Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.

During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Stakeholder Perceptions About Group B Streptococcus Disease and Potential for Maternal Vaccination in Low- and Middle-Income Countries.

BACKGROUND: To inform the World Health Organization's full value of vaccine assessment for group B Streptococcus (GBS) vaccines, a rapid literature appraisal was conducted to inform the operationalization of maternal GBS vaccination. We found limited published information on stakeholder perceptions of the public health importance of GBS disease and vaccination, and we therefore undertook a multicountry survey. METHODS: An online survey was conducted in late 2019 to collect information on stakeholders' awareness of GBS disease and the priority accorded to vaccination. The survey was distributed by email to 395 representatives of national pediatric, gynecology, and obstetrics associations, national immunization technical advisory groups (NITAGs), national regulatory agencies, academia, and United Nations organizations. RESULTS: Among 101 survey respondents from 66 countries, 36% were pediatricians, 25% obstetricians/gynecologists, 21% immunization specialists, and 18% other public health specialists. More than half (58%) of respondents reported being familiar with GBS disease as a public health problem; familiarity decreased by country income level. Knowledge of GBS disease was greatest in the Americas (68%) and Europe (66%) and lowest in Asia (13%-38%). Perception of GBS disease as a public health problem was highest among pediatricians (71%) and lowest among public health policy makers and NITAG members (30%) across country groupings. Approximately half of respondents (49%) considered the introduction of a GBS vaccine as a priority. CONCLUSIONS: The information obtained will inform the appropriate packaging and presentation of information to address stakeholder perceptions and promote evidence-based decision making on GBS vaccination.

A comprehensive group-based cognitive behavioural treatment for blood-injection-injury phobia.

OBJECTIVES: A key feature of blood-injection-injury (BII) phobia is activation of disgust responses, in addition to fear. Yet, standard treatments have largely neglected addressing disgust responses. The disorder is further complicated by fainting in 75% of sufferers. Moreover, treatments have been traditionally delivered in an individual format, which may not be as efficient as group treatment. The aim of this study was to develop a group-based programme for BII phobia, with components targeting fear, disgust, and fainting, to determine feasibility and effectiveness of such an intervention. METHODS: Participants took part in an 8-session, group-based Cognitive Behavioural Therapy (CBT) programme for BII phobia (N = 40). The key outcome measure was the Multidimensional Blood/Injury Phobia Inventory, which assesses a range of phobic stimuli and responses (including fear, disgust, and fainting). RESULTS: There were significant improvements, with large effect sizes, across symptoms over the course of treatment. Participants with higher disgust sensitivity reported higher pre-treatment symptom severity and greater life interference than those with lower disgust scores. Despite this, neither pre-treatment disgust sensitivity nor fainting history impacted on treatment response. For the first time, however, we showed that greater reductions in disgust to BII stimuli were associated with greater overall symptom reductions, highlighting the importance of disgust in the treatment of this disorder, and potentially others. CONCLUSION: Despite the heterogeneous nature of BII phobia, this group-based, modified CBT intervention was effective in reducing a variety of phobic responses, including fear, disgust, and fainting. PRACTITIONER POINTS: Disgust is a key maintaining factor in blood-injection-injury phobia, which clinicians should consider in their assessment and treatment of this disorder. There is little in the existing literature to guide clinicians in this regard. This study examined a novel group treatment for blood-injection-injury phobia which included strategies to target disgust, in addition to traditional CBT strategies to address fear and fainting. The treatment was feasible and acceptable. Symptoms of fear, disgust, and fainting reduced significantly over treatment. Changes in disgust symptoms were associated with overall symptom changes, however a control group is needed to determine the effects of individual treatment components and to make more robust conclusions about the benefits of this enhanced approach.

Insights into vaccine hesitancy from systems thinking, Rwanda.

OBJECTIVE: To investigate vaccine hesitancy leading to underimmunization and a measles outbreak in Rwanda and to develop a conceptual, community-level model of behavioural factors. METHODS: Local immunization systems in two Rwandan communities (one recently experienced a measles outbreak) were explored using systems thinking, human-centred design and behavioural frameworks. Data were collected between 2018 and 2020 from: discussions with 11 vaccination service providers (i.e. hospital and health centre staff); interviews with 161 children's caregivers at health centres; and nine validation interviews with health centre staff. Factors influencing vaccine hesitancy were categorized using the 3Cs framework: confidence, complacency and convenience. A conceptual model of vaccine hesitancy mechanisms with feedback loops was developed. FINDINGS: A comparison of service providers' and caregivers' perspectives in both rural and peri-urban settings showed that similar factors strengthened vaccine uptake: (i) high trust in vaccines and service providers based on personal relationships with health centre staff; (ii) the connecting role of community health workers; and (iii) a strong sense of community. Factors identified as increasing vaccine hesitancy (e.g. service accessibility and inadequate follow-up) differed between service providers and caregivers and between settings. The conceptual model could be used to explain drivers of the recent measles outbreak and to guide interventions designed to increase vaccine uptake. CONCLUSION: The application of behavioural frameworks and systems thinking revealed vaccine hesitancy mechanisms in Rwandan communities that demonstrate the interrelationship between immunization services and caregivers' vaccination behaviour. Confidence-building social structures and context-dependent challenges that affect vaccine uptake were also identified.

Patients' Perceptions of Barriers and Facilitators to the Adoption of E-Hospitals: Cross-Sectional Study in Western China.

BACKGROUND: As an innovative approach to providing web-based health care services from physical hospitals to patients at a distance, e-hospitals (ie, extended care hospitals through the internet) have been extensively developed in China. This closed health care delivery chain was developed by combining e-hospitals with physical hospitals; treatment begins with web-based consultation and registration, and then, patients are diagnosed and treated in a physical hospital. This approach is promising in its ability to improve accessibility, efficiency, and quality of health care. However, there is limited research on end users' acceptance of e-hospitals and the effectiveness of strategies aimed to prompt the adoption of e-hospitals in China. OBJECTIVE: This study aimed to provide insights regarding the adoption of e-hospitals by investigating patients' willingness to use e-hospitals and analyzing the barriers and facilitators to the adoption of this technology. METHODS: We used a pretested self-administered questionnaire and performed a cross-sectional analysis in 1032 patients across three hierarchical hospitals in West China from June to August 2019. Patients' sociodemographic characteristics, medical history, current disease status, proficiency with electronic devices, previous experience with web-based health services, willingness to use e-hospitals, and perceived facilitators and barriers were surveyed. Multiple significance tests were employed to examine disparities across four age groups, as well as those between patients who were willing to use e-hospitals and those who were not. Multivariate logistic regression was also performed to identify the potential predictors of willingness to use e-hospitals. RESULTS: Overall, it was found that 65.6% (677/1032) of participants were willing to use e-hospitals. The significant predictors of willingness to use e-hospitals were employment status (P=.02), living with children (P<.001), education level (P=.046), information technology skills (P<.001), and prior experience with web-based health care services (P<.001), whereas age, income, medical insurance, and familiarity with e-hospitals were not predictors. Additionally, the prominent facilitators of e-hospitals were convenience (641/677, 94.7%) and accessibility to skilled medical experts (489/677, 72.2%). The most frequently perceived barrier varied among age groups; seniors most often reported their inability to operate technological devices as a barrier (144/166, 86.7%), whereas young participants most often reported that they avoided e-hospital services because they were accustomed to face-to-face consultation (39/52, 75%). CONCLUSIONS: We identified the variables, facilitators, and barriers that play essential roles in the adoption of e-hospitals. Based on our findings, we suggest that efforts to increase the adoption of e-hospitals should focus on making target populations accustomed to web-based health care services while maximizing ease of use and providing assistance for technological inquiries.

The pluripotent state in mouse and human.

In the mouse, naïve pluripotent stem cells (PSCs) are thought to represent the cell culture equivalent of the late epiblast in the pre-implantation embryo, with which they share a unique defining set of features. Recent studies have focused on the identification and propagation of a similar cell state in human. Although the capture of an exact human equivalent of the mouse naïve PSC remains an elusive goal, comparative studies spurred on by this quest are lighting the path to a deeper understanding of pluripotent state regulation in early mammalian development.

Countdown to 2015 and beyond: fulfilling the health agenda for women and children.

The end of 2015 will signal the end of the Millennium Development Goal era, when the world can take stock of what has been achieved. The Countdown to 2015 for Maternal, Newborn, and Child Survival (Countdown) has focused its 2014 report on how much has been achieved in intervention coverage in these groups, and on how best to sustain, focus, and intensify efforts to progress for this and future generations. Our 2014 results show unfinished business in achievement of high, sustained, and equitable coverage of essential interventions. Progress has accelerated in the past decade in most Countdown countries, suggesting that further gains are possible with intensified actions. Some of the greatest coverage gaps are in family planning, interventions addressing newborn mortality, and case management of childhood diseases. Although inequities are pervasive, country successes in reaching of the poorest populations provide lessons for other countries to follow. As we transition to the next set of global goals, we must remember the centrality of data to accountability, and the importance of support of country capacity to collect and use high-quality data on intervention coverage and inequities for decision making. To fulfill the health agenda for women and children both now and beyond 2015 requires continued monitoring of country and global progress; Countdown is committed to playing its part in this effort.

From evidence to action to deliver a healthy start for the next generation.

Remarkable progress has been made towards halving of maternal deaths and deaths of children aged 1-59 months, although the task is incomplete. Newborn deaths and stillbirths were largely invisible in the Millennium Development Goals, and have continued to fall between maternal and child health efforts, with much slower reduction. This Series and the Every Newborn Action Plan outline mortality goals for newborn babies (ten or fewer per 1000 livebirths) and stillbirths (ten or fewer per 1000 total births) by 2035, aligning with A Promise Renewed target for children and the vision of Every Woman Every Child. To focus political attention and improve performance, goals for newborn babies and stillbirths must be recognised in the post-2015 framework, with corresponding accountability mechanisms. The four previous papers in this Every Newborn Series show the potential for a triple return on investment around the time of birth: averting maternal and newborn deaths and preventing stillbirths. Beyond survival, being counted and optimum nutrition and development is a human right for all children, including those with disabilities. Improved human capital brings economic productivity. Efforts to reach every woman and every newborn baby, close gaps in coverage, and improve equity and quality for antenatal, intrapartum, and postnatal care, especially in the poorest countries and for underserved populations, need urgent attention. We have prioritised what needs to be done differently on the basis of learning from the past decade about what has worked, and what has not. Needed now are four most important shifts: (1) intensification of political attention and leadership; (2) promotion of parent voice, supporting women, families, and communities to speak up for their newborn babies and to challenge social norms that accept these deaths as inevitable; (3) investment for effect on mortality outcome as well as harmonisation of funding; (4) implementation at scale, with particular attention to increasing of health worker numbers and skills with attention to high-quality childbirth care for newborn babies as well as mothers and children; and (5) evaluation, tracking coverage of priority interventions and packages of care with clear accountability to accelerate progress and reach the poorest groups. The Every Newborn Action Plan provides an evidence-based roadmap towards care for every woman, and a healthy start for every newborn baby, with a right to be counted, survive, and thrive wherever they are born.

Advancing social and economic development by investing in women's and children's health: a new Global Investment Framework.

A new Global Investment Framework for Women's and Children's Health demonstrates how investment in women's and children's health will secure high health, social, and economic returns. We costed health systems strengthening and six investment packages for: maternal and newborn health, child health, immunisation, family planning, HIV/AIDS, and malaria. Nutrition is a cross-cutting theme. We then used simulation modelling to estimate the health and socioeconomic returns of these investments. Increasing health expenditure by just $5 per person per year up to 2035 in 74 high-burden countries could yield up to nine times that value in economic and social benefits. These returns include greater gross domestic product (GDP) growth through improved productivity, and prevention of the needless deaths of 147 million children, 32 million stillbirths, and 5 million women by 2035. These gains could be achieved by an additional investment of $30 billion per year, equivalent to a 2% increase above current spending.

Ontogeny of small RNA in the regulation of mammalian brain development.

BACKGROUND: MicroRNAs (miRNAs) play a pivotal role in coordinating messenger RNA (mRNA) transcription and stability in almost all known biological processes, including the development of the central nervous system. Despite our broad understanding of their involvement, we still have a very sparse understanding of specifically how miRNA contribute to the strict regional and temporal regulation of brain development. Accordingly, in the current study we have examined the contribution of miRNA in the developing rat telencephalon and mesencephalon from just after neural tube closure till birth using a genome-wide microarray strategy. RESULTS: We identified temporally distinct expression patterns in both the telencephalon and mesencephalon for both miRNAs and their target genes. We demonstrate direct miRNA targeting of several genes involved with the migration, differentiation and maturation of neurons. CONCLUSIONS: Our findings suggest that miRNA have significant implications for the development of neural structure and support important mechanisms that if disrupted, may contribute to or drive neurodevelopmental disorders.

From joint thinking to joint action: a call to action on improving water, sanitation, and hygiene for maternal and newborn health.

Yael Velleman and colleagues argue for stronger integration between the water, sanitation, and hygiene (WASH) and maternal and newborn health sectors. Please see later in the article for the Editors' Summary.

Ending of preventable deaths from pneumonia and diarrhoea: an achievable goal.

Global under-5 mortality has fallen rapidly from 12 million deaths in 1990, to 6·9 million in 2011; however, this number still falls short of the target of a two-thirds reduction or a maximum of 4 million deaths by 2015. Acceleration of reductions in deaths due to pneumonia and diarrhoea, which together account for about 2 million child deaths every year, is essential if the target is to be met. Scaling up of existing interventions against the two diseases to 80% and immunisation to 90% would eliminate more than two-thirds of deaths from these two diseases at a cost of US$6·715 billion by 2025. Modelling in this report shows that if all countries could attain the rates of decline of the regional leaders, then cause-specific death rates of fewer than three deaths per 1000 livebirths from pneumonia and less than one death per 1000 livebirths from diarrhoea could be achieved by 2025. These rates are those at which preventable deaths have been avoided. Increasing of awareness of the size of the problem; strengthening of leadership, intersectoral collaboration, and resource mobilisation; and increasing of efficiency through the selection of the optimum mix of a growing set of cost-effective interventions depending on local contexts are the priority actions needed to achieve the goal of ending preventable deaths from pneumonia and diarrhoea by 2025.