Following the West Africa Ebola virus disease (EVD) outbreak (2013–2016), WHO developed a preparedness checklist for its member states. This checklist is currently being applied for the first time on a large and systematic scale to prepare for the cross border importation of the ongoing EVD outbreak in the Democratic Republic of Congo hence the need to document the lessons learnt from this experience. This is more pertinent considering the complex humanitarian context and weak health system under which some of the countries such as the Republic of South Sudan are implementing their EVD preparedness interventions.We identified four main lessons from the ongoing EVD preparedness efforts in the Republic South Sudan. First, EVD preparedness is possible in complex humanitarian settings such as the Republic of South Sudan by using a longer-term health system strengthening approach. Second, the Republic of South Sudan is at risk of both domestic and cross border transmission of EVD and several other infectious disease outbreaks hence the need for an integrated and sustainable approach to outbreak preparedness in the country. Third, a phased and well-prioritized approach is required for EVD preparedness in complex humanitarian settings given the costs associated with preparedness and the difficulties in the accurate prediction of outbreaks in such settings. Fourth, EVD preparedness in complex humanitarian settings is a massive undertaking that requires effective and decentralized coordination.Despite a very challenging context, the Republic of South Sudan made significant progress in its EVD preparedness drive demonstrating that it is possible to rapidly scale up preparedness efforts in complex humanitarian contexts if appropriate and context-specific approaches are used. Further research, systematic reviews and evaluation of the ongoing preparedness efforts are required to ensure comprehensive documentation and application of the lessons learnt for future EVD outbreak preparedness and response efforts. The highly contagious and lethal nature of the Ebola virus disease (EVD) coupled with the negative impact that outbreaks have on the health system, social, cultural and economic development of affected communities ranks the disease as one of the most complex, dreaded and dramatic public health phenomena in recent time. Outbreaks of the disease have become more intense in Africa in terms of frequency, magnitude, duration and impact . As of 1The Republic of South Sudan (RSS) is considered to be at high risk of potential cross border importation from the current outbreak due to its proximity to the epicentres of the outbreak in North Kivu and Ituri provinces . The MinThe importance of instituting effective EVD outbreak preparedness measures for timely detection and containment of outbreaks cannot be overemphasized. The highly effective EVD alert system and preparedness measures instituted in one of the high-risk countries, Uganda ensured that it was able to timely detect and contain two cross border transmissions of the disease which were reported on 11 June and 29 August 2019 , 9. ThisThis article highlights the progress made so far in the EVD preparedness efforts in RSS, the associated challenges, describes the key lessons learnt and proposes recommendations for using these to improve preparedness for the current and future outbreaks in the country and other complex humanitarian settings.RSS is the World’s newest nation, emerging from an almost 50-year-long civil war with its northern neighbour to attain independence in 2011. Unfortunately, the new country soon plunged into a civil war in December 2013 which resulted in the internal and external displacement of over four million South Sudanese thus triggering a major humanitarian crisis. The renewed armed conflict in the country disrupted an already fragile healthcare system culminating in a severe shortage of healthcare workers, a disrupted supply management system for essential medicines and medical supplies, inadequate health financing, weak health governance and oversight system. These continue to constrain access to good quality, sustainable and affordable healthcare services in the country.The health indicators in the country are poor; out of pocket spending on health is 54% while life expectancy at birth is 59 years . The matFollowing the designation of the country as high risk for cross border EVD transmission in August 2018, the Ministry of Health and its partners instituted several EVD preparedness interventions. These include the designation of seven frontline states  as high-risk, the establishment of a national EVD Incident Management System which comprise national and state coordination task forces, establishment of EVD surveillance at major points of entry, communities and health facilities in the high-risk states, activation and training of rapid response teams, establishment of laboratory capacity for diagnosis of EVD at the National Public Health Laboratory (NPHL), risk communication and preventive vaccination of 2974 frontline workers in the high-risk states.Two external joint monitoring missions were conducted in November 2018 and March 2019 to monitor the country’s preparedness for EVD; the results showed improvement in the level of EVD preparedness in the country from 17% in November 2018 to 61% in March 2019 .Despite these achievements, several challenges which continue to constrain timely implementation of EVD preparedness activities persist in the country. Table Against the backdrop of the complex humanitarian setting, achievements and challenges highlighted in Table The appreciable improvements which were observed in the preparedness level for EVD in RSS despite a challenging environment have shown that effective EVD outbreak preparedness is feasible in complex humanitarian settings. This achievement is premised on some success factors which served as guiding principles in the implementation of EVD preparedness interventions. First, the preparedness efforts were based on the principles of bridging the humanitarian-development nexus thus emphasis was on a two-pronged approach. This approach prioritized the rapid establishment of critical outbreak response capacities that did not exist in the country previously while at the same time strengthening the health system capacity for longer-term management of disease outbreaks in general and EVD in particular.For example, instead of depending on a regional referral laboratory (the Uganda Virus Research Institute in Entebbe) for confirmation of the Ebola virus using the reverse transcription polymerase chain reaction (RT-PCR) method, installation of an RT-PCR machine , supporting infrastructure and training of national laboratory technologists to conduct the confirmatory test at the NPHL significantly improved the turnaround time for confirmation of suspected EVD samples in the second half of 2019 (unpublished WHO data). Aside from EVD, this machine is also used to test for other viral diseases such as influenza, Yellow fever, Marburg, Rift Valley fever, and other emerging pathogens. This capacity was rapidly adapted to test for the coronavirus disease 2019 (COVID-19) in early 2020. Likewise, the construction of semi-permanent points of entry screening points and installation of thermal scanners at Juba International Airport and Nimule land crossing border point within the EVD preparedness framework is contributing to screening for other diseases such as the newly detected COVID-19 and longer-term strengthening of the National Port Health System. Furthermore, the semi-permanent all-purpose infectious disease management unit which was originally constructed for EVD preparedness has been adapted and is being used for isolation of suspected and confirmed cases of COVID-19.Second, development and implementation of innovative approaches including the use of appropriate technology were critical in identifying and addressing the key gaps in preparedness. For example, the development of a real-time and health system based integrated health facility supervision tool using digital health technology ensured timely identification of gaps in infection prevention and control and surveillance at the health facility level. This system provides valuable information for decision making for EVD preparedness to the state and national EVD taskforce teams as well as for the broader infection prevention and control in health facilities in the country.Adapting this lesson for use in future EVD preparedness and response efforts requires the incorporation of strategies aimed at bridging the humanitarian-development nexus into EVD preparedness and planning processes in humanitarian settings right from the onset. This will ensure definition, prioritization and implementation of both immediate and long-term health system strengthening interventions for each preparedness and response pillar and phase.Aside from the risk of cross border transmission of EVD from neighbouring countries such as the DRC and Uganda, the southern part of RSS sits in one of the ecological zones of the disease and jointly (with DRC) recorded the first-ever outbreak of EVD in 1976 . The duaAdoption of this lesson requires some critical actions. First, an integrated and sustainable approach to outbreak preparedness in the country which uses the National Action Plan for Health Security (NAPHS) as a platform to strengthen national capacity for surveillance, detection, diagnosis and management of not only EVD but all disease outbreaks is required . Cross bThird, in communities where trust has not been established and the context is volatile, negotiation of the security of EVD preparedness and response assets and access to EVD affected and high-risk areas should be integrated into and implemented as integral part of EVD preparedness and response interventions in all settings similar to RSS and the DRC.Fourth, innovative approaches to overcome the logistic challenges of timely detection, investigation and response to potential outbreaks should be developed as early as possible. Such innovations may include the use of appropriate digital health technologies where feasible, decentralization of preparedness and response interventions and task shifting.From August 2018 to December 2019, an estimated USD 30.5 million was expended on EVD preparedness interventions in RSS . This trThe experiences during the ongoing preparedness efforts in RSS conveyed the challenges associated with predicting the location and timing of outbreaks. Unpublished data from the EVD alert management system showed that many of the alerts were detected outside the four locations where isolation units were sited. Transportation of patients from the site of an outbreak to any of these units would be logistically difficult in the complex humanitarian contexts like RSS. On the other hand, constructing a new EVD treatment centre in an epicentre would take a considerable number of days, during which the absence of proper isolation and infection prevention and control facilities could propagate the outbreak. This lesson calls for more cost-effective and all-hazard approaches to future EVD preparedness.Addressing these lessons requires some key actions. First, evidence-based geographic and programmatic prioritization and phasing of EVD preparedness interventions are required. Each phase should have a clearly defined set of public health interventions. Such interventions should be prioritized according to those required nationwide and those required in the high-risk areas. For instance, preparedness interventions such as EVD risk communication and surveillance are required throughout the country during all phases of preparedness and response while interventions such as case management may be limited in geographic scope to specific locations where the risks of EVD importation are highest. The lessons learnt from neighbouring Uganda on their recent response experience to the importation of cases from the DRC to Kasese district conveyed an innovative approach used to overcome the challenge with predicting the location of a subsequent EVD confirmed case along their western border. A low-cost alternative (less than USD 30 000 per unit) similar to the CUBE  which was developed by The Alliance for International Medical Action (ALIMA)  came in Second, continuous assessment and mapping of the EVD transmission risks would limit the number of interventions and partners required to implement them thus reducing costs.The complexities and the large number of partners required to prepare for EVD transmission into complex humanitarian settings such as RSS is associated with some challenges. The meetings of the national EVD taskforce which was the strategic decision-making body were often long and inconclusive due to limited functionality of its technical working groups which should have discussed and resolved technical issues before presentation to the task force for approval. Inadequate guidance of the several partners most of who were clustered in the capital and one of the high-risk states, Gbudwe resulted in poor adherence to EVD norms and standards in the training of EVD staff and implementation of interventions. For instance, the inability of EVD partners to reach a consensus on the design, specifications and quantities of personal protective equipment, EVD isolation units, and content of EVD training modules and infection prevention and control standards and procedures often compromised the quality and timeliness of essential EVD preparedness interventions. Furthermore, the concentration of most implementing partners in the capital resulted in weak coordination in the high-risk states.The introduction of two streams of coordination namely humanitarian and public health coordination addressed some of these challenges up to an extent. However, the new way of coordination came with its challenges. There was an initial lack of clarity about the allocation of tasks and responsibilities and linkage between the two streams of coordination. This was further complicated by duplication of roles and responsibilities in the national incident management system.Addressing these coordination challenges requires a unified, strong and decentralized coordination platform for EVD preparedness in future. Such a platform should ensure that there is a generic architecture and terms of reference for various streams of EVD preparedness coordination and decentralization of the coordination mechanisms to the high-risk states and potential epicentres of outbreaks . FurtherDespite a very complex humanitarian context, RSS made significant progress in its EVD preparedness efforts which demonstrate that EVD preparedness interventions can be rapidly scaled up in such contexts if appropriate and context-specific approaches are used. Despite the progress, the foregoing lessons show several gaps which should be addressed in the current and future preparedness efforts.Moving forward, the gains made so far in the preparedness efforts for EVD and other outbreaks in RSS should be consolidated and used as an opportunity to build longer-term capacity for national and sub-national outbreak preparedness and response. In this regard, existing plans and systems such as the NAPHS, the Integrated Diseases Surveillance and Response System and the Early Warning, Alert and Response Network should be nurtured to maturity and used as sustainable, integrated and health system-based platforms for outbreak preparedness and response. This has become more pertinent given the emergence of new public health threats such as COVID-19.Furthermore, the lessons highlighted above should be used as evidence to revise the WHO EVD preparedness guidelines. Lastly, further research, systematic reviews, monitoring and evaluation of the ongoing EVD preparedness efforts are required to ensure systematic documentation and application of the lessons learnt from previous and current outbreaks to future EVD preparedness and response efforts. The recently established Lancet Infectious Diseases Commission is a platform for implementing the foregoing recommendations .


Out-of-hospital cardiac arrest patients with pulseless electrical activity are treated by paramedics using basic and advanced life support resuscitation. When resuscitation fails to achieve return of spontaneous circulation, there are limited evidence and national guidelines on when to continue or stop resuscitation. This has led to ambulance services in the United Kingdom developing local guidelines to support paramedics in the resuscitative management of pulseless electrical activity. The content of each guideline is unknown, as is any association between guideline implementation and patient survival. We aim to identify and synthesise local ambulance service guidelines to help improve the consistency of paramedic-led decision-making for the resuscitation of pulseless electrical activity in out-of-hospital cardiac arrest.A systematic review of text and opinion will be conducted on ambulance service guidelines for resuscitating adult cardiac arrest patients with pulseless electrical activity. Data will be gathered direct from the ambulance service website. The review will be guided by the methods of the Joanna Briggs Institute (JBI). The search strategy will be conducted in three stages: 1) a website search of the 14 ambulance services; 2) a search of the evidence listed in support of the guideline; and 3) an examination of the reference list of documents found in the first and second stages and reported using the Preferred Reporting Items for Systematic Reviews and Meta-analyses. Each document will be assessed against the inclusion criteria, and quality of evidence will be assessed using the JBI Critical Appraisal Checklist for Text and Opinion. Data will be extracted using the JBI methods of textual data extraction and a three-stage data synthesis process: 1) extraction of opinion statements; 2) categorisation of statements according to similarity of meaning; and 3) meta-synthesis of statements to create a new collection of findings. Confidence of findings will be assessed using the graded ConQual approach. In the United Kingdom (UK), ambulance services resuscitate over 28,000 adults each year following cardiac arrest , of whicA number of studies have attempted to validate termination of resuscitation rules. However, these studies have reported conflicting results due to a difference in local strategy and the small number of patients who survived when the termination of resuscitation criteria were met . As a reThis review aims to explore and evaluate local clinical guidelines for terminating resuscitation in PEA. However, given the lack of high-quality evidence, it is necessary to utilise evidence derived from clinical expertise and opinion. Therefore, this systematic review will comprise text and opinion .summarise the current variation in treatmentssummarise the evidence cited in support of such treatmentsThe objectives of this review are to:To develop the review protocol, a question was formed using the population, phenomenon of interest and context outcome criteria (PPC) . The popThis review will be guided by the methods of the Joanna Briggs Institute (JBI) . JBI havAn initial Google search for local guidelines was conducted, and two relevant documents were found: the resuscitation policies for the Yorkshire Ambulance NHS Trust  and the This review will consider the local clinical guidelines that manage patients over 18 years old. The population of interest will have suffered a pre-hospital cardiac arrest and present with the non-shockable rhythm PEA.This review will consider local pre-hospital clinical guidelines and cited evidence within, which underpin the resuscitative management of PEA cardiac arrest. Local clinical guidelines are of interest as there is a paucity of national clinical guidance or consensus surrounding the topic area.This review will consider local clinical guidelines from the 14 ambulance services in the UK. The geographical location will capture local-level guidelines, which will contribute to broadening the evidence base and informing future steps towards a national perspective.This review is concerned with local clinical guidelines and the evidence cited in support of them. Only UK clinical guidelines will be considered, as the emergency medical systems in other countries differ from physician to community-led resources and are therefore not comparable to UK-based practice .Local clinical guidelines published from 2015 will be considered. These time parameters reflect the most recent published guidelines from the UK Resuscitation Council and Joint Royal College Ambulance Liaison Committee. If a guideline is found to precede 2015 or is not available via the ambulance service website, a written request will be made to the ambulance service, to ensure the most up-to-date guideline is included. Clinical guidelines that consist of qualitative and quantitative evidence will be considered for synthesis, as often complex health interventions consist of both methodologies .The search strategy will focus on local clinical guidelines and the evidence cited in support of the guideline. It is possible that local guidelines may draw upon a range of sources, including expert opinion, national guidelines and published research. Therefore, reference searches will encompass text, publications and research studies . In addiEast Midlands Ambulance Service NHS TrustEast of England Ambulance Service NHS TrustIsle of Wight NHS TrustLondon Ambulance Service NHS TrustNorth East Ambulance Service NHS TrustNorth West Ambulance Service NHS TrustSouth Central Ambulance Service NHS Foundation TrustSouth East Coast Ambulance Service NHS Foundation TrustSouth Western Ambulance Service NHS Foundation TrustWest Midlands Ambulance Service NHS TrustYorkshire Ambulance Service NHS TrustNorthern Ireland Ambulance ServiceScottish Ambulance ServiceWelsh Ambulance ServiceThe first stage will focus on the UK ambulance services as listed below:Ambulance service websites will be searched. Where local guidelines are unavailable or not found, a written request for the guideline will be made to the National Ambulance Research Steering Group and the National Ambulance Lead Paramedic Group. The use of unpublished literature has caused concern due to the uncontrollable amount and lack of quality assessment . With thThe second stage aims to identify the evidence listed in support of the local guideline. The third stage will examine the reference list of documents found in the first and second stages of the search. This hand search will reduce publication bias by ensuring all documents found are included in the review .https://www.mendeley.com). Duplicates will be removed. The selected documents will be extracted and uploaded to the System for the Unified Management, Assessment and Review of Information from JBI  will be applied .The documents will be screened against the eligibility criteria. To increase the credibility of this review, each document will be screened by two independent reviewers. Disagreement will be resolved by discussion or by introducing a third reviewer . The ratAll documents will be subjected to critical appraisal and include a focus on genuine opinion, driving motivation, the location of the documents, and related conflict to ensure transparency . This apAmbulance Trust (context)Year of publicationReview dateType of textPopulation presentedThe panel who developed the guidelineThe sources cited to inform the guidelineConclusions relevant to the objectives of this reviewReviewer notesThe JBI SUMARI extraction tool will be used to transfer the main conclusions found within the text . Data exType of textThose representedSettingGeographical contentCultural contentLogic of argumentConclusionReviewer commentsFor evidence cited in support of the local guidelines, the data extraction table headings will include:The table format aims to reduce error and to record an accurate account of categorisation and synthesis . This foIdentify when to continue or cease resuscitationIdentify any other related guidanceIdentify the local strategies for terminating resuscitation for cardiac arrest patients with PEA for each ambulance serviceIdentify the evidence cited in support of such treatmentsEach document will be carefully read to identify accurate statements which encompass the main conclusions. The data will be extracted consistently and standardised by using the extraction tool to meet the main outcomes of this review:The quality of evidence will be assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Text and Opinion . The appThe extracted data will be gathered and synthesised using JBI SUMARI . Data syThe summary of findings table, which contains the main elements of the review, will be graded using the ConQual approach. ConQual aims to establish the quality and confidence of synthesis for qualitative reviews. A ConQual score will be provided for each synthesised finding and from the type of research which informs it .The reviewer would like to thank their supervisors for this collaboration and acknowledge the National Institute of Health Research and University of Plymouth for providing the opportunity and funding to complete the JBI systematic review module.AC is the main author of the protocol. RE and SB have made substantial contributions to the protocol search strategy, conducted a critical analysis of the protocol and drafted and approved the final version for submission to the Joanna Briggs Institute and journal publication. SJ proofread, amended and made substantial contributions to the protocol background. RE acts as the guarantor for this article.None declared.Not required.The JBI systematic review training module was undertaken as part of the funded National Institute of Health Research integrated clinical academic award.PROSPERO 2019 (CRD42019138731).


The extraordinary diversity, variability, and complexity of cell types in the vertebrate brain is overwhelming and far exceeds that of any other organ. This complexity is the result of multiple cell divisions and intricate gene regulation and cell movements that take place during embryonic development. Understanding the cellular and molecular mechanisms underlying these complicated developmental processes requires the ability to obtain a complete registry of interconnected events often taking place far apart from each other. To assist with this challenging task, developmental neuroscientists take advantage of a broad set of methods and technologies, often adopted from other fields of research. Here, we review some of the methods developed in recent years whose use has rapidly spread for application in the field of developmental neuroscience. We also provide several considerations regarding the promise that these techniques hold for the near future and share some ideas on how existing methods from other research fields could help with the analysis of how neural circuits emerge. Today, neuroscientists of all stripes, including those working on the development of the nervous system, are taking advantage of the breadth of new methods and technologies that Don Santiago could have only dreamt about. These methods accelerate our capacity to collect and analyse biological information in large and complex specimens. For instance, we can now reconstruct in three dimensions (3D) the complete peripheral nervous system of a cleared lizard embryo2 or obtain a transcriptomic map of gene expression at the single cell (or nucleus) resolution from almost any tissue and species, including humans. Science and technology have been interconnected always, and advances in one historically translate into important progress in the other. Today, the number of available advanced techniques can be overwhelming. In this review, we discuss some recently developed techniques that are currently becoming common in laboratories studying neural development.Long gone are the times when budding neuroscientists would picture themselves working like Don Santiago Ramón y Cajal using only a simple microscope and a shelf full of chemical reagents3, these techniques were very slow and do not scale well to larger embryos or postnatal tissues. This situation began to change with the appearance of light sheet fluorescence microscopy (LSFM). The main advantage of LSFM is the high speed of acquisition and the ability to image large sample sizes that were unpractical to image with conventional microscopes. LSFM was initially used in the field of colloidal chemistry4, and about 30 years ago it was adapted to biology to visualise guinea-pig cochleas in 3D5. LSFM combines the speed of wide-field imaging with optical sectioning and low photobleaching. In conventional fluorescence microscopy, the entire thickness of the sample is illuminated in the same direction as the detection optics, and, as such, the regions outside the detection focal plane of the objective are potentially damaged by extraneous out-of-focus light that increases the photobleaching. In contrast, in LSFM, the sample is illuminated from the side, perpendicular to the direction of observation, thereby placing the excitation light only where it is required. Therefore, this technique enables the visualisation of tissue samples by shining a sheet of light through the specimen, generating a series of images that can then be digitally reconstructed thanks to the development of sophisticated algorithms and huge improvement in the capacity of computers to store and analyse data6. In developmental biology, LSFM was used for the first time to visualise the transparent tissues of zebrafish and Drosophila embryos in 3D in vivo. About 8 years ago, Tomer and colleagues were able to visualise the development of the Drosophila ventral nerve cord for the first time7, and Ahrens and co-authors measured the activity of single neurons in the brain of larval zebrafish embryos8 using in vivo light sheet microscopy.Our capacity to document the 3D organisation of the embryonic brain to understand the basic mechanisms underlying circuit formation has been limited until very recently. Studies on the development of the nervous system of most vertebrates have traditionally relied on histological sectioning methods or open book preparations that enable the visualisation of two-dimensional organisation of the axon tracts in the samples under epifluorescence or confocal microscopes. These approaches, which are based on the observation of selected slices or planes of observation (a process that can inherently introduce biases), provide only partial information about the sample. Even though 3D imaging of small embryos had been performed for many years using wide-field and confocal microscopy11, it is worth mentioning the variants of the CUBIC and DISCO series because their excellent results and easy performance ultimately exalted them as the most widespread methods for brain clearing (see https://idisco.info and http://cubic.riken.jp).However, what eventually enabled LSFM to be used for the analysis of the nervous system was the remarkable improvement in brain clearing techniques. The rapid optimisation of clearing protocols has expanded the application of LSFM in the field of developmental neuroscience in the last 4 to 5 years. Since then, a myriad of different approaches to perform tissue clearing have been developed; these approaches vary based on the type of chemical reagents used and depend on the size of the samples. Although exhaustive reviews about the diversity of clearing protocols have been published12. In axon guidance studies, this combination approach is proving to be extremely useful for visualising neuronal axons growing across the whole embryo and for detecting pathfinding defects in mutants of different members of the main families of axon guidance molecules13. It has been possible to visualise for the first time the development of the peripheral nervous system and the innervation patterns of human embryos14. Now, the power of combining these approaches with axonal tracings15 or antibody staining after functional manipulations  holds the promise of interesting times ahead . In fact, combinations of transcriptomics plus epigenomics or transcriptomics plus proteomics in single cell analyses are rapidly emerging27. Here, we focus our attention on one of the most frequently used modalities in neural development, the transcriptomic characterisation of single cells.The complexity and diversity of cell types is one of the most remarkable characteristics of the mature nervous system. Corticospinal neurons that connect the brain with the spinal cord, sensory neurons that detect and conduct touch information from the skin surrounding our bodies to the central nervous system, or glial cells that modulate neural activity are different examples of the richness and huge variability in cell types that make up the nervous system. Cell specification occurs during development and, until recently, researchers had very limited ways to quantify cell diversity. For many years, the quantification of diversity was constrained by the use of pooling approaches and techniques that require either harvesting cells from the same tissue or combining cells from different individuals to obtain enough material for downstream analysis. For example, in bulk RNA sequencing (RNAseq) approaches, the transcriptomic expression level of a particular gene is not measured from an individual cell but rather as the average level of expression of that gene over many cells present within the same sample. The revolution in the molecular analysis of individual neural progenitors started when the ability to sequence DNA or RNA at the single-cell level became possible. In 2013, the journal 28, and the myriad of protocols that have been developed since then have quickly transformed several research fields and the way developmental studies are performed. The key step in scRNAseq protocols consists of tagging all transcripts inside each cell in such a way that RNA molecules coming from the same cell are easily identifiable and quantifiable29. scRNAseq enables transcriptomic cell types in the sampled tissue to be defined through the analysis of differentially expressed genes in each cell.The first protocol to perform single cell RNAseq (scRNAseq) was published in 200930 as well as the prefrontal cortex of human embryos31 or the temporal changes in the transcriptional landscape of apical progenitors and their successive cohorts of daughter neurons in the cortex32. In general, developing tissues are characterised by the presence of a mix of cells in different stages of differentiation . These stages are captured at the time of scRNAseq processing, thus resulting in a continuous representation of cellular states transitioning from one to another. These transitional stages may be modelled computationally by recapitulating the probable trajectory of the cells through a representation called pseudotime33, which defines the order of the cells through development. This representation therefore enables mapping of particular cell types to different states of the developmental trajectory34.Nowadays, commercialisation of droplet-based sequencing, for example the 10x Genomic Chromium platform, has enabled the widespread use of scRNAseq. In the field of developmental neuroscience, scRNAseq has been used to profile the entire developing mouse brain and spinal cord39, with the latest iteration of the high-definition spatial transcriptomics (HDST) method40 reaching a spatial resolution of 2 µm , thereby yielding cell types that are more representative of the original tissue and less affected by transcriptional artefacts. A potential disadvantage of using nuclei is that lower quantities of messenger RNA are obtained from the nuclei than from whole cells, and consequently fewer genes are typically detected. Nonetheless, recent studies have shown that single nuclei and single cell approaches identify similar cell typesThe heading of this section might sound as if it were taken from a sci-fi movie. However, combining the computational power of modern processors and graphics processing units (GPUs) is exactly what high-throughput methodologies such as those described above require. These techniques generate huge quantities of data that need to be analysed. The simple generation of sequencing results or imaging data by itself does not provide new insights that can advance our understanding of how the nervous system develops. Intense developments in the field of machine learning have generated algorithms that may now be used to deconstruct the complexity of such data.9. Navigating your way through such an enormous amount of information to draw conclusions quickly becomes a dead-end, both in time and in computing requirements. Working with such vast quantities of bytes imposes a heavy processing burden on a lab’s computing capability but also makes analysing such datasets a time-consuming task. To solve this problem, the development of software capable of handling huge quantities of data becomes an urgent requirement; it becomes just as important as the need for the hardware that generates the dataset itself.The imaging of a mouse brain by high-resolution LSFM generates between 20 gigabytes and up to several terabytes of data depending on the resolutionCell Profiler from the Broad Institute, which can easily segment nuclei in dense tissue images, and its more powerful sibling, Cell Profiler Analyst, which makes use of machine learning algorithms to recognise defined cell types from large imaging datasets44. Cell Profiler has been used, for example, to quantify the differences in neuronal numbers between the sulci and gyri of the cortex of Flrt3 mutant mouse embryos45 and to help elucidate the role of PTPRD in neurogenesis46. More recently, the Pachitariu lab47 released a complementary approach for cell segmentation called Cellpose. This is a generalist algorithm for cellular segmentation and is based on the use of a neural network that is trained on thousands of images from different microscope modalities  combined with non-biological images of similar structure. The system creates a platform capable of recognising cells from a wide array of image types. It also enables the generation of researcher-defined custom models by training the algorithm on specific types of images.Paradoxically, even though a researcher spends just a few minutes to image a whole mouse embryo in 3D using LSFM, the quantification of such datasets often relies on tools and systems that require manual and time-consuming annotations. Fortunately, in the last few years, an unparalleled development of informatics tools has begun to help researchers quickly and accurately analyse big datasets in a short period of time. Some examples of these programs are 57. Beyond solving imaging tasks, machine learning approaches may be used in many other applications within the field of neurodevelopment. The quantity of data generated during sequencing experiments such as scRNAseq face the same challenges as those derived from large imaging experiments. Newer and more refined technologies yielding an ever-increasing number of sequenced cells quickly translate not only into larger datasets but also into a higher number of dimensions that need to be non-linearly reduced to define particular cell types. Several packages that allow the processing of sequencing data and perform efficient dimensionality reduction or help to identify defined cell types of interest within the datasets have been released59. While processing of image and sequencing data are both examples from the blooming field of computational biology that are useful for studying neural development, many more developments and applications are predicted to emerge60.The use of machine learning, particularly neural networks trained to recognise structures of interest such as nuclei, cells, blood vessels, noise, etc. in images, has exploded in the last few years, and it is quickly becoming the go-to solution for many biomedical research problems61. Community crowd-sharing of resources such as those mentioned, antibody-related optimisations, and tested protocol modifications and reagents will form the basis for advancing current and future protocols, likely to the point that many antibodies will work for 3D immunostaining applications. Concurrently with advancements in staining methods, parallel development of LSFM will likely enhance the imaging resolution of transparent samples while also reducing the time required for acquisition63.Although transformational technological revolutions are constantly occurring in science, the advances that have been made in the last few years have been spectacular. Here we have highlighted what, in our opinion, are very relevant and novel approaches for investigating the developmental processes that control the formation of neural circuits. It is our belief that we will experience amazing changes in the years to come that will dwarf what we know today. We envision that tissue clearing technologies will evolve into fully applicable methods that will no longer be limited by antibody compatibility. The recent publication of CUBIC-HistoVision points in that direction, as it describes a systematic interrogation of the properties and conditions that preserve antigens and facilitate antibody penetration into fixed animal tissues64. The development of effective and high-throughput approaches in single cell proteomics will aid the quest to fully characterize cells, their functional and developmental states, and the mechanisms involved in transitioning from one state to another. Matched single cell genetic, transcriptomic, and proteomic data will help to elucidate the mechanisms behind the formation of a fully developed nervous system.The most important missing piece for single cell approaches is the development of high-throughput proteomics to individually measure protein content in each cell with enough depth to cover the whole proteome. Beyond basic estimation per cell, single cell DNA or RNA technologies are incapable of measuring the abundance and activity of proteins, which are regulated by both post-translational modifications and degradation. Although single cell proteomic approaches are already available, most of them currently rely on antibodies to detect the proteins of interest; this imposes an important throughput limitation. Methods to quantify thousands of proteins in hundreds of cells through the use of mass spectrometry (MS) are emerging, and improvements in MS are expected to increase the sensitivity of single cell proteomics65, such as those using neural networks trained to identify different types of tumours based on their location and composition in cleared whole mouse bodies66, highlight the possibilities of gathering current computing power so that it can be applied to other fields such as neural development. Similar applications of machine learning algorithms could aid in the recognition of changing mRNA/protein expression patterns in brain development. Labour-intensive tasks commonly used to study the developing nervous system could also greatly benefit from the implementation of tools developed in other neuroscience-related areas. For example, the automated identification and tracking of migrating neurons should be easily adopted following the lead of algorithms like DeepLabCut that behavioural neuroscience labs are using to track the position of different parts of the mouse body without the use of markers67. ClearMap is another algorithm that maps cells automatically in the mouse brain of LSFM datasets, which could be applied to neonate brains24. Another very promising avenue is the algorithm Trailmap, which was recently developed in the Luo lab to automatically identify and extract axonal projections in 3D image volumes68 and may be easily implemented to improve the quantification of axon guidance studies. Adoption of such neural networks will probably require re-training and optimisation to the specific use-case scenario and dataset, which highlights the need for fast-training computational strategies in order to facilitate the broader use of these techniques.The application of machine learning in the field of developmental neuroscience is still in its infancy but will likely explode in the near future. Examples stemming from cancer researchin situ at the same time. Such datasets would contain information about what are considered the main determinants of cell identity while maintaining the intact structure, shape, and form of the tissue. This “fantasy technical improvement” could be pictured even one step further by introducing the fourth dimension into account and analyse datasets of embryos at different stages of development to provide the most detailed description of development progression to date. However, writing a “what will the future look like” piece is bound to fail. History has demonstrated that both the imagination and the driving force of scientists are many orders of magnitude beyond what can be anticipated. As such, while this review will likely become obsolete shortly, it will be a good sign that developmental neuroscience maintains its exponential progression in advancing our understanding of the assembly of neural circuits.Therefore, despite the impressive amount of state-of-the-art technologies developed in the last few years, there is still room for improvement of some of the latest methods available to study the developing nervous system. We could envision a not-so-distant day when 3D embryonic brain imaging will be combined with single-cell technologies to elucidate the chromatin, mRNA, and protein signatures of each cell


In the article titled “Assessment of the Levels of Level of Biomarkers of Bone Matrix Glycoproteins and Inflammatory Cytokines from Saudi Parkinson Patients” , multiplThese should supplement the following sentences in the article text:Osteopontin (OPN) was revealed to be elaborate in inflammatory and degenerative mechanisms of the neurons . OPN plaThe error was introduced during the production process of the article, and Hindawi apologises for causing this error in the article.


Disparities in diabetes care are prevalent, with significant inequalities observed in access to, and outcomes of, healthcare. A population health approach offers a solution to improve the quality of care for all with systematic ways of assessing whole population requirements and treating and monitoring sub-groups in need of additional attention.Collaborative working between primary, secondary and community care was introduced in seven primary care practices in one locality in England, UK, caring for 3560 patients with diabetes and sharing the same community and secondary specialist diabetes care providers. Three elements of the intervention included 1) clinical audit, 2) risk stratification, and 3) the multi-disciplinary virtual clinics in the community.This paper evaluates the acceptability, feasibility and short-term impact on primary care of implementing a population approach intervention using direct observations of the clinics and surveys of participating clinicians.Eighteen virtual clinics across seven teams took place over six months between March and July 2017 with organisation, resources, policies, education and approximately 150 individuals discussed. The feedback from primary care was positive with growing knowledge and confidence managing people with complex diabetes in primary care.Taking a population health approach helped to identify groups of people in need of additional diabetes care and deliver a collaborative health intervention across traditional organisational boundaries. Diabetes is a serious condition that can result in significant morbidity and mortality 23. The c2Diabetes care in the UK compares reasonably well in comparison with other European nations and beyond . However23This paper discusses a project designed to address these specific issues. Firstly, it reflects on the split between primary and specialist diabetes care and the impact this has on the quality of diabetes care in populations. Secondly, it proposes a population health approach as a way of improving organisation of diabetes care through specific interventions integrating primary and specialist care. Thirdly, it evaluates the acceptability, feasibility and short-term impact on primary care of implementing a population approach intervention.In England, care of people with diabetes has been long divided between primary and specialist care. Finding a balance between routine management and specialist input has been an issue since the 1960, up until which time all diabetes care was provided by the specialist services. The growing number of people with T2D prompted the relocation of those suitable for routine management from hospital to primary care 67. The d68912The challenge of developing an optimal model for people with diabetes has been ongoing. In response to the growing prevalence of diabetes and moving care closer to home to support continuity of care, primary care has been tasked with providing both routine and more complex diabetes care. There is clearly a risk of adverse outcomes for people with diabetes if transfer of responsibility to general practices happens without adequate support 1516 and 15171820A population health approach has the potential to improve the quality of care of individuals by introducing solutions targeting groups and sub-groups at risk of developing complications from diabetes 232425. T2324A good indication of general health disparities can be provided by clinical audits which systematically review the structure, processes and outcomes of care against explicit criteria . There a262728While a clinical audit indicates the effectiveness of diabetes healthcare on a national, regional, or service level, it does not enable context informed patient level interventions. The latter is provided by a risk stratification process, which identifies those who do not have their diabetes care processes (key diabetes monitoring measurements) done or treatment targets  achieved and classify them as being at high, medium or low risk of poor outcomes. 3132.31multidisciplinary virtual clinics in the community are one of the options for joint working. The model is for primary care staff to be supported by specialists  through virtual clinics to manage the care of patients with T1D who do not attend the outpatient clinics and of patients with T2D who do not achieve expected treatment outcomes when receiving usual care. ‘Virtual clinic’ refers to face-to-face case conferences between health professionals from primary care and specialists to discuss an individual’s care without their being present [It has been increasingly recognised that a good diabetes care pathway addresses the needs of the local service and is underpinned by a multidisciplinary team working between generalists and specialists, across professional groups, and with specialists reaching into the community . The mul present  as oppos present 35, or ‘i present .In diabetes research, joint working is underrepresented 37. The v4433423338394041434647484950515253The project started in 2014 with the aim of improving diabetes care by developing a fully integrated diabetes service across primary and specialist care in Oxfordshire. The objectives were to find a sustainable solution to the growing demand for diabetes care, provide more person-centred care, improve the outcomes of people with diabetes, and reduce the variation in quality and outcomes of diabetes care across the region while at the same time improving its efficiency. The population health approach was chosen as the ideal way of addressing the unmet needs of the studied population.In the studied diabetes service, the main route to specialist advice was via the referral system with patients being moved to secondary or community care and discharged back to primary care for routine management. The recognition of the need for bringing diabetes expertise into primary care has been ongoing with previous interventions including specialist outpatient clinics in primary care  and primary care health care professional (HCP) education delivered by the local specialists. This was helping some patients but not all. A move towards more inclusive diabetes care to reach those who were not benefiting from the referral system was needed. The population health approach offered tools to help identify those who were slipping through the net.One locality, a locality being a local group of practices led by a GP, piloted the population health approach and the interventions: 1) the diabetes audit , 2) risk stratification – the screening programme to identify patients at risk of developing complications from diabetes, 3) the virtual clinics to plan action in response to issues identified using the audit data and patients’ lists. In the North East Locality, in the time of testing the intervention , there were 365 people with Type 1 diabetes  and 3,195 people with Type 2 and other types of diabetes  . The outThe programme adopted the principles of quality improvement in diabetes care aiming to make care a) safe , b) effective (bringing diabetes expertise to primary care), c) patient-centred, d) timely, e) efficient, and f) equitable (available to everybody) 57.The programme was a) theory driven , b) process oriented (focus on how change happens), c) participatory , d) multidisciplinary and multi-method , and e) meticulously detailed (detailed descriptions of context and processes) .The process of developing the virtual clinics was guided by the principles of co-design in service improvement and continual improvement process. The specialists in diabetes began regular engagement with one surgery in early 2016. Initially the meetings were case discussions with cases identified by primary care healthcare professionals, this was later complemented with the discussions of cases identified in systematic searches of the patients’ list. The systematic searches were refined within a few months. The clinics also included discussions of the surgery’s performance in the National Diabetes Audit Core Audit (NDA). Simultaneously, the interested surgeries across the locality were visited to build engagement and collect their feedback on proposed interventions.This was followed by a survey (August 2016) seeking comments on the individual practices participation in the NDA, factors contributing to the results achieved, and the best way forward; this information was important to understand the primary care perceptions of barriers and facilitators to good quality diabetes care and the contextual factors impacting on the outcomes. Ten participants (eight GPs and two practice managers) completed the survey representing all seven practices in the locality. All participants supported the idea of closer working between generalists and specialists with an easy access to specialist advice and more presence of specialists in the community. The findings from the survey were used to prepare an action plan informed with respondents’ views to feed into the strategy for integrated diabetes care in the pilot locality. The practice managers were approached to schedule the clinics.The use of the National Diabetes Audit was intended as an indicator of the quality of diabetes care in the pilot locality and the individual practices and to help target resources whether at individual practice level (access to practice nurses with expertise) or across locality (access to conveniently timed patient education). The shortcomings of using the audit data were identified by the primary care staff, namely the lack of local indicators of quality of care and a delay in reporting outcomes. This feedback triggered development of a monthly diabetes dashboard which included local indicators of quality of care as well as national indicators. This enabled almost real-time population data monitoring. The suggestion from primary care was to discuss the results in the context of the practice resources and its population. The shortcomings are the same as with any clinical audit – the data depends on the quality of coding and transparency of the analysis and these were discussed at the meetings to build trust and mutual understanding of the audit.Patient lists were screened using criteria agreed by the clinical team to identify adult patients at risk of developing complications from diabetes:patients with HbA1c greater than 9% (75 mmol/mol)patients with HbA1c lower than 6.5% (48 mmol/mol) on insulin or sulphonylureapatients with diabetes and eGFR <30 ml/minThe lists of patients meeting the above criteria were prepared in readiness for the virtual clinics.The clinics were organised around three main items on the agenda,the results of the audit,the care of patients identified in the predefined searches (risk stratification), andthe care of other complex patients as requested by the primary healthcare professionals.The integral part of joint working between the clinics was case management with the discussions of the patients’ care and decisions made at the virtual clinics, followed with a review of a care plan with a patient, and then with a follow-up at the virtual clinic. The GPs and practice nurses had access to the consultant led email advice line and telephone line between the virtual clinics Table .The aim of this work was to validate the population health approach and the interventions proposed. A mixed-methods evaluation, including observations of the clinics and survey with those involved in the delivery of the virtual clinics, was conducted with the aim of assessing acceptability and feasibility of interventions, and the initial impact of the intervention on the primary diabetes care. Acceptability was defined as the willingness of primary care healthcare professionals and practice managers to engage with all three interventions. Feasibility was defined as the ability and capacity to deliver them. The impact was defined as any changes in knowledge and confidence in managing diabetes care in primary care, changes in patterns in communicating with and referring to specialist diabetes services, and any changes introduced to diabetes care in primary care due to the interactions with the specialists.The clinics were observed by a researcher with doctoral training in qualitative methodologies assessing the delivery of interventions to ensure they were provided in accordance with the protocol and delivered consistently, if the participants adhered to the protocol, what modifications were made, and explore participants’ views of and satisfaction with the intervention.In addition, information about each case discussion was recorded to identify how many patients were discussed per clinic, what aspects of their care were discussed, who contributed, what aspects of care were changed, how the decision was made, and if the tasks were assigned. After the first clinic, the following was recorded: were the actions communicated with all interested, were the actions completed, what were the barriers to completing the actions.Recruitment: All practice managers were approached with a request to complete the survey and send the invitation to the GPs and practice nurses in their practices who participated in the clinics. The clinical lead for specialist diabetes nurses was directly invited to participate and asked to invite specialist diabetes nurses participating in the pilot.Method: A survey consisted of a mix of Likert scales to determine the degree of perceived change to the GPs and primary care nurses’ knowledge about diabetes management, confidence in managing patients with diabetes, and behaviours in clinical practice, and open-ended questions to collect further details of experiences and impact of participating in the clinics. Separate surveys with open-ended questions were conducted with the practice managers, specialist diabetes nurses, and consultants in diabetes to collect their feedback on the changes to the management of diabetes care in primary care and processes involved in the clinics. The survey was conducted over three weeks seven months after the pilot concluded (January 2018).Eighteen integrated virtual community diabetes clinics took place between March and July 2017 across seven practices in the North East Locality. Each practice held at least two clinics with one practice holding four. Each clinic was attended by at least one GP, one practice nurse, one specialist diabetes nurse, and one consultant in diabetes. Five clinics were attended by one or more mental health specialists.Approximately 150 patients were discussed, on average eight patients per 60 minute clinic, with the number depending on the complexity of cases, the level of preparation of the case by primary care, the availability of information from the IT system and speed of operating it, the experience of working together and any previous discussions of patients. The majority of patients had type 2 diabetes, 23 had type 1 diabetes, and 3 had a type yet to be confirmed.The interventions changing the course of diabetes care included:change of medicationintroducing new medicationintroducing new intervention, e.g. diet, lifestyle change, referral for consideration of bariatric surgerychange of medication dosereferring to diabetes specialist teams in secondary or community carereferring to allied specialities including dieticians and mental health specialistsreferring to patient structured educationagreeing on delivering specialist care in primary careagreeing on complex care pathway with primary care delivering initial intervention, and if not effective, scaling up to the specialist servicereassuring primary care healthcare professionals about appropriateness of interventions implementedThe feedback from primary care voiced during the virtual clinics was positive with the following noted:the clinics were seen as educational sessions with useful guidelines and adviceknowledge gained at the clinics was applicable and transferable to patient cases not discussed at the virtual clinicsthe clinics increased understanding of diabetes services available and the referral systemThirteen participants responded to the survey including 5 GPs (out of at least 7), 2 primary care nurses (out of at least 7), 4 practice managers (out of 7) and 2 diabetes specialist nurses (out of 3). Five out of 7 participating practices were represented.The self-reported knowledge of diabetes management and referral system gains were reported by all primary care healthcare professionals (GPs and primary care nurses) in all or some aspects of diabetes care following the clinics. The self-reported change in confidence in managing diabetes varied and was different depending on the type of diabetes. Table All primary care healthcare professionals (GPs and primary care nurses) reported an increase in their contacts with the diabetes consultants and majority with the specialist diabetes nurses (DSNs). The impact was noticed in terms of the number of referrals to different specialist services and Table It is unclear if the increase or decrease of referrals is a positive or negative change. The increased awareness of diabetes issues and number of patients identified as in need of further attention increased the referral rates as reported by one GP, while education provided at the clinics reduced referrals to the DSNs as observed by another GP. The DSNs confirmed that the frequency of seeking their advice increased as well as requests for face to face assessment; though the increase was seen as a positive change, there were concerns that the referrals were not always appropriate.Across the practices, the primary care staff  reported changes on the practice level that have been introduced following the clinics:adopting the Year of Care approach promoted at the clinicscontinuing with risk stratification and searching the patient lists for patients at riskfocusing more on blood pressure in patients setting up a new alert for those who did not have microalbuminuria checked in last yearchecking more often whether a low HbA1c could mean regular hypospicking up the issues of unrecognised hypoglycaemia in patients on insulin and gliclazidebringing people with high HbS1c with diabetes in more frequentlyaltering insulin dosesactively tracking and approaching disengaged patientsThere was a shared feeling that the pilot should be extended with further work to refine it. The feedback coming from all participants was positive and the face-to-face format of the meetings was appreciated by both primary care HCPs and DSNs. In overall, all primary care HCPs reported following the decisions made at the clinics but some experienced problems in implementing them due to poor patients’ engagement or internal administration issues. As the practice managers emphasised, the clinics increased focus on diabetes in the practices. Together with other training in diabetes provided by the consultants and DSNs the interventions complemented each other.The key outputs of the pilot, identified from the observations, voiced feedback, and survey, included changes in the processes related to management of diabetes in primary care; identification of the gaps in knowledge of diabetes and its management among GPs and primary care nurses; new ways of working between GPs, primary care nurses and diabetes specialists; and raising awareness of diabetes research. In particular, in each of the areas, the outputs included:management of diabetes in primary careplans to continue with the virtual clinics beyond the pilot; all practices expressed a willingness to do soidentifying groups of patients in need of intervention but previously not perceived as such by primary careproviding primary care practices with a tool (searches) to systematically screen their population and identify patients in need for interventionproducing a protocol for the virtual clinics to be used across Oxfordshiredesigning a new format of the outpatient letter to primary care including relevant information (e.g. care processes delivered) presented in a systematic wayimproving recording of information in primary care for the National Diabetes Audit by exchanging information during the virtual clinics (patient structured education)education of primary care healthcare professionalschallenging diabetes treatments and management not aligned with national or local guidelinesraising awareness of a range of diabetes treatments and interventions available within primary carechanging a narrative about people with high HbA1c – shifting of blame in poorly controlled diabetes from patients to complexity of condition with its physical and mental health aspects, unresponsiveness of the health services and gaps in the servicedeveloping a community of well-linked practitionersimproving the linkage between services with new referrals being made at the virtual clinics to secondary care, community care, patient structured education, mental health specialists, and community type 1 diabetes clinicsplanning together location and level of care in consideration of patients’ needs and not primary and secondary care boundariesresearchincreasing number of people recruited into clinical trialsThe pilot confirmed the acceptability and feasibility of the population health approach and interventions in primary care in an environment with a limited previous experience of joint working. Key gaps in knowledge and confidence in managing patients with diabetes and knowledge of referral practices were identified and addressed through the virtual clinics. The clinics provided a space to explore and address the benefits and problems of joint working.One of the key benefits of the project was the development of a monthly local clinical audit which extracted NDA data from GP surgeries and was able to show improvements in a short period of time. Essential to the success of this audit tool was a learning not blaming atmosphere  with the262628The screening of their population in search of those at risk of poor outcomes enabled the practices to look at the individual patients as part of bigger groups with shared problems and plan for care sharing primary care and specialist resources 3132. The315253The study highlights the importance of focusing on the different processes involved in changing healthcare across a number of different organisations, and acknowledging the multicomponent and multifactorial nature of such interventions 4558. Bei45The interventions tested in the pilot continue to be used in the piloted locality and across the county. The context has changed as the pilot was replaced with a voluntary paid service with practices hosting specialists twice a year. The service has been sustained through the NHS England Diabetes Transformation Funding programme and has been shown to improve not only healthcare professional confidence but also the key care processes delivered to patients. It is currently being refined along the lines of new Primary Care Networks and is now a part of the Locally Commissioned Service for Diabetes within Oxfordshire. Further work is required to refine the processes involved in the virtual clinics including investigating video interactions, a process which has been accelerated by the Covid-19 outbreak, and ongoing review of high risk patients within the GP surgery in between the virtual clinics. A broader impact assessment on the wider health economy including admissions as well as morbidity arising from diabetes is required now that the pilot has been rolled out across the county.This work would benefit from more complex evaluation of the intervention without relying on self-reports only when assessing changes in knowledge and confidence of healthcare professionals. Also, more insights from the participating HCPs on why some of the interventions did not meet their needs and what further improvements are needed. Unfortunately, the reasons for it not happening (e.g. no increase in knowledge or confidence in managing diabetes) for some were not explored further. It is too early to assess the impact of changes in referral patterns following HCP education of the services available for people with diabetes.The implementation of virtual clinics successfully piloted a population health approach in diabetes care, focusing on population screening, risk stratification and assessment (identifying patients at high risk of complications), reviewing patient cases (identifying solutions that are applicable across the population), and improving individual patients’ care as well as at practice and population level. The multidisciplinary virtual clinics in the community enabled the service todiscuss the outcomes of audit taking into consideration the characteristics of the population and plan for improvement,proactively identify groups of patients at risk of complications from diabetes, andplan their care together.Continuing diabetes education in primary care focused on building expertise and skills rather than the dissemination of guidelines. Unnecessary referrals were avoided by encouraging shared decision making and shared responsibility for treatment changes. The intervention had an educational value, improved confidence in primary care in managing diabetes, and improved communication between primary care and specialists. It made the diabetes care for the whole population more cohesive in the piloted area.This project developed and delivered a geographical based intervention to improve care for people with diabetes starting from a baseline of a traditional model of care delivery. Factors which enabled successful adaptation and delivery of a high quality service included the establishment of working relationships and trust developed from engaging with each practice individually to discuss their specific circumstances, allowing for flexibility in the organisation of virtual clinics, working in primary care settings, appreciating primary care familiarity with patients and specialist subject area expertise, and collecting. All of this was underpinned by ongoing reporting of data and acting on ongoing feedback from primary care.Local engagement is essential in the development of the modelRegular updated trusted data is essential to show improvement and understand what changes are effectiveA culture of learning and support is essential rather than blame and performance managementFace to face engagement between GPs, primary care nurses, and specialists outside of the hospital and in the community can transform relationships and break down barriers to joint working


Underground water bearing structures, and the eco-system are being contaminated through seepage of the plumes emanating from the mixtures of the industrial waste materials (IWM), made of moist cemented soil with municipal solid wastes (MSW) dumped at the site. The distribution of the contiminant hazardous plumes emanating from the waste materials' mixtures within the subsurface structural lithological layers was clearly map and delineated within the near-surface structures, using the triplicate technique to collect samples of the soil with the waste mixtures, and the water analysis for the presence of dissolved ions. The deployed method helped to monitor the seepage of the contaminant leachate plumes to the groundwater aquifer units via the ground surface, through the subsurface stratum lithological layers, and hence, estimation of the waste materials' volume was possibly approximated to be 312,000 m•The novel method is transferable, reproduce-able, and most importantly, it is unambiguous technique for the quantification of environmental, industrial and municipal waste materials.•It helps to map the distribution of the plumes emanating from the waste materials' mixtures within the subsurface structural lithological layers that was clearly delineated within the near-surface structures underlain the study site.•The procedure helped in the monitoring of leachate contaminants plumes seepages into the surface water bodies and the groundwater aquifer units, via the ground surface, through to the porous subsurface stratum lithological layers.In summary, the novel method adopted are as presented below:  Specifications tableThe method deployed for recording subsurface parameters from RES2D geophysical survey of the area was adequately distributed within the subsurface stratum as presented in 3 taken the approximate depth to the contaminant plumes as the height to be 15 m [The distributions of the subsurface lithologic layers' depth recorded corresponding to the resistivity distributions was used to quantify volume of the waste materials by means of a rectangular prism model generated using the 3-D Oasis Montaj technique, . The process was accomplished through estimation of the volume covered by the survey area, , as shown in  be 15 m . The conThe study was undertaken due to the harmful effects of the dissolved ions emanated from the hazardous materials deposited at the site on the ecosystems, the environment, and human lives. Most importantly, the growing population around the dumpsite area calls for urgent action. Assessment of the novel research work of this magnitude, on IWM and MSW is most essential to the determinations of the characteristics of these hazardous ions and the movement of contaminant plumes within the subsurface strata which houses the groundwater bodies A closed observation of the huge number of different research methodologies that have been reported in literature on the environmental wastes, management of hazardous contaminant plumes, monitoring of leachable contaminants, delineation of zones susceptible to potential environmental hazards (PEH), and estimation and quantification of the contaminant plumes flows, and risks to human and ecosystems e.g., Results from the method deployed to acquire the RES2D ERT geophysical survey, recorded along the six profiles evenly distributed across the study site, together with the GPS readings for each electrode position, were integrated together using the 3-D Oasis Montaj Software that helped in clear demarcation of the subsurface lithological layers as shown in The novelty of this work lies on the capability of integrated geophysical evaluation of the subsurface depths, and accurate quantification of the municipal, and industrial waste materials within the study area, with the invented 3-D standard rectangular prism. The method deployed for the study is faster and cost effective. The study is significant to the discontinuation, and prevention, of potential environmental hazards, and threats to human, environmental and the ecosystems around the study site, due to the pollutants fumes from the leachate plumes flowing from the mixtures of industrial and municipal waste materials. Knowledge of the hazards associated with landfills contaminants plumes is very relevant to safety of lives, the ecosystems and subsurface structural features. It is note worthy to consider the effects of these hazardous elements no matter how meager their quantity could be. The devastating long-term health effects are not to be permitted in the society ,4.In the prescribed geochemical analysis of the collected soil and water samples that enclosed the mixtures of the IWM and MSW, values of the various hazardous dissolved ions were acquired using the triplicate technique to support the findings from the geoelectrical tools deployed to delineates the zones of the contaminant plumes within the subsurface lithological units. The novel method invented to study the hazards associated with landfills contaminants plumes effect on the ecosystems, and threats to human, environmental and subsurface structural features underlain the dump site area, confirmed the presence of these potential environmental hazardous dissolved ions, except for the recorded values of the Mercury presence in the soil samples that was below the detected level (bdl) The technique for geochemical analysis and assessment of the soil and water samples collected at the study site, followed the laid down world standard provided for, by the 23rd Edition of Waste Water, published in 2017. Determinations of the samples' pH used the HACH Standard Method 8000, with DR 3900 VIS Spectral Photometer, used for the Chemical Oxygen Demand (COD) analysis, certified by the Malaysian Industrial Standard, MS ISO 17025 at an accredited laboratory, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia The results as reported in the main article, e.g.,A generated standard rectangular prism shape block model of the subsurface geophysical characteristics incorporated into the geological situation of the study area are produced with the aid of the 3-D Oasis Motaj modelling allows the quantification of the contaminant plumes' volume presented in 3.The invented novel methods adopted for the generation and quantification of leachate contaminant plumes in the forms of a standard rectangular prism shape block model of the subsurface geophysical characteristics, present a widespread guide for the rapid implementation in any part of the world irrespective of the terrain. The soil and water samples were collected at the same spot with known standardization that uses the triplicate technique of sample collections. Considering the economic gains from the novel method, this makes the novel method for leachate contaminant plumes quantifications less stressful, time saving, and does not require huge financial costs in comparison with other known traditional methods, e.g., the use of borehole wells. However, other methodological concerns for intending future users is in the technical know-how of the RES2DINV and the Oasis Montaj softwares that were deployed to quantify the contaminant plumes from leachate flows approximated to be about 312,000 mThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The initialization of complex cyber-physical systems often requires the interaction of various components that must start up with strict timing requirements on the provision of signals . In order to safely allow an independent development of components, it is necessary to ensure a safe decomposition, i.e. the specification of local timing requirements that prevent later integration errors due to the dependencies. We propose a high-level formalism to model local timing requirements and dependencies. We consider the problem of checking the consistency (existence of an execution satisfying the requirements) and compatibility (absence of an execution that reaches an integration error) of the local requirements, and the problem of synthesizing a region of timing constraints that represents all possible correct refinements of the original specification. We show how the problems can be naturally translated into a model checking and synthesis problem for timed automata with shared variables. Exploiting the linear structure of the requirements, we propose an encoding of the problem into SMT. We evaluate the SMT-based approach using MathSAT and show how it scales better than the automata-based approach using Uppaal and nuXmv.


Of all the various forms of adversity experienced during childhood, childhood maltreatment  is shown to have the largest impacts on mental health and well-being. Yet we still have a limited understanding of why some victims of early maltreatment suffer immense mental health consequences later on in the life course, while others are able to cushion the blow of these early insults. Using two waves of data from the National Survey of Midlife Development in the United States (MIDUS), this study considers change in religiosity as a buffer across three dimensions for victims of childhood abuse: religious importance, attendance, and the specific act of seeking comfort through religion. Results suggest that increases in religious comfort during adulthood are positively associated with adult mental health for victims of abuse, while decreases in religious comfort over time were associated with worse mental health. Changes in religious attendance and religious importance were not significant associated with mental health for victims of abuse. Taken together, my results show that the stress-moderating effects of religion for victims of childhood maltreatment are contingent on the stability or increases or decreases in religiosity over the life course, which has been overlooked in previous work.


There are persistent tensions of both a technical and political nature between Southeast Asia’s two major palm oil producers, Indonesia and Malaysia, and the sustainability governance mechanisms shaping global environmental and trade standards emerging from Europe. The establishment of the national Indonesian Sustainable Palm Oil (ISPO) certification standard in 2011 is a sign of discontent with the transnational Roundtable on Sustainable Palm Oil (RSPO) regime, sparking debate about the legitimacy of private governance models initiated by non-governmental organizations and companies in Europe. This article questions whether the adoption of sustainability norms by Indonesia signals normative convergence or the emergence of rival governance structures that challenge the state. Evidence suggests that elements of norm adoption and rival governance coexist in Indonesia and that ISPO certification is an ambiguous policy with degrees of internal incoherence. The ambiguous nature of ISPO certification gives rise to unresolved disputes over power and authority between various actors. This article shows how these disputes came into being by framing these dynamics as part of a long historical process. Novel insights are gained by employing the state transformation framework and the concept of governance rescaling. Within this framework, we argue that the ambiguous nature of the ISPO results from complex interrelated processes of fragmentation, decentralization and the internationalization of the Indonesian state. In January 2020 Indonesian President Joko Widodo (hereafter Jokowi) accused the European Union (EU) of provoking an unjust trade war that discriminates against palm oil production , a transnational private governance mechanism for sustainable palm oil supported in principle by many EU-based companies and non-governmental organizations (NGOs).This article offers a comprehensive understanding of the ISPO by drawing upon the state transformation framework and the concept of governance rescaling. To better understand the ISPO, we examine the evolution of the Indonesian state and the development of the palm oil industry in the country, including the competition for governance rescaling that follows the transformation of the state in the late 1990s that enabled the establishment of the RSPO and then the ISPO. To elaborate on this central point, this article proceeds in five parts. Part one looks at the existing debate on the nature of ISPO and the relationship between RSPO and ISPO to provide the context in which this article aims to contribute. The second part examines the links between state transformation frameworks and concepts of governance rescaling to conceptualize the ISPO in the context of global trade, followed by an explanation of the data collection methods and evidence. Part three looks at the development of Indonesia’s palm oil industry, especially in the post-1998 democratic transition and liberalization era. Part four examines the establishment of the RSPO as a response from competing networks of actors to the rapid expansion of the-* palm oil sector and the continuous tension within the RSPO. Part five examines the establishment of the ISPO by framing it as part of the continuous transformation of the Indonesian state. The authors conclude that the internationalization of the state apparatus leads to the further fragmentation of the Indonesian state bureaucracy that renders the ISPO an ambiguous and contentious policy that is ineffective on two fronts: sustainability as well as industry protection.There are divergent views on the relationship between the RSPO and the ISPO. By some measures, the establishment of the ISPO in 2011 indicates the proliferation and influence of global sustainability norms. EU normative power influences regulatory standards for global environmental and trade governance in line with renewable energy targets, though there are forms of institutional and networked opposition from Indonesia  urges the government to use the ISPO as a diplomatic instrument to overcome trade barriers against the commodity GAPKI . At the Studies about the nature of the ISPO and its connection to different public and private actors in Indonesia and beyond have touched upon these ambiguities directly and indirectly, although tend not to explicitly identify the ambiguity of the ISPO  and the start of a new era of export risk and contract cancellations. These written sources provide the plot and storyline to be connected and framed through theories of state transformation and governance rescaling.Evidence for this study comes from interviews and observations conducted periodically in Indonesia from 2015 to 2020, crosschecked with a wide range of written sources including statements, in-house publications, minutes of meetings, press releases and position papers from the RSPO, the ISPO, palm oil companies, trade associations such as GAPKI and the palm oil growers’ association (APKASINDO), government sources such as the Ministry of Agriculture, the Ministry of Trade, the Ministry of Foreign Affairs and the Office of the President, as well as civil society actors. The authors analysed selective Indonesian news coverage of the palm oil industry from 2009 to 2020, mainly from sources such as The authors conducted in-depth interviews during different stages of fieldwork in Indonesia  and the UK from 2015 to 2020. Interviewees were purposively chosen based on their expert knowledge of the palm oil industry and agribusiness in Indonesia, especially on the evolution of the RSPO and the ISPO. Semi-structured anonymized interviews were conducted so that participants could express their opinions freely. To obtain a comprehensive picture, the authors sampled elite informants as well as farming communities in Riau, Indonesia’s main palm oil producing province  in the Indonesian palm oil sector. To make it easier to follow, the storyline is presented in a semi-chronological manner.While industry actors often proclaim that palm oil is ‘a gift from God for Indonesia’  emissions being released annually. The report accuses global companies that source palm oil from Indonesia such as Unilever, Nestlé and Procter & Gamble of being complicit with environmental crimes  ran a lipstick from the Rainforest awareness campaign, followed shortly after by a Greenpeace campaign to hold the palm oil industry to account. A 2007 Greenpeace report entitled As consumer awareness about the environmental impacts of the palm oil industry rises in developed countries, so does the pressure placed on global brands which use palm oil in their products. In May 2008, Unilever responded to pressure by declaring its commitment to clean up the company’s supply chain Unilever . In MarcIntensive NGO campaigns and market pressures create a dilemma for producers and the companies that use palm oil in their products. Companies need a steady supply of CPO for their business. Replacing palm oil with rapeseed or soybean oil produced in Europe is challenging because palm oil is generally a more cost-effective and high yielding flex-crop. The question for companies is how to convince conscientious consumers to buy their products while maintaining a steady supply of palm oil. In this context, transnational actors such as international NGOs and multinational companies based in developed countries, especially in Europe, agreed to initiate the Roundtable on Sustainable Palm Oil (RSPO).Transnational private governance in the form of the RSPO has emerged as a significant benchmark for standards of sustainable palm oil, setting out key principles and criteria and developing a system for trade in certified sustainable palm oil Hai , 22. TheThe dominant role of Western NGOs and downstream actors in the palm oil supply chain in the RSPO is evident in the standards-making process. The RSPO works by establishing a standard for sustainable palm oil and creating a system to ensure compliance. As a roundtable, the standard conceptually should come from all the members. While formally this is true as all members have a voice in the process, the standard itself is set by developed countries. The proposed sustainability standard and adaptation strategy that were discussed in the preparatory meetings for the establishment of the RSPO in September 2002 in London were shaped by previous initiatives in Europe. In 1998 for instance, Unilever started to develop the indicators for sustainable palm oil, while in 2000 Migros, with the help of the WWF and Proforest, created supply chain standards that would form most of the RSPO’s principles  during President Yudhoyono’s two terms in office (2004 to 2014) was a powerful agency that pushed for reform in the industry and worked closely with domestic and international NGOs. A similar role, but with a different approach, is played by the Presidential Staff Office (KSP) under the current President Joko Widodo. While the Yudhoyono-era UKP4 approached sustainable palm oil production mostly in the context of international norms to protect the environment, and thus engaged in various international schemes such as reducing emissions from deforestation and forest degradation (the REDD project), the Widodo-era KSP focuses more on land conflicts and agrarian reform.The UKP4 is the agency responsible for the issuance of Presidential Instruction No. 10/2011 on the moratorium on forest conversion, a regulation that was heavily criticized by the palm oil industry. Despite this backlash, the moratorium was extended by President Yudhoyono in 2013 and retained under President Joko Widodo’s administration. UKP4 members intervened in the sector by revising some Ministry of Agriculture regulations, particularly those relating to the issuance of plantation permits Zuhri . This inMutually reinforcing factors of state transformation such as fragmentation and the internationalization of the state apparatus were seen in Yudhoyono’s environmental diplomacy. Indonesia’s high-profile environmental diplomacy was on display at the 2015 Paris climate conference when the country became one of the first developing countries to pledge to cut carbon dioxide emissions by 26%. Indonesia’s voluntary national contribution is incongruent with the realities of unsustainable practices on the ground and ever-growing palm oil production targets Taufik . The conThrough various policy commitments, reformist officials seek to mobilize international resources to support reform initiatives in the forestry sector. Examples include the Australia Forest Carbon Partnership (AU$ 70 million pledged), the German-sponsored emission reduction (REDD) pilot project (EUR 32.4 million), the UN-REDD initiative (US$ 5.6 million), the Korea-sponsored project for the adaptation and mitigation of climate change in forestry  and Australia’s agricultural research fund  , manifested in the 2004 Roundtable on Sustainable Palm Oil (RSPO), in order to sustain their perceived privilege in the national scale of governance. Indonesian trade associations and industry players exploit their connections with relevant government agencies such as the Ministry of Agriculture and the Ministry of Trade. At the same time, reformists within the state bureaucracy internalize global norms of sustainability and attempt to push reforms in the governance of the palm oil industry. This jockeying for power and influence resulted in the establishment the ISPO, an ambiguous policy that internalizes RSPO standards and mimics global and EU norms while simultaneously creating a rival form of governance to protect the palm oil industry from external pressures.The inherent ambiguity of the ISPO causes practical dilemmas on two fronts. The ISPO is generally ineffective in its implementation, to the disappointment of reformists within the Indonesian state, and at the same time is unable to serve as a legitimate alternative to the RSPO in the global market, to the disappointment of protectionists. The low level of acceptance of the ISPO by international markets explains why Indonesian palm oil companies tend to remain in the RSPO despite their criticisms of membership, representation and the interventionist nature of transnational private governance.Active campaigning by industry players with close political connections has elevated palm oil to the status of a strategic priority in Indonesian foreign policy. In October 2019, for instance, during her first speech as Indonesia’s Foreign Minister, Retno Marsudi specifically mentioned palm oil as a diplomatic priority. The foreign minister contends that palm oil is a strategic national commodity and the growth of the sector ‘is a fundamental matter because it is related to the livelihoods of more than 16 million people, especially small-scale farmers and their families’ (CNN Indonesia


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