Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions

Target-Specific Precision of CRISPR-Mediated Genome Editing

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms

Men, rheumatoid arthritis, psychosocial impact and self-management: A narrative review.

Rheumatoid arthritis (RA) is a chronic disease affecting fewer men than women. We systematically reviewed the literature on impact and self-management of RA men. Twenty eight papers were included, and grouped into two categories: Psychosocial impact of RA; and Coping and self-management. This review finds gender differences relating to quality of life; work; distress; self-management; coping; and support. We conclude there is a dearth of literature focussing on RA men only, and mixed gender studies include insufficient men to draw strong conclusions about men. Thus, further research is needed to understand the support needs of men with RA in depth

Absent fathers’, and children’s social and emotional learning: an exploration of the perceptions of ‘positive male role models’ in the primary school sector

This paper focuses on the testimonies of three male primary school staff members who utilised social and emotional learning (SEL) in their everyday practice within their respective schools. The data, collected through individual interviews, illustrate how these three men interpreted SEL, and their role in the development of children’s social, emotional and behavioural (SEB) skills, in response to their perceptions of pupils’ home-life. In particular, the sample identified the children’s fathers’ perceived ability/inability as a main cause of pupils’ SEB deficiencies. Consequently, the three male staff members maintained that in order to advocate and encourage alternative, appropriate behaviours, they should act as ‘replacement fathers’ and become ‘role models’. The findings contribute to existing debates relating to the notion of ‘positive male role models’ in primary schools and the propensity for staff to engage in parental blame. The implications of these findings are discussed, and suggestions that call for a more democratic and cooperative exchange of knowledge between parents and teachers are made

Assessing sustainability in housing LED urban regeneration : insights from a housing association in Northern England

How far do current assessment methods allow the thorough evaluation of sustainable urban regeneration? Would it be useful, to approach the evaluation of the environmental and social impacts of housing regeneration schemes, by making both hidden pitfalls and potentials explicit, and budgeting costs and benefits in the stakeholders’ perspective? The paper aims at answering these questions, by focusing on a case study located in the Manchester area, the City West Housing Trust, a nonprofit housing association. Drawing from extensive fieldwork and including several interviews with key experts from this housing association, the paper first attempts to monetize the environmental and social value of two extant projects – a high-rise housing estate and an environmentally-led program. It then discusses whether and how a stakeholder-oriented approach would allow more engagement of both current and potential funders in the projects at hand. Findings from both the literature and the empirical data that was gathered show how in current housing regeneration processes, room for significant improvements in terms of assessment methods still exist. Findings additionally show that the environmental and social spillovers are largely disregarded because of a gap in the evaluation tools. This may also hinder the potential contributions of further funders in the achievements of higher impacts in terms of sustainability

Proliferation Index: A Continuous Model to Predict Prognosis in Patients with Tumours of the Ewing's Sarcoma Family

The prognostic value of proliferation index (PI) and apoptotic index (AI), caspase-8, -9 and -10 expression have been investigated in primary Ewing's sarcoma family of tumours (ESFT). Proliferating cells, detected by immunohistochemistry for Ki-67, were identified in 91% (91/100) of tumours with a median PI of 14 (range 0–87). Apoptotic cells, identified using the TUNEL assay, were detected in 96% (76/79) of ESFT; the median AI was 3 (range 0–33). Caspase-8 protein expression was negative (0) in 14% (11/79), low (1) in 33% (26/79), medium (2) in 38% (30/79) and high (3) in 15% (12/79) of tumours, caspase-9 expression was low (1) in 66% (39/59) and high (3) in 34% (20/59), and caspase-10 protein was low (1) in 37% (23/62) and negative (0) in 63% (39/62) of primary ESFT. There was no apparent relationship between caspase-8, -9 and -10 expression, PI and AI. PI was predictive of relapse-free survival (RFS; p = 0.011) and overall survival (OS; p = <0.001) in a continuous model, whereas AI did not predict outcome. Patients with tumours expressing low levels of caspase-9 protein had a trend towards a worse RFS than patients with tumours expressing higher levels of caspase-9 protein (p = 0.054, log rank test), although expression of caspases-8, -9 and/or -10 did not significantly predict RFS or OS. In a multivariate analysis model that included tumour site, tumour volume, the presence of metastatic disease at diagnosis, PI and AI, PI independently predicts OS (p = 0.003). Consistent with previous publications, patients with pelvic tumours had a significantly worse OS than patients with tumours at other sites (p = 0.028); patients with a pelvic tumour and a PI≥20 had a 6 fold-increased risk of death. These studies advocate the evaluation of PI in a risk model of outcome for patients with ESFT

How do we improve men’s mental health via primary care? An evaluation of the Atlas Men’s Well-being Pilot Programme for stressed/distressed men

Background Over three-quarters of all suicides are men (England and Wales), this is despite higher levels of anxiety and depression being reported by women. This disparity may in part be explained by atypical presentations of distress in men, and gendered issues around help-seeking. Consequently, the Atlas Men’s Well-being Programme was designed to engage stressed/distressed men who were patients at a London-based GP surgery. Atlas encouraged GPs to identify and refer men for counselling and/or acupuncture by raising their awareness of men’s distress. The aim of this pilot study was to evaluate Atlas in terms of patients’ characteristics, service utilisation, patient outcomes and cost implications. Methods All patients using the Programme were asked to complete a questionnaire before and after their Atlas sessions. Outcome measures included the Hospital Anxiety and Depression scale, Perceived Stress Scale, Warwick-Edinburgh Mental Well-being Scale, a 11-point scale measuring physical health, and the Psychological Outcome Profiles (PSYCHLOPS), a patient-generated outcome measure. Additionally, for cost calculations, participants were asked about their employment, number of days off work due to illness, and their health and social care service use. Results 102 participants were recruited, 82 completed pre- and post-treatment questionnaires. Comparisons pre- and post-treatment revealed a statistically significant improvement in anxious mood (p <0.001), perceived stress (p < 0.001), positive well-being (p = <0.001), PSYCHLOPS (p = <0.001) and physical health (p = 0.001), though not depressed mood (p = 0.660). Additionally, reductions in costs related to lost employment and health and social care use, exceeded the cost of Atlas counselling and acupuncture sessions, with an average saving of nearly £700 per patient. Conclusions Atlas attendance was associated with improvements in patients’ mental and physical health, and demonstrated likely cost savings. It is now important to understand patient and stakeholder perspectives. Further research could compare usual care with the Atlas approach, and investigate full cost-effectiveness

Hands on, hands off: A model of clinical supervision that recognises trainees need for support and independence

Rationale.This article presents a study of junior doctor supervision at a rural hospital. The objective of the present study was to gain insight into the types of supervision events experienced, the quality of supervisory relationships, the frequencies of supervision contact in a rural hospital setting, and the implications of these factors for supervision practice. Methods.A cohort of junior doctors was asked to provide in-depth information about their interactions with their supervisors and other relevant clinical colleagues. The information was filled in on diary sheets to capture the nature, focus and quality of the cohort's supervision experiences over 2 weeks. The information also covered frequency and types of supervisory contacts. Results.The quantitative data reveals that supervisory events occur predominantly as part of ongoing patient care and rarely off-line as part of targeted supervisory practice. The qualitative data analysis reveals that junior doctors value supervisory support of two kinds: assistance from more senior clinicians who are expert in areas where trainees need help, and trust to act independently, without being abandoned. Conclusion.Supervision must be both structured and dynamic. Besides providing a regular forum for discussion and reflection, supervision must accommodate the variable needs of individual junior doctors and navigate between being hands-on and hands-off. Such dynamic approach is necessary to reassure junior doctors they are in a 'zone of safe learning' where they can act with adequate and flexible support and negotiate changes in supervisory attention. What is known about the topic Research is recognising the challenges of treatment complexity and unexpected outcomes faced by junior doctors. These factors mean that supervision needs to include dealing with the experiential and interpersonal aspects of junior doctors' clinical work. It is also recognised that the supervisory relationship remains to be investigated in depth. Further, because supervision guidelines in Australia are still under development, they do not as yet specify senior doctors' or registrar's supervisory accountabilities. Relying on conventional approaches to managing medical supervision, hospitals and associated medical schools are struggling to ensure that supervising doctors' perceptions of and approaches to supervision are aligned with emerging definitions of effective supervision. What does this paper add The 'hands on, hands off' model developed here enriches post-graduate medical curricula on two fronts. First, it advises supervisors that they need to be hands-on, practising 'active supervision'. This involves regular and structured contact with junior doctors to enhance the safety and quality of the care provided by them. Second, it advises supervisors to be hands-off, practising 'passive supervision'. This involves 'trustful' monitoring junior doctors' everyday work and negotiating with them their unique and changing learning trajectories. What are the implications for practitioners The model proposed here has three implications for practitioners. First, the model posits that medical supervision is about 'being there'. Junior doctors set great store by being granted ready access to advice and help if and when that is needed. Second, the model emphasises that junior doctors expect to gain supervisors' trust to act independently albeit with supervisory access and guidance being readily available. Third, junior doctors' needs change, not necessarily in a linear, uni-directional way. For supervisors, this means that they need to devise regular feedback opportunities for their trainees to articulate their developments, concerns and changing needs. © 2010 AHHA