The use of decellularised animal tissue to study disseminating cancer cells

Since the establishment of cell culture, common practice has been to grow adherent cells in 2D monolayers. Although cells behave completely differently when grown under these artificial conditions, the ease of 2D culturing has meant that this practice still prevails, and adopting conditions that more closely reflect the natural microenvironment has been met with substantial inertia. The alternative, animal models that mimic natural human physiology, are less accessible, strictly regulated and require licences and expensive facilities. Although transition from 2D to 3D cell culturing is gathering momentum, there is a clear need for alternative culturing methods that more closely resemble in vivo conditions. Here, we show that decellularised organs gleaned from discarded animal carcasses are ideal biomimetic scaffolds to support secondary tumour initiation in vitro. Further, we describe how to decellularise tissue and perform basic histochemistry and immunofluorescence procedures for cell and matrix detection. Cancer cell behaviour on this matrix is followed by way of an example. Because integration into the traditional work flow is easy and inexpensive, we hope this article will encourage other researchers to adopt this approach

Contrasting Male and Female Dietary Life Histories: A Case Study at an Ancestral Muwekma Ohlone Heritage Site in San Jose, California

Stable carbon and nitrogen isotope analyses of bones and teeth at the ancestral heritage Muwekma Ohlone site of Yakmuy ‘Ooyákma-tka (“Place of the East Ridge Site”; CA-SCL-215) reveal significant differences in the dietary life history of males and females. Overall, isotope data indicate that site inhabitants were primarily dependent on low trophic-level foods, likely plants, and minor amounts of marine food for their main source of dietary protein. From tooth dentin serial samples, we found that males were elevated by 0.6-1.0‰ in δ15N in early childhood (age 1-9 years) relative to females, while δ13C values were similar by sex, indicating boys were accessing slightly greater amounts of higher trophic-level foods, such as meat from game. The sex-biased difference in δ15N diminishes during the second decade of life, as female δ15N values increase and become equal to males. However, a difference in δ13C emerges during the second decade where female δ13C values are elevated relative to males. This could indicate that teenage females consumed higher amounts of low trophic-level, marine-derived protein, such as shellfish. During later adult years, the difference in δ13C disappears, while males again show an increase in δ15N relative to females. Although these differences are small, they reveal important sex-biased life history patterns during childhood and adulthood in this ancient community

Automated pancreatic islet viability assessment for transplantation using bright-field deep morphological signature

Islets transplanted for type-1 diabetes have their viability reduced by warm ischemia, dimethyloxalylglycine (DMOG; hypoxia model), oxidative stress and cytokine injury. This results in frequent transplant failures and the major burden of patients having to undergo multiple rounds of treatment for insulin independence. Presently there is no reliable measure to assess islet preparation viability prior to clinical transplantation. We investigated deep morphological signatures (DMS) for detecting the exposure of islets to viability compromising insults from brightfield images. Accuracies ranged from 98 % to 68 % for; ROS damage, pro-inflammatory cytokines, warm ischemia and DMOG. When islets were disaggregated to single cells to enable higher throughput data collection, good accuracy was still obtained (83-71 %). Encapsulation of islets reduced accuracy for cytokine exposure, but it was still high (78 %). Unsupervised modelling of the DMS for islet preparations transplanted into a syngeneic mouse model was able to predict whether or not they would restore glucose control with 100 % accuracy. Our strategy for constructing DMS' is effective for the assessment of islet pre-transplant viability. If translated into the clinic, standard equipment could be used to prospectively identify non-functional islet preparations unable to contribute to the restoration of glucose control and reduce the burden of unsuccessful treatments.Abbas Habibalahi, Jared M. Campbell, Stacey N. Walters, Saabah B. Mahbub, Ayad G. Anwer, Shane T. Grey, Ewa M. Goldy

Process evaluation protocol for a cluster randomised controlled trial of an intervention to improve the mental health support and training available to secondary school teachers– the Wellbeing in Secondary Education (WISE) project

Background: Secondary school teachers have low levels of wellbeing and high levels of depression compared with the general population. Teachers are in a key position to support students, but poor mental health may be a barrier to doing so effectively. The Wellbeing in Secondary Education (WISE) project is a cluster randomised controlled trial (RCT) of an intervention to improve the mental health support and training available to secondary school teachers through delivery of the training package Mental Health First Aid and a staff peer support service. We will conduct a process evaluation as part of the WISE trial to support the interpretation of trial outcomes and refine intervention theory. The domains assessed will be: the extent to which the hypothesised mechanisms of change are activated; system level influences on these mechanisms; programme differentiation and usual practice; intervention implementation, including any adaptations; intervention acceptability; and intervention sustainability. Methods: Research questions will be addressed via quantitative and qualitative methods. All study schools (n = 25) will provide process evaluation data, with more detailed focus group, interview and observation data being collected from a subsample of case study schools (4 intervention and 4 control). Mechanisms of change, as outlined in a logic model, will be measured via teacher and student surveys and focus groups. School context will be explored via audits of school practice that relate to mental health and wellbeing, combined with stakeholder interviews and focus groups. Implementation of the training and peer support service will be assessed via training observations, training participant evaluation forms, focus groups with participants, interviews with trainers and peer support service users, and peer supporter logs recording help provided. Acceptability and sustainability will be examined via interviews with funders, head teachers, trainers and peer support services users, and focus groups with training participants. Discussion: The process evaluation embedded within the WISE cluster RCT will illuminate how and why the intervention was effective, ineffective or conferred iatrogenic effects. It will contribute to the refinement of the theory underpinning the intervention, and will help to inform any future implementation. Trial registration: International Standard Randomised Controlled Trial Number: ISRCTN95909211 registered on 24 March 2016. Keywords: Mental health, Wellbeing, Schools, Children, Adolescents, Teachers, Process evaluation, Cluster Randomised Controlled Tria

A cluster randomised controlled trial of the Wellbeing in Secondary Education (WISE) Project – an intervention to improve the mental health support and training available to secondary school teachers: protocol for an integrated process evaluation

This is the author accepted manuscript. The final version is available from BioMed Central via the DOI in this record.Background Secondary school teachers have low levels of wellbeing and high levels of depression compared with the general population. Teachers are in a key position to support students, but poor mental health may be a barrier to doing so effectively. The Wellbeing in Secondary Education (WISE) project is a cluster randomised controlled trial (RCT) of an intervention to improve the mental health support and training available to secondary school teachers through delivery of the training package Mental Health First Aid and a staff peer support service. We will conduct a process evaluation as part of the WISE trial to support the interpretation of trial outcomes and refine intervention theory. The domains assessed will be: the extent to which the hypothesised mechanisms of change are activated; system level influences on these mechanisms; programme differentiation and usual practice; intervention implementation, including any adaptations; intervention acceptability; and intervention sustainability. Methods Research questions will be addressed via quantitative and qualitative methods. All study schools (n = 25) will provide process evaluation data, with more detailed focus group, interview and observation data being collected from a subsample of case study schools (4 intervention and 4 control). Mechanisms of change, as outlined in a logic model, will be measured via teacher and student surveys and focus groups. School context will be explored via audits of school practice that relate to mental health and wellbeing, combined with stakeholder interviews and focus groups. Implementation of the training and peer support service will be assessed via training observations, training participant evaluation forms, focus groups with participants, interviews with trainers and peer support service users, and peer supporter logs recording help provided. Acceptability and sustainability will be examined via interviews with funders, head teachers, trainers and peer support services users, and focus groups with training participants. Discussion The process evaluation embedded within the WISE cluster RCT will illuminate how and why the intervention was effective, ineffective or conferred iatrogenic effects. It will contribute to the refinement of the theory underpinning the intervention, and will help to inform any future implementation. Trial registration International Standard Randomised Controlled Trial Number: ISRCTN95909211 registered on 24 March 2016.The work was undertaken with the support of The Centre for the Development and Evaluation of Complex Interventions for Public Health Improvement (DECIPHer), a UKCRC Public Health Research Centre of Excellence. Joint funding (MR/KO232331/1) from the British Heart Foundation, Cancer Research UK, Economic and Social Research Council, Medical Research Council, the Welsh Government and the Wellcome Trust, under the auspices of the UK Clinical Research Collaboration, is gratefully acknowledged. The authors acknowledge the contribution of the WISE Study research administrators Amy Bond and Odell Harris

Pancreatic Islet Viability Assessment Using Hyperspectral Imaging of Autofluorescence

Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising intrinsic and extrinsic factors that contribute to early graft failure, necessitating repeated islet infusions for clinical insulin independence. A lack of reliable pre-transplant measures to determine islet viability severely limits the success of islet transplantation and will limit future beta cell replacement strategies. We applied hyperspectral fluorescent microscopy to determine whether we could non-invasively detect islet damage induced by oxidative stress, hypoxia, cytokine injury, and warm ischaemia, and so predict transplant outcomes in a mouse model. In assessing islet spectral signals for NAD(P)H, flavins, collagen-I, and cytochrome-C in intact islets, we distinguished islets compromised by oxidative stress (ROS) (AUC = 1.00), hypoxia (AUC = 0.69), cytokine exposure (AUC = 0.94), and warm ischaemia (AUC = 0.94) compared to islets harvested from pristine anaesthetised heart-beating mouse donors. Significantly, with unsupervised assessment we defined an autofluorescent score for ischaemic islets that accurately predicted the restoration of glucose control in diabetic recipients following transplantation. Similar results were obtained for islet single cell suspensions, suggesting translational utility in the context of emerging beta cell replacement strategies. These data show that the pre-transplant hyperspectral imaging of islet autofluorescence has promise for predicting islet viability and transplant success.Jared M. Campbell, Stacey N. Walters, Abbas Habibalahi, Saabah B. Mahbub, Ayad G. Anwer, Shannon Handley, Shane T. Grey, and Ewa M. Goldy

Protocol for a cluster randomised controlled trial of an intervention to improve the mental health support and training available to secondary school teachers – the WISE (Wellbeing in Secondary Education) study

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Teachers are reported to be at increased risk of common mental health disorders compared to other occupations. Failure to support teachers adequately may lead to serious long-term mental disorders, poor performance at work (presenteeism), sickness absence and health-related exit from the profession. It also jeopardises student mental health, as distressed staff struggle to develop supportive relationships with students, and such relationships are protective against student depression. A number of school-based trials have attempted to improve student mental health, but these have mostly focused on classroom based approaches and have failed to establish effectiveness. Only a few studies have introduced training for teachers in supporting students, and none to date have included a focus on improving teacher mental health. This paper sets out the protocol (version 4.4 20/07/16) for a study aiming to address this gap. METHODS: Cluster randomised controlled trial with secondary schools as the unit of randomisation. Intervention schools will receive: i) Mental Health First Aid (MHFA) training for a group of staff nominated by their colleagues, after which they will set up a confidential peer support service for colleagues ii) training in MHFA for schools and colleges for a further group of teachers, which will equip them to more effectively support student mental health iii) a short mental health awareness raising session and promotion of the peer support service for all teachers. Comparison schools will continue with usual practice. The primary outcome is teacher wellbeing measured using the Warwick Edinburgh Mental Wellbeing Scale (WEMWBS). Secondary outcomes are teacher depression, absence and presenteeism, and student wellbeing, mental health difficulties, attendance and attainment. Measures will be taken at baseline, one year follow up (teachers only) and two year follow up. Economic and process evaluations will be embedded within the study. DISCUSSION: This study will establish the effectiveness and cost-effectiveness of an intervention that supports secondary school teachers’ wellbeing and mental health, and improves their skills in supporting students. It will also provide information regarding intervention implementation and sustainability.This research study is funded by the National Institute for Health Research Public Health Research (NIHR PHR) Programme (project number 13/164/06). The views and opinions expressed in the paper are those of the authors and do not necessarily reflect those of the NIHR PHR Programme or the Department of Health. The intervention is jointly funded by Public Health Wales, Public Health England and Bristol City Council. The pilot study that led to this RCT was funded by the National Institute for Health Research’s School for Public Health Research (NIHR SPHR)

Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments