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To foster a "wellbeing curriculum" in a climate with an increasingly competitive graduate jobs market, we believe it is critical to support undergraduate career development and to develop positive peer and educator relationships, particularly for non-vocational degree programs. However, these relationships between undergraduate wellbeing and their career development or peer/educator relationships have not been specifically examined. This study used a mixed methods approach to examine if poor career development or university engagement (quality of relationships with peers or educators, use of the university careers and counselling services, time studying) were associated with psychological distress for students in non-vocational degree programs. Undergraduates (biomedical science; n=1100) from five Australian universities participated in a survey to investigate relationships between psychological distress, as determined by their responses to the Depression, Anxiety and Stress Scales, and their career development or university engagement. Almost half of the students lacked confidence in their "future employment and job prospects." Students? psychological distress was significantly correlated with lack of confidence with their career development, poor relationships with their peers and educators and little use of the counselling service. Further exploration of these factors in student focus groups highlighted stress associated with academic competition between students and a critical need for undergraduate career development, especially industry placements. We provide pivotal recommendations to promote undergraduate and educator wellbeing, by developing a „wellbeing curriculum? that supports career development and positive relationships between students and their peers and educators, particularly vital for non-vocational degrees.

Background Young adults represent a third of the United Kingdom prison population and are at risk of poor health outcomes, including drug and alcohol misuse, self-harm and suicide. Court diversion interventions aim to reduce the negative consequences of criminal sanctions and address the root causes of offending. However, evidence of their effectiveness has not yet been established. The Gateway programme, issued as a conditional caution, aimed to improve the life chances of young adults committing low-level offences. Participants agreed not to reoffend during the 16-week caution and, following a needs assessment, received individual support from a Gateway navigator and attended two workshops encouraging analysis of own behaviour and its consequences. Objective To evaluate the effectiveness and cost-effectiveness of Gateway in relation to health and well-being of participants compared to usual process (court summons or a different conditional caution). Design, setting and participants Pragmatic, multisite, parallel-group, superiority randomised controlled trial with two 6-month internal pilots and a target sample size of 334. Randomisation between Gateway and usual process was on a 1 : 1 basis. Four Hampshire Constabulary sites recruited 18- to 24-year-old residents of Hampshire and Isle of Wight who were questioned for an eligible low-level offence. Semistructured interviews were also held with a sample of Gateway programme participants, staff and police study recruiters. Main outcome measures Primary outcome was the Warwick-Edinburgh Mental Wellbeing Scale score at 12 months. Secondary outcomes included health status, alcohol and drug use, recidivism and resource use. Results Recruitment commenced in October 2019 and the trial stopped in April 2021. A total of 191 participants were recruited, with 109 randomised to Gateway and 82 to usual process. Due to an initial overestimation of potentially eligible young people and low retention rates, recruitment targets were adjusted, and a range of mitigating measures introduced. Although recruitment broadly met study progression criteria [35/50 (70%) Pilot 1: 64/74 (86%) Pilot 2], retention was low throughout (overall: data collected at week 4 was 50%: at week 16 it was 50%: 1-year 37%). Low retention was multifactorial, with one of the main barriers being difficulties contacting participants. It was therefore not possible to complete the randomised controlled trial or the health economics analyses. Qualitative interviews held with 58 individuals yielded rare insights into the benefits and limitations of this type of intervention, as well as barriers and facilitators in relation to recruitment in this setting. Limitations Despite close collaboration with the police to address recruitment and consent issues, expansion of the inclusion criteria and recruitment area and introducing other measures, the researchers were unable to collect sufficient data within an acceptable timeframe. Conclusions The Gateway study was a unique endeavour to gather evidence for a potentially life-changing intervention for an underserved population. The experience gained indicates that randomised controlled trials of interventions, with a health-related outcome, are possible in this setting but point towards the need for conservative recruitment and retention estimates in this target population. Other study designs should be considered. The qualitative evaluation provided a range of valuable lessons for those seeking to design similar interventions or conduct research in similar settings. Study registration This study is registered as ISRCTN11888938. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Public Health Research programme (NIHR award ref: 16/122/20) and is published in full in Public Health Research; Vol. 12, No. 7. See the NIHR Funding and Awards website for further award information. Plain language summary Young adults who commit low-level offences often have many health and social needs, making them vulnerable to physical and mental health problems. The Gateway programme was a conditional caution developed to address the underlying causes of low-level offending in young people aged 18–24 years and hence improve their life chances. In Gateway, a mentor assessed the young person’s needs and supported them, signposting to healthcare, housing or other services as required. The young people also participated in two workshops, analysing the causes and consequences of their behaviour. To find out if Gateway improved health and reoffending rates, a group of those who received a Gateway conditional caution were compared with a group of those receiving a court summons or a different conditional caution. Of the 191 participants recruited to the study, 109 were randomised to Gateway and 82 to the usual process. However, the researchers had significant difficulties getting hold of the study participants on the phone and they were unable to collect enough information from them to be able to say whether Gateway worked. The researchers introduced various changes to overcome this, but in the end had to stop the study early. As part of the study, the researchers interviewed 28 Gateway programme participants, 17 Gateway project staff and 13 police officers and staff who had been recruiting into the study. From the interviews the study discovered the perceived benefits of Gateway, how programmes like this could be improved and which factors helped or got in the way of doing research in the police setting. The Gateway study aimed to provide evidence for a potentially life-changing intervention for vulnerable young adults. Although it proved impossible to complete the study, the lessons learnt from running it should help colleagues design similar programmes or plan research studies with similar populations or in similar settings. Scientific summary Background Young adults represent a third of the United Kingdom (UK) prison population and are at risk of poor health outcomes including drug and alcohol misuse, self-harm and suicide. Those aged between 18 and 24, who have been questioned as suspects in relation to a low-level offence, may need to attend court and, if convicted, face penalties such as imprisonment. However, other means aimed at preventing young adults from reoffending exist. Court diversion interventions aim to reduce the negative consequences of some types of criminal sanctions and focus resources on addressing the root causes of offending. Although diversions are widely used in the UK, evidence of their effectiveness in terms of health outcomes has not yet been established using robust research methods. Hampshire Constabulary (HC), working with local charities, developed the Gateway programme, an out-of-court disposal aimed at improving the life chances of young adults. Objectives The aim of this study was to evaluate the effectiveness and cost-effectiveness of the Gateway programme issued as a conditional caution (intervention) compared to usual process (court appearance or a different conditional caution). The study objectives were to: Examine the effectiveness of the Gateway intervention on: (1) health and well-being including alcohol and substance use, (2) access to and use of health and social services and (3) quality of life, among young adult offenders. Explore the views and experiences of victims. Assess the quantity and quality of the Gateway intervention as delivered in the study and the generalisability of the findings. Identify and measure relevant consequences, both cost and benefits, of the Gateway intervention compared with usual process. Examine the effectiveness of the Gateway intervention on recidivism. Methods Design The study undertook a pragmatic, superiority RCT with two 6-month internal pilot phases and qualitative evaluation: an economic evaluation was planned. Participants were randomised using a 1 : 1 allocation ratio to either the Gateway conditional caution (intervention) or disposal as usual to a court summons or a different conditional caution to Gateway (usual process). The qualitative evaluation aimed to capture the experiences and perceptions of the impact of the intervention on participants, the police, victims and those delivering the intervention. Participants, setting and recruitment Eligible participants were those aged 18–24 who had committed low-level offences and resided within Hampshire and Isle of Wight (IoW) area. Participants were recruited by Police investigators during processing for an offence. Potential participants were offered a chance by the police to receive the Gateway caution, and those interested were invited to take part in the study. Police officers obtained Stage 1 consent and carried out an eligibility check, after which those eligible were automatically randomised to receive either a Gateway caution or follow the usual process, such as court appearance or a different conditional caution. Qualitative interviews were undertaken with trial participants, police officers and those delivering the intervention. Sample size There is no widely accepted and established minimal clinically significant difference for the primary outcome, Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS). A change of three or more points is likely to be important to individuals. There is also variation in the standard deviation (SD) of the WEMWBS with estimates ranging from 6 to 10.8 with the pooled estimate of 10 across all studies. Assuming 90% power, 5% 2-sided statistical significance, mean difference of 4 points on WEMWBS and a SD of 10, 266 participants were required. Conservatively, assuming a 20% attrition rate, the study aimed to recruit and randomise 334 participants. Interventions The Gateway programme, issued as a conditional caution, required participants to undertake a health and social care needs assessment, attend workshops encouraging analysis of their behaviour and its consequences, and agree not to reoffend during the 16-week caution. Usual process was disposal to a court summons or an alternative conditional caution. Outcomes The primary outcome measure was the WEMWBS. Participants self-reported WEMWBS at 4-weeks, 16-weeks and 1-year post-randomisation. Secondary outcomes were health status [Short Form 12 questionnaire (SF-12)]; alcohol [Alcohol Use Disorders Identification Test (AUDIT)] and drug [Adolescent Drug Involvement Scale (ADIS)] use; type and frequency of reoffending (police data); and health and social care resource use (self-reported). Statistical methods The original plan, pre-specified in version 1.0 of the SAP, was to carry out a repeated measures, mixed-effects linear regression model with the WEMWBS score at 4-weeks, 16-weeks and 1-year post-randomisation as the dependent variable, adjusting for treatment group, time, group by time interaction, total number of records management system incidents and police national computer convictions 1-year prerandomisation, age at randomisation, Index of Multiple Deprivation quintile at randomisation, pandemic time period and standard of usual process available as fixed effects. Recruiting site was to be adjusted for as a random effect. However, due to the study closing early because of issues with retaining participants, a descriptive analysis was undertaken with no formal hypothesis testing. All outcomes were summarised descriptively by a randomised treatment group. Qualitative evaluation A qualitative evaluation was conducted to assess the implementation of the Gateway programme, including any related issues and observed benefits to the clients. Focusing on implementation, mechanisms and context, the research questions were: How is Gateway being implemented? What are the barriers to the implementation and effects of the Gateway programme? What are the mechanisms through which the intervention brings about change? How do different delivery methods (face-to-face/virtual/telephone) influence the above questions? We conducted qualitative interviews and focus groups with a range of stakeholders across three time periods during the implementation of the Gateway programme. Economic evaluation A formal health economic analysis was not feasible. Health economic data are summarised descriptively with the trial data. Results Randomised controlled trial We recruited 191 participants; 109 were randomised to Gateway and 82 to usual process. Although recruitment rates were within acceptable limits, the number of participants providing data [94 (32%) at week 4; 95 (34%) at week 16; 43 (28%) at 1-year] was insufficient to undertake any formal hypothesis testing and the trial was closed early. The groups were generally well balanced in terms of characteristics and percentage providing data, but more of those providing valid data had a previous conviction than those who did not provide data. Similar percentages from each arm provided data with those attending interviews completing all sections. Telephone interviews were acceptable to those willing to share an active telephone number. Rates for those who were non-contactable were similar between the groups at all three time points. Eighty-one of the 101 allocated to Gateway complied with the intervention. Reasons for non-compliance were reoffending and non-attendance at the LINX workshops. Qualitative evaluation Across 3 time points, 69 in-depth interviews were conducted with: 28 young people, 25 Gateway staff, 13 police recruiters and 3 focus groups with navigators. The researchers were unable to pursue interviews with victims as there were few offences with a victim. Our findings showed that, following engagement with the Gateway programme, young people reported being better able to make decisions after engagement with the LINX workshops, while navigators played a significant role in enabling compliance and change among young people. The role of the navigator was akin to that of the mentor, providing practical support towards improved health including, for example, making and attending doctors’ appointments with clients, as well as offering a listening role. Young people felt that, for them, the wider determinants (or ‘areas’) addressed with navigators, such as access to employment or improved health, were of greater importance than a reduction in reoffending. There was a polarity of needs among young people, which meant that all stakeholders valued the ability to tailor and adapt the programme to individual client’s needs, also giving clients a sense of agency and control over their lives. The independence of the Gateway intervention team, from the police, was highly valued by young people. Factors related to communication were a concern for all, particular at the point of recruitment (by police) and between multiple delivery agencies. Discussion The problems encountered throughout this trial and the researchers’ endeavours to overcome these problems provide valuable insights for colleagues seeking to design similar interventions and/or conduct studies with vulnerable populations in the police setting. Co-production is essential for studies in the police setting. By working in close collaboration with HC, and their two project dedicated officers, the researchers were able to make pragmatic adjustments to the study design as issues arose. However, training an entire police force is fraught with difficulty even when supported by senior officers; competing interests precluded mandatory training for research purposes. The use of a two-stage consent process and a web-based eligibility and randomisation tool facilitate recruitment in the police setting, but frequent, regular need to use is required to maintain the study profile. Young people who have committed an offence are known to be a difficult group to engage with generally, let alone in research. The study identified and tested implementation of different approaches to overcome this problem. Switching to telephone interviews produced a positive response. Persistence and engagement paid off, but the study was unable to solve the problem of inactive mobile telephone numbers; an issue shared by the navigators and police. Independence of those delivering the intervention is important as perceived links with the police caused some disengagement. The collected data provide valuable information on attrition rates for health studies. Interestingly, allocation did not appear to make any difference to participation. The study’s qualitative evaluation highlighted the unmet health needs for this group of 18- to 24-year-olds, and the need to address the wider determinants of reoffending through individualised assessment. The Gateway programme was, however, developed for those with higher needs, which meant that flexibility and adaptability to suit individual needs was essential. It further highlighted the invaluable role of mentors in rehabilitative programmes such as Gateway. The study has demonstrated that it is possible to recruit and randomise to a RCT in the police setting. The data that the study presents should be used to inform the planning of future trials, including anticipated attrition rates, and setting conservative targets for retention as well as recruitment rates. Internal pilots should be long enough to confirm recruitment and data collection rates are achievable over an adequate follow-up period. Given the challenges encountered, alternative study designs should be considered for the evaluation of interventions with a health-related outcome. These include: cluster RCTs where processes at individual cluster sites could be simplified; post hoc cohort studies which may address non-response and attrition bias; and regression discontinuity design (RDD), a quasi-experimental approach at lower risk of bias which has the potential to equate to a RCT. Conclusions This ambitious RCT and qualitative study provides information about an out-of-court intervention aimed at improving life chances, health and well-being and recidivism in young adults. Challenges encountered in participation and retention in this setting and the ways in which these may be addressed are described and will be useful in planning future research. Study registration This study is registered as ISRCTN11888938. Funding This award was funded by the National Institute for Health and Care Research (NIHR) Public Health Research programme (NIHR award ref: 16/122/20) and is published in full in Public Health Research; Vol. 12, No. 7. See the NIHR Funding and Awards website for further award information.

Summary: Leukemia niche impacts quiescence; however, culturing patient-derived samples ex vivo is technically challenging. Here, we present a protocol for in vitro co-culture of patient-derived xenograft acute lymphoblastic leukemia (PDX-ALL) cells with human mesenchymal stem cells (MSCs). We describe steps for labeling PDX-ALL cells with CellTrace Violet dye to demonstrate MSC-primed PDX-ALL cycling. We then detail procedures to identify MSC-primed G0/quiescent PDX-ALL cells via Hoechst-33342/Pyronin Y live cell cycle analysis.For complete details on the use and execution of this protocol, please refer to Pal et al.1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Becker muscular dystrophy (BMD) is characterised by fiber loss and expansion of fibrotic and adipose tissue. Several cells interact locally in what is known as the degenerative niche. We analysed muscle biopsies of controls and BMD patients at early, moderate and advanced stages of progression using Hyperion imaging mass cytometry (IMC) by labelling single sections with 17 markers identifying different components of the muscle. We developed a software for analysing IMC images and studied changes in the muscle composition and spatial correlations between markers across disease progression. We found a strong correlation between collagen-I and the area of stroma, collagen-VI, adipose tissue, and M2-macrophages number. There was a negative correlation between the area of collagen-I and the number of satellite cells (SCs), fibres and blood vessels. The comparison between fibrotic and non-fibrotic areas allowed to study the disease process in detail. We found structural differences among non-fibrotic areas from control and patients, being these latter characterized by increase in CTGF and in M2-macrophages and decrease in fibers and blood vessels. IMC enables to study of changes in tissue structure along disease progression, spatio-temporal correlations and opening the door to better understand new potential pathogenic pathways in human samples.

Abstract Immune-mediated inflammatory diseases (IMIDs) are typically characterised by relapsing and remitting flares of inflammation. However, the unpredictability of disease flares impedes their study. Addressing this critical knowledge gap, we use the experimental medicine approach of immunomodulatory drug withdrawal in rheumatoid arthritis (RA) remission to synchronise flare processes allowing detailed characterisation. Exploratory mass cytometry analyses reveal three circulating cellular subsets heralding the onset of arthritis flare – CD45RO+PD1hi CD4+ and CD8+ T cells, and CD27+CD86+CD21- B cells – further characterised by single-cell sequencing. Distinct lymphocyte subsets including cytotoxic and exhausted CD4+ memory T cells, memory CD8+CXCR5+ T cells, and IGHA1+ plasma cells are primed for activation in flare patients. Regulatory memory CD4+ T cells (Treg cells) increase at flare onset, but with dysfunctional regulatory marker expression compared to drug-free remission. Significant clonal expansion is observed in T cells, but not B cells, after drug cessation; this is widespread throughout memory CD8+ T cell subsets but limited to the granzyme-expressing cytotoxic subset within CD4+ memory T cells. Based on our observations, we suggest a model of immune dysregulation for understanding RA flare, with potential for further translational research towards novel avenues for its treatment and prevention.

Background Young adults who commit low-level offences commonly have a range of health and social needs and are significantly over-represented in the criminal justice system. These young adults may need to attend court and potentially receive penalties including imprisonment. Alternative routes exist, which can help address the underlying causes of offending. Some feel more should be done to help young adults entering the criminal justice system. The Gateway programme was a type of out-of-court disposal developed by Hampshire Constabulary, which aimed to address the complex needs of young adults who commit low-level crimes. This study aimed to evaluate the effectiveness and cost-effectiveness of the Gateway programme, issued as a conditional caution, compared with usual process.Methods The Gateway study was a pragmatic, parallel-group, superiority randomised controlled trial that recruited young adults who had committed a low-level offence from four sites covering Hampshire and Isle of Wight. The primary outcome was mental health and well-being measured using the Warwick-Edinburgh Mental Well-being Scale. Secondary outcomes were quality of life, alcohol and drug use, and recidivism. Outcomes were measured at 4, 16 and 52 weeks postrandomisation.Results Due to issues with retention of participants and low data collection rates, recruitment ended early, with 191 eligible participants randomised (Gateway 109; usual process 82). The primary outcome was obtained for 93 (48.7%) participants at 4 weeks, 93 (48.7%) at 16 weeks and 43 (22.5%) at 1 year. The high attrition rates meant that effectiveness could not be assessed as planned.Conclusions Gateway is the first trial in a UK police setting to have a health-related primary outcome requiring individual data collection, rather than focusing solely on recidivism. We demonstrated that it is possible to recruit and randomise from the study population, however follow-up rates were low. Further work is needed to identify ways to facilitate engagement between researchers and vulnerable populations to collect data.Trial registration number ISRCTN11888938.

Introduction In response to COVID-19, the South African government banned the sale of tobacco products for 20 weeks. Before the ban, the illicit cigarette market was well-entrenched and smoking cessation services were not widely available. Several surveys conducted to ascertain cigarette smokers’ responses to the ban reported substantial differences in the proportion of smokers who quit. This study provides a broadly nationally representative ex-post investigation into cigarette smokers’ quitting behavior related to the sales ban. Methods We used data from wave three of NIDS-CRAM (the National Income Dynamics Study-Coronavirus Rapid Mobile Survey) conducted in November– December 2020. We first investigated the proportion of people who quit and who continued smoking during and after the sales ban. We subsequently linked the NIDS-CRAM survey to the fifth wave of NIDS (2017) to identify a subset of established smokers, and considered whether their quitting behavior differed from that of all smokers who smoked at the start of the sales ban. Results The cross-sectional analysis showed that 7.8% of cigarette smokers quit during the sales ban, but that 55% of these quitters relapsed after it was lifted. Of the pre-ban smokers, 3.5% indicated that they did not smoke both during and after the sales ban, and 3.7% quit after the ban was lifted. The longitudinal analysis showed that 7% of people who were smoking in 2017, quit smoking cigarettes during the tobacco sales ban, but that >70% of quitters relapsed after it was lifted. Only 2% of pre-ban established smokers indicated that they did not smoke during or after the ban. Conclusions The sales ban did not have the intended objective of encouraging large-scale smoking cessation. This reflects policy failures to provide smokers with appropriate cessation support and to effectively control the illicit market both prior to and during the sales ban.

Abstract Mitochondrial dysfunction has been suggested to contribute to Parkinson’s disease pathogenesis, though an understanding of the extent or exact mechanism of this contribution remains elusive. This has been complicated by challenging nature of pathway-based analysis and an inability simultaneously study multiple related proteins within human brain tissue. We used imaging mass cytometry (IMC) to overcome these challenges, measuring multiple protein targets, whilst retaining the spatial relationship between targets in post-mortem midbrain sections. We used IMC to simultaneously interrogate subunits of the mitochondrial oxidative phosphorylation complexes, and several key signalling pathways important for mitochondrial homoeostasis, in a large cohort of PD patient and control cases. We revealed a generalised and synergistic reduction in mitochondrial quality control proteins in dopaminergic neurons from Parkinson’s patients. Further, protein-protein abundance relationships appeared significantly different between PD and disease control tissue. Our data showed a significant reduction in the abundance of PINK1, Parkin and phosphorylated ubiquitinSer65, integral to the mitophagy machinery; two mitochondrial chaperones, HSP60 and PHB1; and regulators of mitochondrial protein synthesis and the unfolded protein response, SIRT3 and TFAM. Further, SIRT3 and PINK1 did not show an adaptive response to an ATP synthase defect in the Parkinson’s neurons. We also observed intraneuronal aggregates of phosphorylated ubiquitinSer65, alongside increased abundance of mitochondrial proteases, LONP1 and HTRA2, within the Parkinson’s neurons with Lewy body pathology, compared to those without. Taken together, these findings suggest an inability to turnover mitochondria and maintain mitochondrial proteostasis in Parkinson’s neurons. This may exacerbate the impact of oxidative phosphorylation defects and ageing related oxidative stress, leading to neuronal degeneration. Our data also suggest that that Lewy pathology may affect mitochondrial quality control regulation through the disturbance of mitophagy and intramitochondrial proteostasis.

Summary: Background: Lung damage in severe COVID-19 is highly heterogeneous however studies with dedicated spatial distinction of discrete temporal phases of diffuse alveolar damage (DAD) and alternate lung injury patterns are lacking. Existing studies have also not accounted for progressive airspace obliteration in cellularity estimates. We used an imaging mass cytometry (IMC) analysis with an airspace correction step to more accurately identify the cellular immune response that underpins the heterogeneity of severe COVID-19 lung disease. Methods: Lung tissue was obtained at post-mortem from severe COVID-19 deaths. Pathologist-selected regions of interest (ROIs) were chosen by light microscopy representing the patho-evolutionary spectrum of DAD and alternate disease phenotypes were selected for comparison. Architecturally normal SARS-CoV-2-positive lung tissue and tissue from SARS-CoV-2-negative donors served as controls. ROIs were stained for 40 cellular protein markers and ablated using IMC before segmented cells were classified. Cell populations corrected by ROI airspace and their spatial relationships were compared across lung injury patterns. Findings: Forty patients (32M:8F, age: 22–98), 345 ROIs and >900k single cells were analysed. DAD progression was marked by airspace obliteration and significant increases in mononuclear phagocytes (MnPs), T and B lymphocytes and significant decreases in alveolar epithelial and endothelial cells. Neutrophil populations proved stable overall although several interferon-responding subsets demonstrated expansion. Spatial analysis revealed immune cell interactions occur prior to microscopically appreciable tissue injury. Interpretation: The immunopathogenesis of severe DAD in COVID-19 lung disease is characterised by sustained increases in MnPs and lymphocytes with key interactions occurring even prior to lung injury is established. Funding: UK Research and Innovation/Medical Research Council through the UK Coronavirus Immunology Consortium, Barbour Foundation, General Sir John Monash Foundation, Newcastle University, JGW Patterson Foundation, Wellcome Trust.

Abstract Background Aggression is a near-universal behaviour with substantial influence on and implications for human and animal social systems. The neurophysiological basis of aggression is, however, poorly understood in all species and approaches adopted to study this complex behaviour have often been oversimplified. We applied targeted expression profiling on 40 genes, spanning eight neurological pathways and in four distinct regions of the brain, in combination with behavioural observations and pharmacological manipulations, to screen for regulatory pathways of aggression in the zebrafish (Danio rerio), an animal model in which social rank and aggressiveness tightly correlate. Results Substantial differences occurred in gene expression profiles between dominant and subordinate males associated with phenotypic differences in aggressiveness and, for the chosen gene set, they occurred mainly in the hypothalamus and telencephalon. The patterns of differentially-expressed genes implied multifactorial control of aggression in zebrafish, including the hypothalamo-neurohypophysial-system, serotonin, somatostatin, dopamine, hypothalamo-pituitary-interrenal, hypothalamo-pituitary-gonadal and histamine pathways, and the latter is a novel finding outside mammals. Pharmacological manipulations of various nodes within the hypothalamo-neurohypophysial-system and serotonin pathways supported their functional involvement. We also observed differences in expression profiles in the brains of dominant versus subordinate females that suggested sex-conserved control of aggression. For example, in the HNS pathway, the gene encoding arginine vasotocin (AVT), previously believed specific to male behaviours, was amongst those genes most associated with aggression, and AVT inhibited dominant female aggression, as in males. However, sex-specific differences in the expression profiles also occurred, including differences in aggression-associated tryptophan hydroxylases and estrogen receptors. Conclusions Thus, through an integrated approach, combining gene expression profiling, behavioural analyses, and pharmacological manipulations, we identified candidate genes and pathways that appear to play significant roles in regulating aggression in fish. Many of these are novel for non-mammalian systems. We further present a validated system for advancing our understanding of the mechanistic underpinnings of complex behaviours using a fish model.

Introduction Current research aimed at understanding and preventing stillbirth focuses almost exclusively on the role of the placenta. The underlying origins of poor placental function leading to stillbirth, however, remain poorly understood. There is evidence demonstrating that the endometrial environment in which the embryo implants impacts not only the establishment of pregnancy but also the development of some pregnancy outcomes. Menstrual fluid has recently been applied to the study of menstrual disorders such as heavy menstrual bleeding or endometriosis, however, it has great potential in the study of adverse pregnancy outcomes. This study aims to identify differences in menstrual fluid and menstrual cycle characteristics of women who have experienced preterm stillbirth and other associated adverse pregnancy outcomes, compared with those who have not. The association between menstrual fluid composition and menstrual cycle characteristics will also be determined.Methods and analysis This is a case–control study of women who have experienced a late miscarriage, spontaneous preterm birth or preterm stillbirth or a pregnancy complicated by placental insufficiency (fetal growth restriction or pre-eclampsia), compared with those who have had a healthy term birth. Cases will be matched for maternal age, body mass index and gravidity. Participants will not currently be on hormonal therapy. Women will be provided with a menstrual cup and will collect their sample on day 2 of menstruation. Primary exposure measures include morphological and functional differences in decidualisation of the endometrium (cell types, immune cell subpopulations and protein composition secreted from the decidualised endometrium). Women will complete a menstrual history survey to capture menstrual cycle length, regularity, level of pain and heaviness of flow.Ethics and dissemination Ethics approval was obtained from Monash University Human Research Ethics Committee (27900) on 14/07/2021 and will be conducted in accordance with these conditions. Findings from this study will be disseminated through peer-reviewed publications and conference presentations.

Imaging flow cytometry (IFC) provides single‐cell imaging data at a high acquisition rate. It is increasingly used in image‐based profiling experiments consisting of hundreds of thousands of multi‐channel images of cells. Currently available software solutions for processing microscopy data can provide good results in downstream analysis, but are limited in efficiency and scalability, and often ill‐adapted to IFC data. In this work, we propose Scalable Cytometry Image Processing (SCIP), a Python software that efficiently processes images from IFC and standard microscopy datasets. We also propose a file format for efficiently storing IFC data. We showcase our contributions on two large‐scale microscopy and one IFC datasets, all of which are publicly available. Our results show that SCIP can extract the same kind of information as other tools, in a much shorter time and in a more scalable manner. [ABSTRACT FROM AUTHOR]

Background and Aims: Pre-implantation genetic testing (PGT) is a technology utilised in assisted reproductive technologies (ART) to aid in embryo selection and reduce time to pregnancy. However, the impact on PGT outcomes of different fertilisation techniques, to date, has been the subject of a limited number of studies. Therefore, we investigated the effect of three different fertilisation methods - intracytoplasmic sperm injection (ICSI), PIEZO-ICSI, and standard in vitro fertilisation (IVF) - on PGT outcomes, with further analysis performed for day of biopsy. Method: Retrospective analysis of 760 embryos biopsied for PGT from 290 patients undergoing IVF treatment in 2021. Fertilisation was achieved via ICSI, PIEZO-ICSI, or IVF, followed by embryo culture using a sequential media system. Trophectoderm biopsy samples were analysed using a Next Generation Sequencing based assay (Illumina VeriSeq PGS). Ploidy outcomes from PGT (euploid, aneuploid, mosaic, inconclusive and no result) were interrogated for differences between fertilisation method or day of biopsy (Day 5 or 6). For analyses, frequency data of ploidy outcomes were reported as percentages and divided into two maternal age groups: 38 years old (n=431) PIEZO-ICSI 61% vs ICSI 58% vs IVF 51%; p>0.05, and [Formula: see text]38 years old (n=329) PIEZO-ICSI 37% vs ICSI 40% vs IVF 39%; p>0.05). For both age groups, no significant differences were observed in PGT outcomes for Day 5 biopsy compared to Day 6 (% euploid embryos: 0.05, and [Formula: see text]38 years old (n=329) Day 5 45% vs Day 6 37%; p>0.05). Conclusion: The study suggests that different fertilisation techniques or the day of biopsy may not influence PGT outcomes.

The purpose of the Beginning Teacher Evaluation Study (BTES) was to identify teaching activities and classroom conditions that foster student learning in elementary schools. The study focused on instruction in reading and mathematics at grades two and five. During the multi-year series of substudies comprising BTES, a variety of issues were addressed, and data from several samples of teachers and students were collected and analyzed. Depending on the question being asked, various data collection techniques were used, including: ethnography, stimulated recall, interviews, teacher and student self-report, objective observation, and testing. As the study progressed, a model of classroom instruction and student learning evolved and provided the conceptual framework that guided the final empirical stage of the study. The development of the model is in itself one of the more important outcomes of the study. The discussion presented in this article will present: (1) a brief description of the model as it applies to the acquisition of reading and mathematics skills in elementary schools; (2) an overview of the methods used in the final field study; (3) the major findings of the study; and (4) some implications of the study for the practice of teaching.

Background Post mortem examination of lung and heart tissue has been vital to developing an understanding of COVID-19 pathophysiology; however studies to date have almost uniformly used tissue obtained from hospital-based deaths where individuals have been exposed to major medical and pharmacological interventions. Methods In this study we investigated patterns of lung and heart injury from 46 community-based, pre-hospital COVID-19-attributable deaths who underwent autopsy. Results The cohort comprised 22 females and 24 males, median age 64 years (range 19–91) at time of death with illness duration range 0–23 days. Comorbidities associated with poor outcomes in COVID-19 included obesity (body mass index >30 kg·m−2) in 19 out of 46 cases (41.3%). Diffuse alveolar damage in its early exudative phase was the most common pattern of lung injury; however significant heterogeneity was identified with bronchopneumonia, pulmonary oedema consistent with acute cardiac failure, pulmonary thromboembolism and microthrombosis also identified and often in overlapping patterns. Review of clinical records and next of kin accounts suggested a combination of unexpectedly low symptom burden, rapidly progressive disease and psychosocial factors may have contributed to a failure of hospital presentation prior to death. Conclusions Identifying such advanced acute lung injury in community-based deaths is extremely unusual and raises the question why some with severe COVID-19 pneumonitis were not hospitalised. Multiple factors including low symptom burden, rapidly progressive disease trajectories and psychosocial factors provide possible explanations.

Persistence of residual disease in acute lymphoblastic leukemia (ALL) during the initial stages of chemotherapy is associated with inferior survival. To better understand clonal evolution and mechanisms of chemoresistance, we used multiparameter mass cytometry, at single-cell resolution, to functionally characterize pediatric B-ALL cells at disease presentation and those persisting during induction therapy. Analysis of ALL cells from presentation samples (n=42) showed that the most abundant phosphosignals were pCREB, pH2AX and pHH3 and we identified JAK-STAT and RAS pathway activation in five of six patients with JAK or RAS genetic aberrations. The clonal composition of ALL was heterogeneous and dynamic during treatment but all viable cell clusters showed pCREB activation. Levels of pCREB in ALL cells were increased or maintained during therapy and high dimensional analysis revealed a subpopulation of ALL cells at presentation that was positive for pCREB/pHH3/pS6 which increased during treatment in some patients, implicating this signaling node in conferring a survival advantage to multi-agent induction therapy. The small molecule CREB inhibitor, 666-15, was shown to reduce CREB transcriptional activity and induce apoptosis in ALL patient-derived xenograft cells of varying cytogenetic subtypes in vitro, both in the presence and absence of stromal support. Together, these data suggest that the cAMP signaling pathway may provide an opportunity for minimal residual disease-directed therapy for many patients at high risk of relapse.

Background: Young adults represent a third of the United Kingdom prison population and are at risk of poor health outcomes, including drug and alcohol misuse, self-harm and suicide. Court diversion interventions aim to reduce the negative consequences of criminal sanctions and address the root causes of offending. However, evidence of their effectiveness has not yet been established. The Gateway programme, issued as a conditional caution, aimed to improve the life chances of young adults committing low-level offences. Participants agreed not to reoffend during the 16-week caution and, following a needs assessment, received individual support from a Gateway navigator and attended two workshops encouraging analysis of own behaviour and its consequences. Objective: To evaluate the effectiveness and cost-effectiveness of Gateway in relation to health and well-being of participants compared to usual process (court summons or a different conditional caution). Design, setting and participants: Pragmatic, multisite, parallel-group, superiority randomised controlled trial with two 6-month internal pilots and a target sample size of 334. Randomisation between Gateway and usual process was on a 1 : 1 basis. Four Hampshire Constabulary sites recruited 18- to 24-year-old residents of Hampshire and Isle of Wight who were questioned for an eligible low-level offence. Semistructured interviews were also held with a sample of Gateway programme participants, staff and police study recruiters. Main outcome measures: Primary outcome was the Warwick-Edinburgh Mental Wellbeing Scale score at 12 months. Secondary outcomes included health status, alcohol and drug use, recidivism and resource use. Results: Recruitment commenced in October 2019 and the trial stopped in April 2021. A total of 191 participants were recruited, with 109 randomised to Gateway and 82 to usual process. Due to an initial overestimation of potentially eligible young people and low retention rates, recruitment targets were adjusted, and a range of mitigating measures introduced. Although recruitment broadly met study progression criteria [35/50 (70%) Pilot 1: 64/74 (86%) Pilot 2], retention was low throughout (overall: data collected at week 4 was 50%: at week 16 it was 50%: 1-year 37%). Low retention was multifactorial, with one of the main barriers being difficulties contacting participants. It was therefore not possible to complete the randomised controlled trial or the health economics analyses. Qualitative interviews held with 58 individuals yielded rare insights into the benefits and limitations of this type of intervention, as well as barriers and facilitators in relation to recruitment in this setting. Limitations: Despite close collaboration with the police to address recruitment and consent issues, expansion of the inclusion criteria and recruitment area and introducing other measures, the researchers were unable to collect sufficient data within an acceptable timeframe. Conclusions: The Gateway study was a unique endeavour to gather evidence for a potentially life-changing intervention for an underserved population. The experience gained indicates that randomised controlled trials of interventions, with a health-related outcome, are possible in this setting but point towards the need for conservative recruitment and retention estimates in this target population. Other study designs should be considered. The qualitative evaluation provided a range of valuable lessons for those seeking to design similar interventions or conduct research in similar settings. Study registration: This study is registered as ISRCTN11888938. Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Public Health Research programme (NIHR award ref: 16/122/20) and is published in full in Public Health Research; Vol. 12, No. 7. See the NIHR Funding and Awards website for further award information. Plain language summary: Young adults who commit low-level offences often have many health and social needs, making them vulnerable to physical and mental health problems. The Gateway programme was a conditional caution developed to address the underlying causes of low-level offending in young people aged 18–24 years and hence improve their life chances. In Gateway, a mentor assessed the young person's needs and supported them, signposting to healthcare, housing or other services as required. The young people also participated in two workshops, analysing the causes and consequences of their behaviour. To find out if Gateway improved health and reoffending rates, a group of those who received a Gateway conditional caution were compared with a group of those receiving a court summons or a different conditional caution. Of the 191 participants recruited to the study, 109 were randomised to Gateway and 82 to the usual process. However, the researchers had significant difficulties getting hold of the study participants on the phone and they were unable to collect enough information from them to be able to say whether Gateway worked. The researchers introduced various changes to overcome this, but in the end had to stop the study early. As part of the study, the researchers interviewed 28 Gateway programme participants, 17 Gateway project staff and 13 police officers and staff who had been recruiting into the study. From the interviews the study discovered the perceived benefits of Gateway, how programmes like this could be improved and which factors helped or got in the way of doing research in the police setting. The Gateway study aimed to provide evidence for a potentially life-changing intervention for vulnerable young adults. Although it proved impossible to complete the study, the lessons learnt from running it should help colleagues design similar programmes or plan research studies with similar populations or in similar settings. Scientific summary: Background: Young adults represent a third of the United Kingdom (UK) prison population and are at risk of poor health outcomes including drug and alcohol misuse, self-harm and suicide. Those aged between 18 and 24, who have been questioned as suspects in relation to a low-level offence, may need to attend court and, if convicted, face penalties such as imprisonment. However, other means aimed at preventing young adults from reoffending exist. Court diversion interventions aim to reduce the negative consequences of some types of criminal sanctions and focus resources on addressing the root causes of offending. Although diversions are widely used in the UK, evidence of their effectiveness in terms of health outcomes has not yet been established using robust research methods. Hampshire Constabulary (HC), working with local charities, developed the Gateway programme, an out-of-court disposal aimed at improving the life chances of young adults. Objectives: The aim of this study was to evaluate the effectiveness and cost-effectiveness of the Gateway programme issued as a conditional caution (intervention) compared to usual process (court appearance or a different conditional caution). The study objectives were to: Examine the effectiveness of the Gateway intervention on: (1) health and well-being including alcohol and substance use, (2) access to and use of health and social services and (3) quality of life, among young adult offenders. Explore the views and experiences of victims. Assess the quantity and quality of the Gateway intervention as delivered in the study and the generalisability of the findings. Identify and measure relevant consequences, both cost and benefits, of the Gateway intervention compared with usual process. Examine the effectiveness of the Gateway intervention on recidivism. Methods: Design: The study undertook a pragmatic, superiority RCT with two 6-month internal pilot phases and qualitative evaluation: an economic evaluation was planned. Participants were randomised using a 1 : 1 allocation ratio to either the Gateway conditional caution (intervention) or disposal as usual to a court summons or a different conditional caution to Gateway (usual process). The qualitative evaluation aimed to capture the experiences and perceptions of the impact of the intervention on participants, the police, victims and those delivering the intervention. Participants, setting and recruitment: Eligible participants were those aged 18–24 who had committed low-level offences and resided within Hampshire and Isle of Wight (IoW) area. Participants were recruited by Police investigators during processing for an offence. Potential participants were offered a chance by the police to receive the Gateway caution, and those interested were invited to take part in the study. Police officers obtained Stage 1 consent and carried out an eligibility check, after which those eligible were automatically randomised to receive either a Gateway caution or follow the usual process, such as court appearance or a different conditional caution. Qualitative interviews were undertaken with trial participants, police officers and those delivering the intervention. Sample size: There is no widely accepted and established minimal clinically significant difference for the primary outcome, Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS). A change of three or more points is likely to be important to individuals. There is also variation in the standard deviation (SD) of the WEMWBS with estimates ranging from 6 to 10.8 with the pooled estimate of 10 across all studies. Assuming 90% power, 5% 2-sided statistical significance, mean difference of 4 points on WEMWBS and a SD of 10, 266 participants were required. Conservatively, assuming a 20% attrition rate, the study aimed to recruit and randomise 334 participants. Interventions: The Gateway programme, issued as a conditional caution, required participants to undertake a health and social care needs assessment, attend workshops encouraging analysis of their behaviour and its consequences, and agree not to reoffend during the 16-week caution. Usual process was disposal to a court summons or an alternative conditional caution. Outcomes: The primary outcome measure was the WEMWBS. Participants self-reported WEMWBS at 4-weeks, 16-weeks and 1-year post-randomisation. Secondary outcomes were health status [Short Form 12 questionnaire (SF-12)]; alcohol [Alcohol Use Disorders Identification Test (AUDIT)] and drug [Adolescent Drug Involvement Scale (ADIS)] use; type and frequency of reoffending (police data); and health and social care resource use (self-reported). Statistical methods: The original plan, pre-specified in version 1.0 of the SAP , was to carry out a repeated measures, mixed-effects linear regression model with the WEMWBS score at 4-weeks, 16-weeks and 1-year post-randomisation as the dependent variable, adjusting for treatment group, time, group by time interaction, total number of records management system incidents and police national computer convictions 1-year prerandomisation, age at randomisation, Index of Multiple Deprivation quintile at randomisation, pandemic time period and standard of usual process available as fixed effects. Recruiting site was to be adjusted for as a random effect. However, due to the study closing early because of issues with retaining participants, a descriptive analysis was undertaken with no formal hypothesis testing. All outcomes were summarised descriptively by a randomised treatment group. Qualitative evaluation: A qualitative evaluation was conducted to assess the implementation of the Gateway programme, including any related issues and observed benefits to the clients. Focusing on implementation, mechanisms and context, the research questions were: How is Gateway being implemented? What are the barriers to the implementation and effects of the Gateway programme? What are the mechanisms through which the intervention brings about change? How do different delivery methods (face-to-face/virtual/telephone) influence the above questions? We conducted qualitative interviews and focus groups with a range of stakeholders across three time periods during the implementation of the Gateway programme. Economic evaluation: A formal health economic analysis was not feasible. Health economic data are summarised descriptively with the trial data. Results: Randomised controlled trial: We recruited 191 participants; 109 were randomised to Gateway and 82 to usual process. Although recruitment rates were within acceptable limits, the number of participants providing data [94 (32%) at week 4; 95 (34%) at week 16; 43 (28%) at 1-year] was insufficient to undertake any formal hypothesis testing and the trial was closed early. The groups were generally well balanced in terms of characteristics and percentage providing data, but more of those providing valid data had a previous conviction than those who did not provide data. Similar percentages from each arm provided data with those attending interviews completing all sections. Telephone interviews were acceptable to those willing to share an active telephone number. Rates for those who were non-contactable were similar between the groups at all three time points. Eighty-one of the 101 allocated to Gateway complied with the intervention. Reasons for non-compliance were reoffending and non-attendance at the LINX workshops. Qualitative evaluation: Across 3 time points, 69 in-depth interviews were conducted with: 28 young people, 25 Gateway staff, 13 police recruiters and 3 focus groups with navigators. The researchers were unable to pursue interviews with victims as there were few offences with a victim. Our findings showed that, following engagement with the Gateway programme, young people reported being better able to make decisions after engagement with the LINX workshops, while navigators played a significant role in enabling compliance and change among young people. The role of the navigator was akin to that of the mentor, providing practical support towards improved health including, for example, making and attending doctors' appointments with clients, as well as offering a listening role. Young people felt that, for them, the wider determinants (or 'areas') addressed with navigators, such as access to employment or improved health, were of greater importance than a reduction in reoffending. There was a polarity of needs among young people, which meant that all stakeholders valued the ability to tailor and adapt the programme to individual client's needs, also giving clients a sense of agency and control over their lives. The independence of the Gateway intervention team, from the police, was highly valued by young people. Factors related to communication were a concern for all, particular at the point of recruitment (by police) and between multiple delivery agencies. Discussion: The problems encountered throughout this trial and the researchers' endeavours to overcome these problems provide valuable insights for colleagues seeking to design similar interventions and/or conduct studies with vulnerable populations in the police setting. Co-production is essential for studies in the police setting. By working in close collaboration with HC , and their two project dedicated officers, the researchers were able to make pragmatic adjustments to the study design as issues arose. However, training an entire police force is fraught with difficulty even when supported by senior officers; competing interests precluded mandatory training for research purposes. The use of a two-stage consent process and a web-based eligibility and randomisation tool facilitate recruitment in the police setting, but frequent, regular need to use is required to maintain the study profile. Young people who have committed an offence are known to be a difficult group to engage with generally, let alone in research. The study identified and tested implementation of different approaches to overcome this problem. Switching to telephone interviews produced a positive response. Persistence and engagement paid off, but the study was unable to solve the problem of inactive mobile telephone numbers; an issue shared by the navigators and police. Independence of those delivering the intervention is important as perceived links with the police caused some disengagement. The collected data provide valuable information on attrition rates for health studies. Interestingly, allocation did not appear to make any difference to participation. The study's qualitative evaluation highlighted the unmet health needs for this group of 18- to 24-year-olds, and the need to address the wider determinants of reoffending through individualised assessment. The Gateway programme was, however, developed for those with higher needs, which meant that flexibility and adaptability to suit individual needs was essential. It further highlighted the invaluable role of mentors in rehabilitative programmes such as Gateway. The study has demonstrated that it is possible to recruit and randomise to a RCT in the police setting. The data that the study presents should be used to inform the planning of future trials, including anticipated attrition rates, and setting conservative targets for retention as well as recruitment rates. Internal pilots should be long enough to confirm recruitment and data collection rates are achievable over an adequate follow-up period. Given the challenges encountered, alternative study designs should be considered for the evaluation of interventions with a health-related outcome. These include: cluster RCTs where processes at individual cluster sites could be simplified; post hoc cohort studies which may address non-response and attrition bias; and regression discontinuity design (RDD), a quasi-experimental approach at lower risk of bias which has the potential to equate to a RCT. Conclusions: This ambitious RCT and qualitative study provides information about an out-of-court intervention aimed at improving life chances, health and well-being and recidivism in young adults. Challenges encountered in participation and retention in this setting and the ways in which these may be addressed are described and will be useful in planning future research. Study registration: This study is registered as ISRCTN11888938. Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Public Health Research programme (NIHR award ref: 16/122/20) and is published in full in Public Health Research; Vol. 12, No. 7. See the NIHR Funding and Awards website for further award information. [ABSTRACT FROM AUTHOR]