A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens.

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.

A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility.

We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in ∼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and ∼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types. We develop a technique for mapping single cell gene expression data to single-cell chromatin accessibility data, facilitating the comparison of atlases. By intersecting mouse chromatin accessibility with human genome-wide association summary statistics, we identify cell-type-specific enrichments of the heritability signal for hundreds of complex traits. These data define the in vivo landscape of the regulatory genome for common mammalian cell types at single-cell resolution.

Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin.

Nucleosome positioning varies between cell types. By deep sequencing cell-free DNA (cfDNA), isolated from circulating blood plasma, we generated maps of genome-wide in vivo nucleosome occupancy and found that short cfDNA fragments harbor footprints of transcription factors. The cfDNA nucleosome occupancies correlate well with the nuclear architecture, gene structure, and expression observed in cells, suggesting that they could inform the cell type of origin. Nucleosome spacing inferred from cfDNA in healthy individuals correlates most strongly with epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how nucleosome footprints can be used to infer cell types contributing to cfDNA in pathological states such as cancer. Since this strategy does not rely on genetic differences to distinguish between contributing tissues, it may enable the noninvasive monitoring of a much broader set of clinical conditions than currently possible.

The single-cell transcriptional landscape of mammalian organogenesis.

Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution. Using single-cell combinatorial indexing, we profiled the transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting 'mouse organogenesis cell atlas' (MOCA) provides a global view of developmental processes during this critical window. We use Monocle 3 to identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. We explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle.

Can the delivery of behavioural support be improved in the NHS England Low-Calorie Diet Programme? An observational study of behaviour change techniques.

BACKGROUND: Previous research has illustrated a drift in the fidelity of behaviour change techniques (BCTs) during the design of the pilot NHS England Low-Calorie Diet (NHS-LCD) Programme. This study evaluated a subsequent domain of fidelity, intervention delivery. Two research questions were addressed: (1) To what extent were BCTs delivered with fidelity to providers programme plans? (2) What were the observed barriers and facilitators to delivery? METHODS: A mixed-methods sequential explanatory design was employed. Remote delivery of one-to-one and group-based programmes were observed. A BCT checklist was developed using the BCT Taxonomy v1; BCTs were coded as present, partially delivered, or absent during live sessions. Relational content analysis of field notes identified observed barriers and facilitators to fidelity. RESULTS: Observations of 122 sessions across eight samples and two service providers were completed. Delivery of the complete NHS-LCD was observed for five samples. Fidelity ranged from 33% to 70% across samples and was higher for group-based delivery models (64%) compared with one-to-one models (46%). Barriers and facilitators included alignment with the programme's target behaviours and outcomes, session content, time availability and management, group-based remote delivery, and deviation from the session plan. CONCLUSIONS: Overall, BCTs were delivered with low-to-moderate fidelity. Findings indicate a dilution in fidelity during the delivery of the NHS-LCD and variation in the fidelity of programmes delivered across England. Staff training could provide opportunities to practice the delivery of BCTs. Programme-level changes such as structured activities supported by participant materials and with sufficient allocated time, might improve the delivery of BCTs targeting self-regulation.

Supporting Weight Management during COVID-19 (SWiM-C): twelve-month follow-up of a randomised controlled trial of a web-based, ACT-based, guided self-help intervention.

OBJECTIVES: We developed a guided self-help intervention (Supporting Weight Management during COVID-19, "SWiM-C") to support adults with overweight or obesity in their weight management during the COVID-19 pandemic. This parallel, two-group trial (ISRCTN12107048) evaluated the effect of SWiM-C on weight and determinants of weight management over twelve months. METHODS: Participants (≥18 years, body-mass-index ≥25 kg/m2) were randomised to the SWiM-C intervention or to a standard advice group (unblinded). Participants completed online questionnaires at baseline, four months, and twelve months. The primary outcome was change in self-reported weight from baseline to twelve months; secondary outcomes were eating behaviour (uncontrolled eating, emotional eating, cognitive restraint of food intake), experiential avoidance, depression, anxiety, stress, wellbeing and physical activity. INTERVENTIONS: SWiM-C is based on acceptance and commitment therapy (ACT). Participants had access to an online web platform with 12 weekly modules and email and telephone contact with a trained, non-specialist coach. Standard advice was a leaflet on managing weight and mood during the COVID-19 pandemic. RESULTS: 388 participants were randomised (SWiM-C: n = 192, standard advice: n = 196). The baseline-adjusted difference in weight change between SWiM-C (n = 119) and standard advice (n = 147) was -0.81 kg (95% CI: -2.24 to 0.61 kg). SWiM-C participants reported a reduction in experiential avoidance (-2.45 [scale:10-70], 95% CI: -4.75 to -0.15), uncontrolled eating (-3.36 [scale: 0-100], 95% CI: -5.66 to -1.06), and emotional eating (-4.14 [scale:0-100], 95% CI: -7.25 to -1.02) and an increase in physical activity (8.96 [MET-min/week], 95% CI: 0.29 to 17.62) compared to standard advice participants. We found no evidence of an effect on remaining outcomes. No adverse events/side effects were reported. CONCLUSIONS: Whilst we were unable to conclude that the intervention had an effect on weight, SWiM-C improved eating behaviours, experiential avoidance and physical activity. Further refinement of the intervention is necessary to ensure meaningful effects on weight prior to implementation in practice. TRIAL REGISTRATION NUMBER: ISRCTN 12107048.

The need for future research into the assessment and monitoring of eating disorder risk in the context of obesity treatment.

In adolescents and adults, the co-occurrence of eating disorders and overweight or obesity is continuing to increase, and the prevalence of eating disorders is higher in people with higher weight compared to those with lower weight. People with an eating disorder with higher weight are more likely to present for weight loss than for eating disorder treatment. However, there are no clinical practice guidelines on how to screen, assess, and monitor eating disorder risk in the context of obesity treatment. In this article, we first summarize current challenges and knowledge gaps related to the identification and assessment of eating disorder risk and symptoms in people with higher weight seeking obesity treatment. Specifically, we discuss considerations relating to the validation of current self-report measures, dietary restraint, body dissatisfaction, binge eating, and how change in eating disorder risk can be measured in this setting. Second, we propose avenues for further research to guide the development and implementation of clinical and research protocols for the identification and assessment of eating disorders in people with higher weight in the context of obesity treatment. PUBLIC SIGNIFICANCE: The number of people with both eating disorders and higher weight is increasing. Currently, there is little guidance for clinicians and researchers about how to identify and monitor risk of eating disorders in people with higher weight. We present limitations of current research and suggest future avenues for research to enhance care for people living with higher weight with eating disorders.

Eating Disorders In weight-related Therapy (EDIT) Collaboration: rationale and study design.

The cornerstone of obesity treatment is behavioural weight management, resulting in significant improvements in cardio-metabolic and psychosocial health. However, there is ongoing concern that dietary interventions used for weight management may precipitate the development of eating disorders. Systematic reviews demonstrate that, while for most participants medically supervised obesity treatment improves risk scores related to eating disorders, a subset of people who undergo obesity treatment may have poor outcomes for eating disorders. This review summarises the background and rationale for the formation of the Eating Disorders In weight-related Therapy (EDIT) Collaboration. The EDIT Collaboration will explore the complex risk factor interactions that precede changes to eating disorder risk following weight management. In this review, we also outline the programme of work and design of studies for the EDIT Collaboration, including expected knowledge gains. The EDIT studies explore risk factors and the interactions between them using individual-level data from international weight management trials. Combining all available data on eating disorder risk from weight management trials will allow sufficient sample size to interrogate our hypothesis: that individuals undertaking weight management interventions will vary in their eating disorder risk profile, on the basis of personal characteristics and intervention strategies available to them. The collaboration includes the integration of health consumers in project development and translation. An important knowledge gain from this project is a comprehensive understanding of the impact of weight management interventions on eating disorder risk.

Improving programme-led and focused interventions for eating disorders: An experts' consensus statement-A UK perspective.

OBJECTIVE: Eating disorders are associated with significant illness burden and costs, yet access to evidence-based care is limited. Greater use of programme-led and focused interventions that are less resource-intensive might be part of the solution to this demand-capacity mismatch. METHOD: In October 2022, a group of predominantly UK-based clinical and academic researchers, charity representatives and people with lived experience convened to consider ways to improve access to, and efficacy of, programme-led and focused interventions for eating disorders in an attempt to bridge the demand-capacity gap. RESULTS: Several key recommendations were made across areas of research, policy, and practice. Of particular importance is the view that programme-led and focused interventions are suitable for a range of different eating disorder presentations across all ages, providing medical and psychiatric risk are closely monitored. The terminology used for these interventions should be carefully considered, so as not to imply that the treatment is suboptimal. CONCLUSIONS: Programme-led and focused interventions are a viable option to close the demand-capacity gap for eating disorder treatment and are particularly needed for children and young people. Work is urgently needed across sectors to evaluate and implement such interventions as a clinical and research priority.

Analysis of protein-coding genetic variation in 60,706 humans.

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.

Behavioural activation to prevent depression and loneliness among socially isolated older people with long-term conditions: The BASIL COVID-19 pilot randomised controlled trial.

BACKGROUND: Older adults, including those with long-term conditions (LTCs), are vulnerable to social isolation. They are likely to have become more socially isolated during the Coronavirus Disease 2019 (COVID-19) pandemic, often due to advice to "shield" to protect them from infection. This places them at particular risk of depression and loneliness. There is a need for brief scalable psychosocial interventions to mitigate the psychological impacts of social isolation. Behavioural activation (BA) is a credible candidate intervention, but a trial is needed. METHODS AND FINDINGS: We undertook an external pilot parallel randomised trial (ISRCTN94091479) designed to test recruitment, retention and engagement with, and the acceptability and preliminary effects of the intervention. Participants aged ≥65 years with 2 or more LTCs were recruited in primary care and randomised by computer and with concealed allocation between June and October 2020. BA was offered to intervention participants (n = 47), and control participants received usual primary care (n = 49). Assessment of outcome was made blind to treatment allocation. The primary outcome was depression severity (measured using the Patient Health Questionnaire 9 (PHQ-9)). We also measured health-related quality of life (measured by the Short Form (SF)-12v2 mental component scale (MCS) and physical component scale (PCS)), anxiety (measured by the Generalised Anxiety Disorder 7 (GAD-7)), perceived social and emotional loneliness (measured by the De Jong Gierveld Scale: 11-item loneliness scale). Outcome was measured at 1 and 3 months. The mean age of participants was aged 74 years (standard deviation (SD) 5.5) and they were mostly White (n = 92, 95.8%), and approximately two-thirds of the sample were female (n = 59, 61.5%). Remote recruitment was possible, and 45/47 (95.7%) randomised to the intervention completed 1 or more sessions (median 6 sessions) out of 8. A total of 90 (93.8%) completed the 1-month follow-up, and 86 (89.6%) completed the 3-month follow-up, with similar rates for control (1 month: 45/49 and 3 months 44/49) and intervention (1 month: 45/47and 3 months: 42/47) follow-up. Between-group comparisons were made using a confidence interval (CI) approach, and by adjusting for the covariate of interest at baseline. At 1 month (the primary clinical outcome point), the median number of completed sessions for people receiving the BA intervention was 3, and almost all participants were still receiving the BA intervention. The between-group comparison for the primary clinical outcome at 1 month was an adjusted between-group mean difference of -0.50 PHQ-9 points (95% CI -2.01 to 1.01), but only a small number of participants had completed the intervention at this point. At 3 months, the PHQ-9 adjusted mean difference (AMD) was 0.19 (95% CI -1.36 to 1.75). When we examined loneliness, the adjusted between-group difference in the De Jong Gierveld Loneliness Scale at 1 month was 0.28 (95% CI -0.51 to 1.06) and at 3 months -0.87 (95% CI -1.56 to -0.18), suggesting evidence of benefit of the intervention at this time point. For anxiety, the GAD adjusted between-group difference at 1 month was 0.20 (-1.33, 1.73) and at 3 months 0.31 (-1.08, 1.70). For the SF-12 (physical component score), the adjusted between-group difference at 1 month was 0.34 (-4.17, 4.85) and at 3 months 0.11 (-4.46, 4.67). For the SF-12 (mental component score), the adjusted between-group difference at 1 month was 1.91 (-2.64, 5.15) and at 3 months 1.26 (-2.64, 5.15). Participants who withdrew had minimal depressive symptoms at entry. There were no adverse events. The Behavioural Activation in Social Isolation (BASIL) study had 2 main limitations. First, we found that the intervention was still being delivered at the prespecified primary outcome point, and this fed into the design of the main trial where a primary outcome of 3 months is now collected. Second, this was a pilot trial and was not designed to test between-group differences with high levels of statistical power. Type 2 errors are likely to have occurred, and a larger trial is now underway to test for robust effects and replicate signals of effectiveness in important secondary outcomes such as loneliness. CONCLUSIONS: In this study, we observed that BA is a credible intervention to mitigate the psychological impacts of COVID-19 isolation for older adults. We demonstrated that it is feasible to undertake a trial of BA. The intervention can be delivered remotely and at scale, but should be reserved for older adults with evidence of depressive symptoms. The significant reduction in loneliness is unlikely to be a chance finding, and replication will be explored in a fully powered randomised controlled trial (RCT). TRIAL REGISTRATION: ISRCTN94091479.

On the design of CRISPR-based single-cell molecular screens.

Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.

Cost-effectiveness of a proportionate universal offer of free exercise: Leeds Let's Get Active.

BACKGROUND: The purpose of this paper is to assess the cost-effectiveness of a proportionate universal programme to reduce physical inactivity (Leeds Let us Get Active (LLGA)) in adults. METHODS: A continuous-time Markov chain model was developed to assess the cost implications and QALY gains associated with increases in physical activity levels across the adult population. A parametric survival analysis approach was applied to estimate the decay of intervention effect over time. Baseline model data were obtained from previous economic models, population-based surveys and other published literature. A cost-utility analysis was conducted from a health care sector perspective over the programme duration (39 months). Scenario and probabilistic sensitivity analyses were performed to test the robustness of cost-effectiveness results. RESULTS: In total, 51 874 adult residents registered to the programme and provided baseline data,19.5% of which were living in deprived areas. Under base case assumptions, LLGA was found to be likely to be cost-effective. However, variations in key structural assumptions showed sensitivity of the results. CONCLUSIONS: Results from this study suggest a non-negligible level of uncertainty regarding the effectiveness, and therefore, cost-effectiveness of a universal offer of free leisure centre-based exercise that targets hard to reach groups. Further data collection and a shift towards prospective evaluations are needed.

Split cGAL, an intersectional strategy using a split intein for refined spatiotemporal transgene control in Caenorhabditis elegans.

Bipartite expression systems, such as the GAL4-UAS system, allow fine manipulation of gene expression and are powerful tools for interrogating gene function. Recently, we established cGAL, a GAL4-based bipartite expression system for transgene control in Caenorhabditis elegans, where a single promoter dictates the expression pattern of a cGAL driver, which then binds target upstream activation sequences to drive expression of a downstream effector gene. Here, we report a split strategy for cGAL using the split intein gp41-1 for intersectional control of transgene expression. Split inteins are protein domains that associate, self-excise, and covalently ligate their flanking peptides together. We split the DNA binding domain and transcriptional activation domain of cGAL and fused them to the N terminal of gp41-1-N-intein and the C terminal of gp41-1-C-intein, respectively. In cells where both halves of cGAL are expressed, a functional cGAL driver is reconstituted via intein-mediated protein splicing. This reconstitution allows expression of the driver to be dictated by two promoters for refined spatial control or spatiotemporal control of transgene expression. We apply the split cGAL system to genetically access the single pair of MC neurons (previously inaccessible with a single promoter), and reveal an important role of protein kinase A in rhythmic pharyngeal pumping in C. elegans Thus, the split cGAL system gives researchers a greater degree of spatiotemporal control over transgene expression, and will be a valuable genetic tool in C. elegans for dissecting gene function with finer cell-specific resolution.

Multimodal single-cell analysis reveals distinct radioresistant stem-like and progenitor cell populations in murine glioma.

Radiation therapy is part of the standard of care for gliomas and kills a subset of tumor cells, while also altering the tumor microenvironment. Tumor cells with stem-like properties preferentially survive radiation and give rise to glioma recurrence. Various techniques for enriching and quantifying cells with stem-like properties have been used, including the fluorescence activated cell sorting (FACS)-based side population (SP) assay, which is a functional assay that enriches for stem-like tumor cells. In these analyses, mouse models of glioma have been used to understand the biology of this disease and therapeutic responses, including the radiation response. We present combined SP analysis and single-cell RNA sequencing of genetically-engineered mouse models of glioma to show a time course of cellular response to radiation. We identify and characterize two distinct tumor cell populations that are inherently radioresistant and also distinct effects of radiation on immune cell populations within the tumor microenvironment.

The relationship between obesity and tertiary education outcomes: a systematic review.

Previous reviews have documented an overall weak or uncertain association between obesity and school-based educational attainment in children and young people. However, up to half of young adults will go on to further college or university education by age 30. The study aim was to systematically review evidence on the association between obesity and tertiary education outcomes in young men and women. A search of multiple databases, including Embase, Global Health, ERIC, Medline, PsycInfo, and Science Citation Index was conducted in March 2018. Cross-sectional and longitudinal studies were included that reported on young people aged 16+, an association between obesity and academic achievement, and a comparison to healthy weight students. Risk of bias was assessed using criteria from the STROBE checklist. From 1297 records, 16 studies met all inclusion criteria. All six cross-sectional studies and 8/10 longitudinal studies reported lower educational achievement by students with obesity. All longitudinal studies were at low risk of bias but four cross-sectional studies were at medium risk and two at high risk of bias. Three of four studies showed reduced enrolment, in 6/8 graduation was less likely, and all 6 studies reporting on performance showed this was lower in those with obesity. Five of nine studies reported that obesity had a greater impact on educational achievement for women. Overall, there is compelling evidence of weight bias in that students with obesity do less well in tertiary education than their healthy weight peers. It is likely that university/college attainment is less impacted by socio-economic factors than school-based achievement. A better understanding of the processes that underpin this weight bias is needed, including stakeholder (student, staff) experiences of weight stigma, perceived or enacted. Responsive actions could mirror those to address disability or gender bias, or in health promotion in tertiary education settings.

Nutrition transition, overweight and obesity among rural-to-urban migrant women in Kenya.

OBJECTIVE: To assess the effect of rural-to-urban migration on nutrition transition and overweight/obesity risk among women in Kenya. DESIGN: Secondary analysis of data from nationally representative cross-sectional samples. Outcome variables were women's BMI and nutrition transition. Nutrition transition was based on fifteen different household food groups and was adjusted for socio-economic and demographic characteristics. Stepwise backward multiple ordinal regression analysis was applied. SETTING: Kenya Demographic and Health Survey 2014. PARTICIPANTS: Rural non-migrant, rural-to-urban migrant and urban non-migrant women aged 15-49 years (n 6171). RESULTS: Crude data analysis showed rural-to-urban migration to be associated with overweight/obesity risk and nutrition transition. After adjustment for household wealth, no significant differences between rural non-migrants and rural-to-urban migrants for overweight/obesity risk and household consumption of several food groups characteristic of nutrition transition (animal-source, fats and sweets) were observed. Regardless of wealth, migrants were less likely to consume main staples and legumes, and more likely to consume fruits and vegetables. Identified predictive factors of overweight/obesity among migrant women were age, duration of residence in urban area, marital status and household wealth. CONCLUSIONS: Our analysis showed that nutrition transition and overweight/obesity risk among rural-to-urban migrants is apparent with increasing wealth in urban areas. Several predictive factors were identified characterising migrant women being at risk for overweight/obesity. Future research is needed which investigates in depth the association between rural-to-urban migration and wealth to address inequalities in diet and overweight/obesity in Kenya.

MRI diagnosis of suprascapular neuropathy using spinoglenoid notch distension.

PURPOSE: To assess the use of a spinoglenoid notch distension measurement as a radiographic marker on MRI to aid the diagnosis of suprascapular neuropathy. METHODS: Spinoglenoid notch distension was compared on MRI by blinded independent observers for two patient cohorts: one group with an electromyography/nerve conduction study confirmed diagnosis of suprascapular neuropathy who underwent arthroscopic suprascapular nerve decompression, and a control group of patients aged 18-30 years with a normal shoulder MRI. RESULTS: Sixty suprascapular nerve patients (average age 52 years) were compared to 47 control patients (average age 24 years). Intra-rater and inter-rater reliability showed excellent agreement between reviewers for all measurements. There was a significant difference in the mean spinoglenoid notch distension for the SSN group (m = 8.36, SD = 2.42) compared to the control group (m = 5.7, SD = 1.56); [t(212) = 9.40, p < 0.0001]. CONCLUSION: The spinoglenoid notch distension is significantly increased in patients with suprascapular neuropathy. We hypothesize that hypertrophy of the transverse scapular ligament creates a venous obstruction resulting in varicosities of the suprascapular vein which runs with the nerve under the ligament. This distends the spinoglenoid notch and can be enlarged in cases of suprascapular neuropathy which is evident on MRI.