Experimental investigation of the control and function of the muscle segment homeobox genes during vertebrate embryological development

The control and function of the msh-class (muscle segment homeobox) genes, Msxl and Msx2, was investigated during vertebrate embryonic development. These genes are widely expressed in the embryo, often in structures which are elaborations on the presumed ancestral chordate body-plan. It is thought that duplication events of a single ancestral msh gene, and subsequent divergence early in the vertebrate line, may have given the genes a special role in development of vertebrate-specific structures.The limb was chosen as a model developmental system in which to analyse patterns of Msx gene expression by whole -mount in situ hybridisation in mouse and chicken embryos, to relate these patterns to developmental processes known to be occurring and hence to elucidate potential roles for the genes. Expression of Msx genes in a number of areas of cell death, proliferation and differentiation suggested that they are involved in a wide range of developmental decisions in several tissues, although their molecular role is still uncertain. The potential for functional redundancy between the genes was addressed. There are distinct differences between the expression patterns of Msxl and Msx2 during limb development; these differences are largely quantitative, however, and the broad similarities between the genes suggest that a large degree of functional redundancy may be possible.At all stages of limb development the expression of the Msx genes and the reported expression of bone morphogenetic protein -4 (BMP4) coincide. By implanting beads, soaked in BMP4, into the chick wing in ovo, I showed that BMP4 is an upstream activator of Msxl and Msx2 in the limb. This made it possible to suggest elements of the genetic pathways which may be involved in epithelial-mesenchymal interactions at disparate sites around the body, and to suggest a scenario for the evolution of Msx gene expression patterns.The control of Msxl was further investigated using two reporter genes which express 13- Gal/Msxl chimaeric protein under control of Msxl regulatory sequences. Both reporters transcribe lacZ from 4.7kb of Msxl 5' promoter, but one also contains 7kb of .Msxl sequence 3' of lacZ, including the intron, 3'UTR and 3' genomic sequence.An assay was developed, by means of which the reporter constructs could be introduced into cells in culture by microinjection, then grafted into the developing chicken limb in ovo, using X -Gal staining to visualise expression of the gene. The primary aim was to find sequences in the promoter of Msxl which are responsible for induction of Msxl expression in response to the signal from the apical ectodermal ridge (AER) of the limb.1OT' /2 cells were injected with these constructs; the shorter construct, pH7lacA3', was expressed constitutively, whereas the longer one, pH7lacT, was infrequently expressed, showing that there are elements 3' of the transcription start site which had a negative regulatory effect on the expression of lacZ. The experiments demonstrated that the 4.7kb is almost certainly not the whole of the Msxl promoter; in grafted cells, expression of pH7lacT was not induced in response to the AER. The failure of the AER response could have been due to deficiencies within the promoter or to a failure of the signal transduction pathway within the cells. Radioactive in situ hybridisation showed endogenous Msxl and Msx2 were not induced in 10TV, cells grafted underneath the AER. However, inability to express endogenous Msxl need not preclude expression of the introduced promoter, so further work was necessary to determine whether the reporter is capable of responding to the AER.The behaviour of the 4.7kb Msxl /lacZ reporter gene was therefore further investigated in embryonic tissue from several lines of transgenic mouse which contained the gene. Analysing expression of the transgene in these mice confirmed that the 4.7kb was not the whole of the promoter, although it could reproducibly drive an expression pattern similar to elements of expression of Msxl. Grafting transgenic limb mesenchyme under the AER of chicken limbs did not induce lacZ, providing further evidence that the AER -response element was not in the 4.7kb.The transgene was strongly expressed in the cells of the ventrolateral dermamyotome of the somites which are fated to form limb muscles. Expression of Pax3 was previously the only known molecular marker for these cells. Expression of the transgene is retained in a subset of the developing muscles in the anterior of the limb. Hence the transgene provides a molecular marker for these differentiating myoblasts which can be used in experimental situations, and which I used to show that mouse myoblasts can contribute to chicken muscle.BMP4 could neither induce nor maintain expression of the transgene in cultured limb mesenchyme. The missing AER/BMP4 response element(s) may be one reason why expression of the transgene differs from that of the endogenous Msxl, but it is not the only reason. Although transgenic mouse limb tissue is initially responsive to position -specific signals within the chicken which can maintain its expression, it later becomes refractory to induction by these same signals (at stages when the endogenous gene is still responsive), and non -expressing transgenic mesenchyme does not reinitiate expression when grafted into appropriate areas of the chicken or the mouse.This has the characteristics of an epigenetic silencing effect; it raises the possibility that chromosomal location is important even for regulation of non -clustered homeobox- containing genes, but again suggests that sequences missing from the 4.7kb allow the promoter to fall under the control of repressor proteins which do not affect the endogenous gene

Requirement of Pax6 for the integration of guidance cues in cell migration

Data accessibility. Cell trajectories data and a summary of directedness and angle values are deposited at Dryad: http://dx.doi.org/10.5061/dryad.53512. Funding MA was funded by an Alban International Research Studentship (code: E07D400602UY).Peer reviewedPublisher PD

Widespread tissue hypoxia dysregulates cell and metabolic pathways in SMA

Open Access via the Wiley Jisc Agreement Acknowledgments: SHP, EH‐G, INF, SD’A, and JMC were funded by SMA Europe (SMA UK and Prinses Beatrix Spierfonds). Thanks to Prof Andy Welch for helpful discussions on imaging.Peer reviewedPublisher PD

Chemical characteristics of macroscopic pyrogenic carbon following millennial-scale environmental exposure

Pyrogenic Carbon (PyC) is ubiquitous in global environments, and is now known to form a significant, and dynamic component of the global carbon cycle, with at least some forms of PyC persisting in their depositional environment for many millennia. Despite this, the factors that determine the turnover of PyC remain poorly understood, as do the physical and chemical changes that this material undergoes when exposed to the environment over tens of thousands of years. Here, we present the results of an investigation to address these knowledge gaps through chemical and physical analysis of a suite of wood PyC samples exposed to the environment for varying time periods, to a maximum of >90,000 years. This includes an assessment of the quantity of resistant carbon, known as Stable Polyaromatic Carbon (SPAC) versus more chemically labile carbon in the samples. We find that, although production temperature is likely to determine the initial ‘degradation potential’ of PyC, an extended exposure to environmental conditions does not necessarily mean that remaining PyC always progresses to a ‘SPAC-dominant’ state. Instead, some ancient PyC can be composed largely of chemical components typically thought of as environmentally labile, and it is likely that the depositional environment drives the trajectory of preservation versus loss of PyC over time. This has important implications for the size of global PyC stocks, which may have been underestimated, and also for the potential loss of previously stored PyC, when its depositional environment alters through environmental or climatic changes

Jography: Exploring meanings, experiences and spatialities of recreational road-running

Jogging is a relatively under-researched mobile practice with much existing literature focusing on ‘serious’ and competitive running. In this paper, we provide an account of some of the movements, meanings and experiences that together help produce the practice of jogging in the south-western English city of Plymouth. Drawing upon participant diaries and interviews, we uncover rich detail about how joggers ascribe not one but a number of meanings to their practice. Some of these are positive, some are negative; some complement each other and some compete with each other. We also consider how the experiences of joggers can be shaped by their ongoing need to develop tactics capable of enabling them to negotiate space with non-joggers. This is in some contrast to more competitive running that occurs in the separated space of an athletics track. Our sense is that better awareness of the meanings and experiences of jogging will be of value if the advertised health and sustainability benefits of the practice are to be more effectively encouraged and promoted

Enhanced feedback interventions to promote evidence-based blood transfusion guidance and reduce unnecessary use of blood components:The AFFINITIE research programme including two cluster factorial RCTs

Background: Blood transfusion is a common but costly treatment. Repeated national audits in the UK suggest that up to one-fifth of transfusions are unnecessary when judged against recommendations for good clinical practice. Audit and feedback seeks to improve patient care and outcomes by comparing clinical care against explicit standards. It is widely used internationally in quality improvement. Audit and feedback generally has modest but variable effects on patient care. A considerable scope exists to improve the impact that audit and feedback has, particularly through head-to-head trials comparing different ways of delivering feedback. Objectives: The AFFINITIE (Development & Evaluation of Audit and Feedback INterventions to Increase evidence-based Transfusion practIcE) programme aimed to design and evaluate enhanced feedback interventions, within a national blood transfusion audit programme, to promote evidence-based guidance and reduce the unnecessary use of blood components. We developed, piloted and refined two feedback interventions, ‘enhanced content’ and ‘enhanced follow-on’ (workstream 1), evaluated the effectiveness and cost-effectiveness of the two feedback interventions compared with standard feedback practice (workstream 2), examined intervention fidelity and contextual influences (workstream 3) and developed general implementation recommendations and tools for other audit and feedback programmes (workstream 4). Design: Interviews, observations and documentary analysis in four purposively sampled hospitals explored contemporary practice and opportunities for strengthening feedback. We developed two interventions: ‘enhanced content’, to improve the clarity and utility of feedback reports, and ‘enhanced follow-on’, to help hospital staff with action-planning (workstream 1). We conducted two linked 2 × 2 factorial cross-sectional cluster-randomised trials within transfusion audits for major surgery and haematological oncology, respectively (workstream 2). We randomised hospital clusters (the organisational level at which hospital transfusion teams operate) to enhanced or standard content or enhanced or standard follow-on. Outcome assessment was masked to assignment. Decision-analytic modelling evaluated the costs, benefits and cost-effectiveness of the feedback interventions in both trials from the perspective of the NHS. A parallel process evaluation used semistructured interviews, documentary analyses and web analytics to assess the fidelity of delivery, receipt and enactment and to identify contextual influences (workstream 3). We explored ways of improving the impact of national audits with their representatives (workstream 4). Setting and participants: All NHS hospital trusts and health boards participating in the National Comparative Audit of Blood Transfusions were invited to take part. Among 189 hospital trusts and health boards screened, 152 hospital clusters participated in the surgical audit. Among 187 hospital trusts and health boards screened, 141 hospital clusters participated in the haematology audit. Interventions: ‘Enhanced content’ aimed to ensure that the content and format of feedback reports were consistent with behaviour change theory and evidence. ‘Enhanced follow-on’ comprised a web-based toolkit and telephone support to facilitate local dissemination, planning and response to feedback. Main outcome measures: Proportions of acceptable transfusions, based on existing evidence and guidance and algorithmically derived from national audit data. Data sources: Trial primary outcomes were derived from manually collected, patient-level audit data. Secondary outcomes included routinely collected data for blood transfusion. Results: With regard to the transfusions in the major surgery audit, 135 (89%) hospital clusters participated from 152 invited. We randomised 69 and 66 clusters to enhanced and standard content, respectively, and 68 and 67 clusters to enhanced and standard follow-on, respectively. We analysed a total of 2222 patient outcomes at 12 months in 54 and 58 (enhanced and standard content, respectively) and 54 and 58 (enhanced and standard follow-on, respectively) hospital clusters. With regard to the haematology audit, 134 hospital clusters (95%) participated from 141 invited. We randomised 66 and 68 clusters to enhanced and standard content, respectively, and 67 clusters to both enhanced and standard follow-on. We analysed a total of 3859 patient outcomes at 12 months in 61 and 61 (enhanced and standard content, respectively) and 63 and 59 (enhanced and standard follow-on) hospital clusters. We found no effect of either of the enhanced feedback interventions in either trial across all outcomes. Incremental enhanced intervention costs ranged from £18 to £248 per site. The enhanced feedback interventions were dominated by the standard intervention in cost-effectiveness analyses. The interventions were delivered as designed and intended, but subsequent local engagement was low. Although the enhancements were generally acceptable, doubts about the credibility of the blood transfusion audits undermined the case for change. Limitations: Limitations included the number of participating clusters; loss to follow-up of trial clusters, reducing statistical power and validity; incomplete audit and cost data contributing to outcome measures; participant self-selection; reporting; missing data related to additional staff activity generated in response to receiving feedback; and recall biases in the process evaluation interviews. Conclusions: The enhanced feedback interventions were acceptable to recipients but were more costly and no more effective than standard feedback in reducing unnecessary use of blood components, and, therefore, should not be recommended on economic grounds. Future work: We have demonstrated the feasibility of embedding ambitious large-scale rigorous research within national audit programmes. Further head-to-head comparisons of different feedback interventions are needed in these programmes to identify cost-effective ways of increasing the impact of the interventions

Polygenic in vivo validation of cancer mutations using transposons

The in vivo validation of cancer mutations and genes identified in cancer genomics is resource-intensive because of the low throughput of animal experiments. We describe a mouse model that allows multiple cancer mutations to be validated in each animal line. Animal lines are generated with multiple candidate cancer mutations using transposons. The candidate cancer genes are tagged and randomly expressed in somatic cells, allowing easy identification of the cancer genes involved in the generated tumours. This system presents a useful, generalised and efficient means for animal validation of cancer genes.Link_to_subscribed_fulltex

Integration of robotic surgery into routine practice and impacts on communication, collaboration, and decision making: A realist process evaluation protocol

Background: Robotic surgery offers many potential benefits for patients. While an increasing number of healthcare providers are purchasing surgical robots, there are reports that the technology is failing to be introduced into routine practice. Additionally, in robotic surgery, the surgeon is physically separated from the patient and the rest of the team, with the potential to negatively impact teamwork in the operating theatre. The aim of this study is to ascertain: how and under what circumstances robotic surgery is effectively introduced into routine practice; and how and under what circumstances robotic surgery impacts teamwork, communication and decision making, and subsequent patient outcomes. Methods and design: We will undertake a process evaluation alongside a randomised controlled trial comparing laparoscopic and robotic surgery for the curative treatment of rectal cancer. Realist evaluation provides an overall framework for the study. The study will be in three phases. In Phase I, grey literature will be reviewed to identify stakeholders' theories concerning how robotic surgery becomes embedded into surgical practice and its impacts. These theories will be refined and added to through interviews conducted across English hospitals that are using robotic surgery for rectal cancer resection with staff at different levels of the organisation, along with a review of documentation associated with the introduction of robotic surgery. In Phase II, a multi-site case study will be conducted across four English hospitals to test and refine the candidate theories. Data will be collected using multiple methods: the structured observation tool OTAS (Observational Teamwork Assessment for Surgery); video recordings of operations; ethnographic observation; and interviews. In Phase III, interviews will be conducted at the four case sites with staff representing a range of surgical disciplines, to assess the extent to which the results of Phase II are generalisable and to refine the resulting theories to reflect the experience of a broader range of surgical disciplines. The study will provide (i) guidance to healthcare organisations on factors likely to facilitate successful implementation and integration of robotic surgery, and (ii) guidance on how to ensure effective communication and teamwork when undertaking robotic surgery

A 'synthetic-sickness' screen for senescence re-engagement targets in mutant cancer backgrounds.

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments