quotes, based on best practices for rapid qualitative data analysis.To facilitate this phase, the team created a data matrix, with each row representing an individual participant, and each column representing an analytic category. One matrix was created for each of the two stakeholder groups. The analytic team collaboratively and iteratively reviewed the data to refine the themes and subthemes and to identify illustrative participant quotations. Themes included prominent sentiments within each category, as well as any significant divergences between and within stakeholder groups . Multiple subthemes were identified for each theme. Given participant difficulty in distinguishing ITU and HRV items in the questionnaire, findings were reported in aggregate  unless noted.n = 14) predominantly identified as White (79%) and male (57%) with a mean age of 68 years. Colorectal cancer was the most common malignancy (64%), followed by pancreatic (14%) and neuroendocrine (14%). The majority of patients were diagnosed with stage III or stage IV disease . Clinician participants (n = 12) included GI physicians (50%), nurse practitioners (33%), and registered nurses (17%), and two-thirds had >15 years professional experience in their specialty. Patients  clinical utility of questionnaire, (2) barriers to questionnaire implementation, (3) considerations and strategies for modifying the questionnaire, and (4) considerations and strategies for questionnaire implementation. Themes captured important information about incorporating this questionnaire into clinical practice. Responses from all participants supported and clarified each theme and each theme's properties .Patients and clinicians overwhelmingly agreed the questionnaire was useful and clinically meaningful, especially in highlighting the “whole personhood” of patients, creating opportunities for patients to receive tailored, person-centered care, and increasing opportunities for concordance with patient goals. For example, patients reported that when their clinician understands their HRV (per questionnaire responses), their treatment recommendations are subsequently customized and individualized. Participants also shared that the questionnaire invited patient participation in care by considering their emotions, and sometimes supported caregiver involvement in conversations .Participants described the questionnaire as a way to “anchor” conversations and overall communication between patients and clinicians. For example, patients and clinicians stated that questionnaire responses supported follow-up conversations or sparked ideas for topics to cover during appointments. Importantly, all participants noted the questionnaire helped serious illness conversations occur more frequently than they would naturally arise, and that questionnaire information gave clinicians a greater sense of patient preferences for information delivery during those conversations . Clinicians noted that questionnaire responses offered insight about patients' understanding of clinicians' previously communicated information.Some respondents agreed that completing the questionnaire could be anxiety-provoking, but that the information is critical to discuss and best support the patient. Most patients stated that they did not experience negative emotions when completing the questionnaire, but rather, it helped them feel included in important care discussions.Patient participants did not speak specifically to implementation barriers. However, clinician respondents pointed to three primary barriers to implementing the questionnaire in clinical practice. Clinicians noted a lack of continuity of care and information flow between clinics, inpatient care settings, and other outpatient care settings, which they believed limits the successful integration of the questionnaire into care delivery. Clinicians especially noted that without effective information flow between inpatient and outpatient settings, patients' preferences and responses may not be accurately communicated—or communicated at all—across settings. Clinicians noted a need for improved awareness of the questionnaire, as well as associated conversations among other clinicians across the institution.Clinicians also described the challenge of integrating the questionnaire data into their clinic workflow due to time constraints; sometimes they were simply “too busy” to incorporate the questionnaire and associated conversations. For example, clinicians described sometimes having as little as 15 minutes with each patient, then stated that discussion of patient information from the questionnaire could be “cut” in favor of more time-sensitive patient needs, such as immediate symptom management. Clinicians also discussed the challenge of needing to interrupt or stop HRV-related patient conversations due to limited appointment time, at the expense of reducing participation for patients who are particularly engaged in the discussion of their HRV.Clinicians described lack of institutional support as an implementation barrier, noting that full integration of the questionnaire would require a shift across disciplines—including physicians, nurses, and social workers—to actively prioritize the questionnaire and the conversations it supports. Clinicians further noted that without overarching institutional support, it could be difficult for even the strongest champions to actively integrate the questionnaire or prioritize the “face time” required for effective HRV conversations. Participants pointed to the importance of participation and endorsement of the questionnaire “from the top.”Participants addressed several directions for improving the questionnaire. Participants, primarily patients, described the need for concrete explanations about the purpose of the questionnaire to the patient . Several patients needed help to complete the questionnaire because they lacked clarity about the intent of the questions. Patients also advised that the questionnaire could include additional language about the nature of the questions .Participants recommended that questionnaire language should be modified to provide structured guidance about how to complete it. For example, some patient participants reported confusion about how to answer open-ended questions on the questionnaire and what type of information was being requested. Participants noted that, especially in a major cancer center where patients are asked to complete many questionnaires and surveys, clarification and details about overall intent would help them differentiate from other questionnaires.Participants noted that very few patients broached the questionnaire with their clinicians in-person, thus clinician awareness of the questionnaire remained mixed. As a result, participants provided guidance to develop specific strategies for staged implementation. Participants noted that using the patient portal was generally an effective modality for patients to complete the questionnaire. Patients, specifically, stated that the portal gave them the opportunity to review the questions, respond in advance of their clinic visit, and process-related information at home before their appointment. Clinicians shared that receiving patient responses through the portal reduced time that would be needed for them to answer questions during the appointment. Although a few patients reported that they were inclined to give shorter responses when completing the questionnaire on their smartphone (vs. on a computer), there was significant agreement among patients and clinicians that completing the questions through the portal supported patients in having time to reflect and provide honest answers, without the pressure of responding during an actual appointment.Respondents emphasized the importance of the timing of questionnaire administration and possible follow-up conversations. Patients and clinicians pointed to the benefit of multiple, repeated questionnaires, spaced in a way that would allow changes in prognosis and treatment status to inform conversations during appointments. Many participants believed that spacing the questionnaires apart by 6–12 months would be most useful, as every 3 months felt too repetitive. Patients recommended offering the questionnaire after major changes to care  would be appropriate.Participants also offered suggestions for making the questionnaire more inclusive and even more individualized, including by cancer type, treatment stage, and patient age. For example, a small number of patients endorsed distress upon completing the questions, and clinicians noted that some patients might indeed experience such distress. Participants therefore suggested addressing potential distress as part of the survey. Respondents also pointed to the need to offer alternative modalities of questionnaire delivery for patients not using the portal.Clinicians offered recommendations to streamline the process for workflow purposes. For example, clinician participants noted that practicing clinicians who understood the questionnaire and were aware of its integration into the patient portal would be more motivated to incorporate them into their typical workflow. To achieve increased clinician awareness and greater workflow integration, participants suggested providing clinicians with email or chart reminders around the patient's second or third appointment with the clinician. Participants also recommended a nurse triage approach, wherein nurses would review patients' responses in the portal in advance of the follow-up clinic visit and communicate results to the oncologist. Clinician participants also provided suggestions about how to distribute the questionnaire information during an appointment .This qualitative study obtained patient and clinician perspectives on the workflow, processes, and usability of the PERSON questionnaire that elicited both patient ITU and HRV. Findings suggest that questions about ITU and HRV presented through the patient portal can be clinically useful in achieving goal-concordant care while improving overall clinical communication and helping patients to feel cared for, regardless of available treatment options. Insight from patients and clinicians provided our team with a data-based roadmap to further refine our portal questionnaire, in terms of timing and user friendliness, the infrastructure within which the questionnaire is delivered, and opportunities to strengthen both system-level and interprofessional understanding of intervention aims and integration.4 patients in our study noted that ITU and HRV questions and conversations were usually not occurring in clinical settings independent of the pilot strategy, and furthermore, the introduction of the PERSON questionnaire was the first time clinicians explored in-depth key elements of their personhood. Some patients expressed surprise at being asked this type of information, either expecting a sole clinical focus on biomedical interventions or not understanding how these aspects of their individuality were relevant to their treatment plan. However, many participants shared that the PERSON questionnaire helped them to “feel heard and understood,” which is considered a patient-reported measure of care quality.30 Thus, our current stakeholder data suggest that the portal-elicited ITU and HRV questions will have positive and measurable impact on care quality outcomes as the PERSON approach is refined and scaled institutionally.Although guidelines suggest that oncology clinicians consistently incorporate person-centered communication throughout clinical encounters,31 and shared decision making,33 and is distinct from goals of care discussions, which have been less clearly defined34 and tend to focus on treatment planning or care preferences related to an episode of care or care transitions.35–37 Addressing HRV can shed light on more self-defined and stable aspects of personhood beyond end-of-life wishes, including a patient's sources of strength, hopes, concerns, and their definition of acceptable quality of life.38–40 Despite mixed empirical evidence on the associations between ACP and cancer care at the end of life, the communication aspects of ACP tend to have more influence than other components, such as documents, on related outcomes.41 Using the PERSON approach to support person-centered communication may support clinicians in leveraging technology and implementation science methods to facilitate iterative ACP throughout the lifespan and disease course.15The PERSON questionnaire is positioned to become a reliable intervention to promote goal-concordant and person-centered care delivery through portal-based ACP. HRV elicitation is a core component of ACP42For instance, participants in our study recommended that regularly administering the PERSON questionnaire ∼6–12 months apart through patient portal would support patient reflection and ongoing discussions with clinicians in the context of prognostic and treatment changes. Similar approaches to early nurse-led palliative care integration have reflected core aspects of our intervention, including leveraging interdisciplinary skills and practice priorities with goals of facilitating cross-team collaboration and communication, integrating patients' personal narratives, and moving from a disease-focused model to the promotion of living well with cancer.43 allowing the coding team to identify salient themes. Although we purposively sampled patients and primary oncology clinicians, future research should include the perspectives of caregivers who may assist patients with portal completion of ITU and HRV questionnaire and are active participants in clinic encounters where the bulk of communication occurs. Most patient participants were also White and Christian, limiting the inclusion of more diverse group perspectives. Additional research should focus on capturing the experiences of minoritized groups related to ITU and HRV. Other interdisciplinary clinicians who find ITU and HRV data beneficial to fulfilling their professional responsibilities  should also provide future insights to best translate portal elicited information into care delivery.23 In addition, more than 90% of the patients interviewed were diagnosed with stages III or IV disease.Results should be considered in the context of the following limitations. Ours was a relatively limited sample that hinders generalizability. Despite the smaller sample size, we reached data saturation within the allotted number of interviews,Since person-centered communication should ideally begin at the time of cancer diagnosis, it will be important to gain further insights from patients earlier in their disease process, facing different and possibly less complexity in decision making and treatment options. Finally, future elicitation of broader patient portal implementation barriers from the patient perspective would assist to identify other elements not captured in the current study  and to further refine our intervention.In sum, the patients and clinicians who participated in our intervention shared positive feedback that supported its clinical utility and actionable guidance to further strengthen our approaches to integrated portal questionnaire of ITU and HRV. Our ongoing research is addressing barriers to implementation that participants noted, including the timing of portal questionnaire delivery and workflow streamlining for clinicians. This innovative approach to portal-elicited ITU and HRV data presents a promising intervention to enhance clinician–patient relationships throughout the cancer trajectory, use technology to improve person-centered communication, and promote tailored, value- and goal-concordant cancer care in the context of emerging therapeutic and scientific advances.


The activities and role of the cerebellum in sleep have, until recently, been largely ignored by both the sleep and cerebellum fields. Human sleep studies often neglect the cerebellum because it is at a position in the skull that is inaccessible to EEG electrodes. Animal neurophysiology sleep studies have focussed mainly on the neocortex, thalamus and the hippocampus. However, recent neurophysiological studies have shown that not only does the cerebellum participate in the sleep cycle, but it may also be implicated in off-line memory consolidation. Here we review the literature on cerebellar activity during sleep and the role it plays in off-line motor learning, and introduce a hypothesis whereby the cerebellum continues to compute internal models during sleep that train the neocortex. While sleep plays important roles in metabolic regulation, energy conservation and cellular restoration, it has been described as an activity “of the brain, by the brain and for the brain” , taking During waking movements the cerebellum is thought to compute an internal model of the motor apparatus by providing predictive feedback to on-going motor output . The proThe sleep cycle is segmented into rapid eye movement (REM) and non-REM phases, with the latter conventionally divided into three stages. The EEG activity from the neocortex (largely used to define different sleep stages) shows a progressive increase of neuronal synchrony as the brain falls deeper into sleep from stage 1 to 3. Stage 1 (light sleep) occurs at the beginning of sleep and only forms around 5% of total sleep duration. It is characterised by low amplitude mixed frequency EEG signals. Deepening sleep progresses to stage 2, comprising 45% of total sleep time and is defined by the presence of sleep spindles and/or K complexes . Sleep sMuch of the neuroscience of sleep has been concerned with sleep-dependent consolidation of declarative memories , thoughtThe cerebellum has long been implicated in the acquisition of procedural skills , for exaFor example, Imaging studies also combining fMRI with motor sequence, visuomotor and timing tasks also suggest a link between cerebellum-dependent procedural learning and sleep. For example, the dentate nucleus of the cerebellum shows increased functional connectivity with the superior temporal sulcus after sleep while learning a pursuit task, but this is reduced after sleep deprivation . IncreasInvasive animal neurophysiological studies have focussed mainly on the motor-control functions of the cerebellum  while stWith hindsight, these findings collectively should have produced compelling evidence already by the late 1970s that the cerebellum may play an important role in shaping sleep architecture and the sleep-wake cycle . HoweverTo address this gap in knowledge, we carried out a study in monkeys which showed that primary motor cortex (M1) and cerebellar activity during sleep exhibited correlations at several frequency bands . Using aOur study also revealed a surprising directionality of causal influence between the cerebellum, thalamus and neocortex [These results place the cerebellum in an important position in the sleep process and complicate the decades-old sleep-research doctrine that neocortical sleep spindles arise solely from the thalamus , 1987. MHow might the cerebellum generate oscillations in the sleep spindle frequency range (9–16 Hz) The spindle-generating mechanism is fairly well-described in the thalamus, via reciprocal interactions between thalamocortical and thalamic reticular neurons. Activation of the GABAergic thalamic reticular neurons activates GABAa and GABAb receptors on thalamocortical neurons, leading to hyperpolarisations which then causes a rebound excitation. Conversely, rebound excitation in thalamocortical neurons can then cause excitation and spike bursting in thalamic reticular neurons, thereby creating reverberating activity in the loop at spindle frequencies [for detailed review see Are there similar circuits in the cerebellum that could plausibly generate spindle-band oscillations? There certainly exists reverberating loops within the cerebellar cortex and between the cerebellar cortex and its related nuclei. For example, cerebellar Purkinje cells inhibit neurons of the deep cerebellar nuclei which then show rebound excitation . InhibitIt has previously been shown that low frequency M1 neuronal dynamics during movements are similar to those during low frequency oscillations in sleep, i.e., in the delta and low range (<4 Hz) . A recenIf the cerebellum is active in sleep, and is receiving inputs from the motor cortex that resemble those seen during movement, this raises the possibility that the sleeping cerebellum is performing some analogous computation to that which it performs in the awake state.In this section we offer some tentative suggestions as to what this computation may be and how it could contribute to off-line learning. Influential theories of cerebellar function confer responsibility for learning internal models of the body and its interactions with the environment. Broadly these can be categorised as forward models that map causes (movements) to consequences (sensory predictions), or inverse models that map consequences (desired states) to causes (actions) . During Recent evidence suggests that cerebellar forward models may continue to generate sensory predictions in sleep. In adults, neocortical activity in sleep rarely causes overt movement and resultant sensory feedback. However, we hypothesise that fictive goal-directed motor commands (that resemble those seen during waking movement) can nevertheless be processed by cerebellar forward models to generate predicted sensory consequences. The discrepancy between fictive goals and predicted consequences could act as a simulated reward signal (relayed via sleep spindles) to drive off-line optimisation of the controller in sleep. As a result, daytime learning stored in cerebellar forward models could during sleep be transferred and transformed into improved control policies (learned patterns of motor output) represented in the neocortex.The concept of using a previously learnt predictive model to optimise control policies is long-standing in the computational literature. Forward mappings  are in general well-posed and, crucially, sensory predictions can be compared directly to actual consequences. As a result, forward models can be learned through efficient error-based mechanisms wherein sensory feedback from the world acts as a teaching signal. By contrast, inverse control policies  are often ill-posed  and the world provides only an indirect training signal (an unsuccessful outcome does not directly tell you what action you should have taken instead). The distal supervised learning architecture of This scheme could also be extended to learning that does not depend on the neocortex, such as eye-blink conditioning . EvidencThe cerebellum seems to be an active participant in the sleep process and is emerging as an important player in sleep-dependent memory formation. While confirmed by recent experiments, it appears that disjointed evidence for a cerebellar contribution to sleep processes has been present for decades. With this in mind future targetted experiments will hopefully shed more light on this contribution. While cerebellar lesions can disrupt sleep architecture (see above), it is unclear whether cerebro-cerebellar signals, for example at sleep spindle frequencies, influence the time spent in different sleep stages, for example stage 2 sleep. Closed-loop perturbations are increasingly used to explore the role of hippocampal ripples , and a sOur hypothesis that the cerebellum acts as an off-line “simulator” enabling off-line practice of motor control policies begs speculation about a role in dreams. However, some caution is warranted here since the strongest evidence for a role of sleep in cerebellar-dependent procedural learning relates to stage 2 sleep. While it is true that dreams can take place in stage 2 sleep , most ocIndeed, there are many disorders for which cerebellar damage, sleep disorders  and impaired off-line consolidation are overlapping, from autism to schizophrenia . The nonBoth authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Advocates of value-based insurance design (VBID) have argued based on break-even economic modeling that VBID can be effective but acknowledge that direct medical savings from increased use of services with strong evidence of clinical benefit are unlikely to finance the entire [VBID] investment in the short term. Thus, these VBID proponents support investments in processes to define low-value care and a benefit design that couples cost-sharing reductions for high-value services with cost-sharing increases for services not identified as high value. This suggestion has led to an important emerging issue for employers considering VBID adoption, provider and patient acceptance of higher levels of cost-sharing for services deemed low-value. Since publication of our earlier observations about deficiencies in research on VBID, new copayment reduction studies using nonrandomized comparison groups have been published, and an additional study measured the association between medication adherence and all-cause health care costs. Although all the VBID studies provided evidence that implementation of VBID might have a small favorable effect on medication adherence, none provided the information that payers need to make an informed decision about VBID because of serious problems in reporting, program design, and effect calculation. None of the new VBID studies reported its effect on generic drug utilization, and none reported the cost of the intervention to the payer. There are still no randomized controlled trials of VBID. Thus, nearly a decade after pharmacy benefit copayment reduction was initially proposed as a means to improve the outcomes of patients with chronic health conditions, health plan sponsors have little of the information that they need to assess the costs and benefits of VBID.A follow-up article on the subject of this editorial is available here:  VBID, the PPACA, and FREEE Medications: Did Politics Trump the Evidence About Cost Sharing?


IntroductionRacism is a pervasive social problem that influences medicine, highlighting the need for interventions. One promising educational technique, referred to as edutainment, utilizes clips from television shows as an instructive strategy. The objective of this study was to examine the acceptability of edutainment around anti-racist curricula for residents.MethodsWe conducted a survey of underrepresented in medicine (URM) medical faculty to inform content for subsequent focus groups with medicine, psychiatry, and pediatrics residents. For the survey, URM faculty were randomly assigned to view four of eight clips and responded to close- and open-ended items. Focus group participants viewed selected clips and provided feedback. All study procedures occurred in 2020-2021. We calculated descriptive statistics for close-ended survey items and employed thematic analysis for open-ended items and focus group transcripts.ResultsTwelve URM faculty completed the survey. Feedback was uniformly positive so we included all eight clips in the resident focus groups. For each of the three participating specialties, we conducted two focus groups  with participants viewing four of the eight clips. Analysis of focus group transcripts found that participants were receptive to the edutainment approach. Feedback as to the realism and acceptability of certain clips differed by specialty. Triangulation of survey and focus group results found differences in the acceptability of specific clips between residents and faculty.ConclusionEdutainment with medical television shows may be a promising avenue for anti-racist curricular content for residents. The educational methods described here are being incorporated into a multi-pronged, hospital system wide graduate medical education anti-racist curriculum. Systemic racism continues to be a major problem plaguing every facet of American life; healthcare is no exception. Centuries of discrimination toward Black Americans have resulted in less access to appropriate education, funding, and encouragement, preventing them from having equal access to medical education . AlthougWhile medical systems and training programs have focused heavily on the recruitment and retention of a racially and ethnically diverse workforce, there has been less emphasis on addressing workplace burdens experienced by trainees from racial and ethnic groups underrepresented in medicine  and consulted with Hollywood, Health & Society to identify a set of 15 storylines on popular medical dramas  addressing key concepts for anti-racist curricula identified in the literature, such as the minority tax ,16,17. TFinally, the group discussed the compiled feedback and eliminated clips deemed unrealistic leaving a final set of eight clips with a plan to pair complementary clips  Table . The fouFor each clip, respondents were presented with a brief synopsis and a link to view the clip in their browser. To assess acceptability, we asked respondents to rate their agreement with four items on a Likert scale of one (strongly disagree) to five (strongly agree) and to complete two open-ended questions about each clip. We also asked one open-ended item after each pair about using the clips together. The full survey instrument is available in Table Resident focus group sample selectionWe coordinated with internal medicine (IM), psychiatry, and pediatric residency leadership at our institution to send recruitment emails via residency program listservs at our institution. Focus groups were conducted during normal residency didactic times and approved by each program. All residents in each of the three programs were eligible to participate in the focus groups.Resident focus group proceduresWe conducted two small focus group discussions with residents in each specialty  from October to December 2021. No personnel affiliated with residency leadership attended the discussions. Focus groups were facilitated by the first author with one other investigator present to take notes and lasted approximately one hour. Focus groups were conducted in person for IM and pediatrics and via Zoom videoconferencing software for psychiatry. Audio was recorded for transcription.Focus group participants independently completed a brief socio-demographic survey and then viewed two pairs of clips  as a group, with one focus group per specialty viewing the first two pairs and the other viewing the last two. Focus group questions were developed in consultation with the University of Pittsburgh Qualitative, Evaluation, and Stakeholder Engagement Research Service.AnalysisFor the survey, we calculated descriptive statistics for close-ended items using Stata 17  and used thematic analysis by two independently working researchers for the open-ended items . Two resWe transcribed focus group recordings using Otter.ai and entered the transcripts and notes into NVivo software for deductive content analysis . Two resURM faculty surveyTwelve URM faculty completed the survey, with five respondents viewing the first four clips and seven viewing the second four. Percent agreement for each statement was >80% for most items Figure ; the onlResident focus groupsFocus group sizes ranged from two to nine participants (median=4), with a total of 25 participants across the three specialties. The focus groups where participants viewed the first two pairs of clips included two, four, and four participants , and the focus groups where participants viewed the second two pairs included two, four, and nine participants . The majority  were either PGY-1 or PGY-2, 26-30 years of age , and female . Approximately half  identified as White and 12% (n=3) as Black or African-American.Overall, participants supported using the clips as part of training Table . One theRegarding realism, some participants thought certain clips were overly dramatic , while others were realistic  Table . Of noteResults from this mixed-methods study support the use of edutainment for GME anti-racist curricular content. Resident participants felt the edutainment approach was impactful, largely realistic, and created psychological safety for the discussion of emotionally sensitive concepts. Interestingly, we found some differences between faculty and resident participants. The most notable difference between faculty and resident participants was related to the clip from Scrubs, with faculty less supportive of using this clip compared to residents. This clip was the only one in our set from a comedy program, and it may be that faculty were unsure if the comedic approach to a serious topic would be appropriate in the educational context. Faculty participants may have been less familiar with the characters from Scrubs, and therefore less likely to identify the humorous banter between characters on the show. Given the popularity of Scrubs among residents and health sciences students, future research could examine support for edutainment with clips from comedies compared to dramas .We also found differences in perceptions of realism among residents of different specialties, supporting the importance of tailored approaches to curricular design. The varied feedback suggests that further curriculum development could be enhanced by co-creating content with individuals from the specific target groups of faculty and residents. Including learners in the design and conduction of educational initiatives has been recommended by the International Association for Health Professions Education (AMEE) as a technique for increasing learner motivation .Given increasing societal discussions about race bias and race disparities in healthcare as well as the increasing call from medical education leaders to address these disparities in training, it is essential to identify effective ways to teach these concepts . SpecifiEdutainment, in contrast, aims to first engage participants with relatable scenarios before highlighting biases that may be relevant to the learner and their team. However, to the best of our knowledge clinician educators have not yet employed an edutainment approach for race bias education among resident trainees; furthermore, we did not identify any similar curricula for medical students or faculty learners. This edutainment approach may be one aspect of a successful and comprehensive multi-pronged approach to promoting racial justice and reducing health inequity in GME. Results from the current study, specifically resident focus group participants stating the clips were a helpful jumping-off point for discussion, suggest that an edutainment approach may “disarm learner defensiveness” and promote the psychological safety called for by Blanchard et al. (2022) to discuss these challenging topics . AdditioMoreover, given that medical professionals on television are more racially diverse than in real-life, the edutainment approach may be particularly impactful in reducing implicit bias among residents by exposing them to diverse characters and storylines that challenge preconceived notions and stereotypes . This exDespite the numerous possible advantages to an edutainment approach in anti-racism curricula, educators undertaking this work must also employ a framework of cultural competence to navigate potentially challenging and emotionally charged discussions that may follow edutainment clips that begin such discussions. With diverse groups of participants, educators facilitating discussion need to understand various perspectives, defuse conflict, and have a trauma-informed approach to racist and microaggressive comments. Depending on the pre-existing skills and interests of the educators, it is expected such a curriculum would require faculty development for group facilitators. Furthermore, the authors recommend that educators for these sessions maintain confidentiality and emphasize that they are not evaluating participants for competency or promotion in a summative way, as this may undermine psychological safety for group discussion. Finally, edutainment curricula are limited by the television clips that are available in terms of content/topic, emotional depth, and diversity of actors portraying characters; the extent of the clips has not yet limited our work in this area but is a conceivable limitation for future work.Our results should be interpreted with the following limitations. First, while the use of snowball sampling for recruitment was deemed most feasible given the COVID-19 pandemic and increased URM physician workforce demands, it limited our sample size and limits the generalizability of our survey findings. Moreover, although we had no reason to suspect receipt of survey responses from individuals who did not meet our inclusion criteria, we did not conduct formal eligibility screening. Future work could use convenience or random sampling to both increase the number of respondents and the generalizability of our findings to the broader URM medical faculty workforce. Similarly, several of our focus groups were small and our focus groups included residents from only three specialties. We attempted to involve six large specialties but were unsuccessful in engaging three of them during the COVID-19 era with its clinical demands and workforce challenges. Larger system-wide efforts have been more successful in engaging other programs and specialties in a multimodal GME effort incorporating our approach. While thematic saturation was achieved with the open-ended survey responses and focus groups, future research could expand this work to additional specialties. Additionally, participants who opted into the study may have been more likely to watch medical television programs and more engaged in race bias mitigation efforts than those who did not. Future research could employ a randomized design whereby participants watch these clips or another instructional tool, to compare the two modalities. A mandatory requirement for participation may also reduce the effects of selection bias among participants.Our findings suggest edutainment may be a promising avenue for GME anti-racist curricular content, and specifically that edutainment may offer a way to promote psychological safety when discussing this potentially sensitive content area. Our study also highlights the value of formative research in developing such curricula, with differences observed between faculty and resident participants and residents of different specialties supporting the use of gathering input from a diverse group of participants and tailored approaches to curricular design. Future work could use these findings to pilot anti-racist faculty development sessions and educational workshops for residents that incorporate the clips utilized in this study; our team is currently conducting such work as part of a multi-pronged, hospital system wide GME anti-racist curriculum. Future research could also conduct focus groups with trainees from other specialties or identify and obtain feedback on different clips or media content .


In oncology, geriatric medicine represents a topic with contrasting perspectives. On the one hand, it is familiar to many of practicing physicians, on the other hand, high-level evidence to guide our treatment decisions for this patient population is lacking. Although accumulating data show that treatment benefit can be retained with advancing age in fit patients, it is not unusual that when facing an older adult, even in good health, a less intensive approach is prioritized to avoid excessive toxicity . While tGatta et al.). Although a substantial proportion of deaths in the elderly population is due to non-cancer related competing mortality, wide-reaching improvements in anticancer management of elderly people is required with some aspects being covered in this article collection.These shortcomings seem to be even more pronounced in head and neck cancer (HNC) patients. Specific anatomic characteristics in the head and neck area contribute to the complexity of radiotherapy planning, which may be associated with further challenges regarding toxicity in senior persons. Moreover, well-known carcinogenic risk factors including alcohol consumption and tobacco use lead to collateral damages in terms of multifaceted comorbidities, especially after a long-term exposure being typical for elderly people. Finally, any deformities in a visually exposed region as is the area of the head and neck may have far-reaching impact on quality of life in this patient population with specific considerations in older adults as explained below. This background motivated conceptualization of the present article collection focusing on different aspects of anticancer care in elderly HNC patients and discussing the evolving epidemiological landscape as well. In this respect, absolute and relative numbers of HNC cases in the elderly have been steadily rising, particularly in high-income countries where almost 50% of newly diagnosed HNC patients are older than 65 years of age. Nevertheless, their 5-year overall survival rates still fall behind outcomes seen in younger patients in European countries, ranging between 60% in salivary gland and laryngeal cancers to only 22% in hypopharyngeal cancers . The investigators received answers from 132 respondents with only half of them declaring that radiotherapy target volumes corresponded to current treatment guidelines irrespective of the patient age, while 28% reported to adapt target volume definition in dependency of age and comorbidities and 22% in dependency of the tumour stage. Although the respondents administered concurrent chemotherapy regardless of chronological age, they were far more restrictive in terms of altered fractionation regimens, including hypofractionation. The latter schedule defined by using at least 2.2 Gy fractions was a subject of a systematic review by Piras et al. aiming to find out whether hypofractionation is a safe and feasible option in patients of 65 years of age or older. Based on data from 17 included papers, the authors conclude that a dose of 55 Gy delivered in 20 fractions represents a viable alternative to the standard fractionated radiotherapy, although the choice of a specific regimen needs to be individualized.Representing the mainstay of anticancer management in HNC patients, radiotherapy has been used across different clinical settings and patient populations. As mentioned above, the heterogeneity of elderly individuals can lead to treatment modifications towards less intensive schedules. Therefore, good understanding of current clinical practice patterns is welcome in order to identify areas necessitating better targeting in clinical trials, treatment guidelines, and awareness programs. Colombo et al. point out some of the common practice patterns in elderly patients and relate them to recommended procedures including but not limited to geriatric assessment and screening tests. Elderly patients with salivary gland cancers are diagnosed more frequently at advanced stages with a higher proportion of more aggressive (high-grade) histotypes and lower 5-year overall survival rates per disease stage in comparison with their younger counterparts. Interestingly, it seems that chronological age plays a less important role in elderly patients with salivary gland cancers than with other types of HNC when it comes to surgery, probably due to anatomic differences influencing feasibility of resection and a generally lower sensitivity of salivary gland cancers to radiation and systemic therapies. The authors present a very workable algorithm for the clinical assessment and treatment and give a clear summary of cytotoxic and non-cytotoxic systemic therapy options readapted to the elderly population.Another topic that has recently been brought to the spotlight of researchers concerns rare diagnoses. Interestingly, they share some characteristics attributed to the elderly cancer patients, namely paucity of high-level evidence and the resulting uncertainty about appropriate management. Typical examples are salivary gland cancers and nasopharyngeal carcinoma, particularly in non-endemic regions. Now, what happens if the two categories, elderly population and rare diseases, are merged? You will find possible answers in two contributions to our collection. In a comprehensive review about management of salivary gland cancers, Chan et al. in their review article about management of elderly people with nasopharyngeal carcinoma. Involvement of this anatomic site leads to specific challenges in delivery of local treatment owing to its complex geometry and the fact that older persons are more likely to develop treatment-related toxicities. In addition, nasopharyngeal carcinoma affects only about 10-15% of patients of 70 years of age or older, i.e., about a half of what is observed in HNC of other subsites , and this raises a further barrier to data availability . Due to the increasing prevalence of frailty and vulnerability with advancing age, elderly people are prone to acquire such a complex image of HNC. Nevertheless, fit older adults self-report greater resilience in health-related quality of life and psychosocial outcomes than younger individuals. It is also of note that judging by data from studies about variations in unmet needs by age in HNC, either no difference was observed or a lower burden was seen in senior persons who also stated less unmet needs for individual domains such as sexual, psychological, emotional, and spiritual needs .The third area covered in this Research Topic has been addressed by In conclusion, we believe that this Research Topic will provide interested readers with a comprehensive overview of HNC in the elderly ranging from practice-oriented topics over areas touching up on rare entities all the way to important considerations gaining attention only in recent years, including psychosocial aspects and unmet needs. Altogether, the ever-growing population of older cancer patients requires individualized measures adapted to their biological and not chronological age. Undertreatment, underrepresentation in clinical trials, and disregarding specific patient requirements represent the major issues in daily practice we will have to address in the near future to move forward.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.