Primary health care offers a cost–effective route to achieving universal health coverage (UHC). However, primary health-care systems are weak in many low- and middle-income countries and often fail to provide comprehensive, people-centred, integrated care. We analysed the primary health-care systems in 20 low- and middle-income countries using a semi-grounded approach. Options for strengthening primary health-care systems were identified by thematic content analysis. We found that: (i) despite the growing burden of noncommunicable disease, many low- and middle-income countries lacked funds for preventive services; (ii) community health workers were often under-resourced, poorly supported and lacked training; (iii) out-of-pocket expenditure exceeded 40% of total health expenditure in half the countries studied, which affected equity; and (iv) health insurance schemes were hampered by the fragmentation of public and private systems, underfunding, corruption and poor engagement of informal workers. In 14 countries, the private sector was largely unregulated. Moreover, community engagement in primary health care was weak in countries where services were largely privatized. In some countries, decentralization led to the fragmentation of primary health care. Performance improved when financial incentives were linked to regulation and quality improvement, and community involvement was strong. Policy-making should be supported by adequate resources for primary health-care implementation and government spending on primary health care should be increased by at least 1% of gross domestic product. Devising equity-enhancing financing schemes and improving the accountability of primary health-care management is also needed. Support from primary health-care systems is critical for progress towards UHC in the decade to 2030. Health systems around the world are facing increasingly complex challenges, such as the growing burden of chronic noncommunicable disease and related commercial determinants of health , new epidemics and antimicrobial resistance. As a result, the focus has shifted from curative care to health promotion and disease prevention, and new models of primary health-care service delivery, financing and governance have been developed. ,–Primary health-care systems are fundamental for responding to pandemics, such as the coronavirus disease 2019 (COVID-19) pandemic, and for maintaining essential health services. Moreover, primary health-care policies and interventions can enhance equity and support disadvantaged groups that have little coverage by essential services.In response to the 2018 Declaration of Astana,Traditionally, the core primary health-care principles of community engagement and empowerment, and of strengthening local health systems have been neglected at the expense of innovative and rapid interventions.In low- and middle-income countries, the policies and system reforms needed to develop a more comprehensive and responsive primary health-care system must be supported by context-appropriate evidence. At present, there are gaps in knowledge about key aspects of primary health-care systems, including: quality, safety and performance management; policies and governance; organization and models of care; and financing.Our aim was to use data from these 20 case studies to conduct a multicountry analysis of the system-level determinants of primary health-care performance and to derive lessons for primary health-care implementation, policy-making and systems reform that can aid efforts to strengthen primary health care in low- and middle-income countries.In 2015, the Alliance for Health Policy and Systems Research convened an expert consultation on primary health-care systems in low- and middle-income countries, which involved global and country experts, including policy-makers and researchers from low- and middle-income countries.The case studies were developed using a mixed-methods approach, which involved secondary data sets and primary data collected through engagement with primary health-care stakeholders, including in-depth interviews and focus group discussions. When primary health-care-specific indicators were not available as secondary data, the country teams produced estimates using the latest available validated metrics. Studies were reported using a standardized template.We carried out the data analysis using a semi-grounded approach and applied two primary health-care frameworks as coding matrices in the deductive component: the PRIMASYS framework and the Primary Health Care Performance Initiative framework.Box 1The PRIMASYS framework included: (i) primary health-care policy and systems domains ; (ii) system governance and architecture; (iii) financing; (iv) human resources; (v) planning, implementation, monitoring, evaluation and information systems; (vi) regulatory processes; (vii) community participation; (viii) service delivery; (ix) infrastructure and supplies; and (x) primary health-care system strengthening priorities.The Primary Health Care Performance Initiative framework was used to assess data on the domains of: (i) primary health-care systems ; (ii) inputs, such as drugs, supplies, infrastructure, health management information systems, workforce and funds; and (iii) service delivery, including access to care, care availability, people-centred care, organization and management.PRIMASYS: Primary Health Care Systems.In 11 of the 20 countries studied (55%), urbanization was growing and overall mortality was decreasing as incomes rose. In all countries, noncommunicable diseases and associated mortality were both increasing. The proportion of deaths due to noncommunicable disease was greater in countries with higher incomes . In 12 cBox 2In Georgia, the cost of medicines for outpatients traditionally made up a high proportion of out-of-pocket health expenditure. In response, the government expanded the UHC benefits package in 2017 to cover noncommunicable disease drugs for four chronic conditions that accounted for more than 80% of the disease burden in the country, thereby including the prevention and management of noncommunicable diseases in primary health-care services.UHC: universal health coverage.In 70% of countries (14/20), primary health-care services were fragmented: they involved a wide range of public and private providers, nongovernmental organizations, faith-based organizations and traditional medicine providers . ServiceBox 3In Bangladesh, different government departments ran primary health-care services in urban and rural areas with little coordination between them, and the private sector was very strong in urban areas.In Mexico, despite ongoing efforts to integrate the three main, primary health-care, public sector institutions using health-care service exchange agreements, fragmentation remained.In Nigeria, the coordination of primary health care was limited by the presence of a wide array of providers, including faith-based organizations, nongovernmental organizations, the private sector, local government authorities, and national and state public institutions.Commonalities in primary health-care financing across countries included: (i) policy commitments to reducing catastrophic health expenditure and improving equity; and (ii) movements towards financial protection and UHC and towards increasing primary health-care coverage using a risk-pooling mechanism. In 2014, out-of-pocket expenditure was over 40% of total health expenditure in 10 of the 20 countries .24 In 65Current strategies to reduce out-of-pocket and catastrophic health expenditure are listed in Box 4abolish user fees and implement free maternal, newborn and child health services as a priority;fund services and infrastructure in poor, rural or remote areas;provide government-funded health insurance for poor people and for vulnerable population groups, such as refugees;level health insurance premiums in a progressive way;regulate health professionals’ service fees;ensure providers of contracted-out services  provide services at prescribed rates; andestablish a cash transfer system that targets certain services  or poorer households and ensure government resources are distributed on an equitable basis (e.g. according to need or remoteness).Overall, government funding as a percentage of gross domestic product was generally low and often health systems’ budgets were not aligned with indicators of workload or resource needs.The licensing of primary health-care facilities presented a mixed picture. Good processes were reported in some countries, whereas systems were very weak or nonexistent in others . In 70% Box 5In Ghana, different bodies were responsible for regulating health personnel, drugs and facilities, there was poor coordination between institutions, and monitoring and enforcement were difficult.In Kenya, under-resourced regulators were not able to prevent facilities operating without licences or with unqualified staff.Box 6In Indonesia, only accredited facilities could receive payments from the national insurance body and primary health-care centres were accredited only if their staff had undergone special training in midwifery emergencies.In Lebanon, contractual agreements between the health ministry and primary health-care facilities covered the supply of essential medicines. The contractual process was linked to the accreditation of primary health-care providers and dispensaries, which had to maintain care quality.Fifteen countries (75%) had a national oversight body (similar to the United States Food and Drug Administration) responsible for regulating the manufacture, importation, registration, distribution and pricing of drugs and, sometimes, equipment. In 45% (9/20), the regulatory system was weak as there was limited capacity to monitor or enforce regulations, which led to problems with substandard and counterfeit drugs. Most countries (13/20) had issues with supply chain management that affected drug stocks and, in many countries, the regulation of traditional medicine was an emerging challenge. Improvements in the effectiveness of regulation have occurred where financial incentives were linked to regulation and quality improvement (e.g. in Indonesia and Lebanon) and where there was strong community involvement and oversight (e.g. in Ethiopia). In all countries, quality improvement was an area of weakness: little effort was being made to improve primary health-care performance and there were few incentives. In 70% (14/20), quality improvement structures and processes were in place but there were problems with benchmarking standards, funding for quality improvements and achieving gains in outcomes and performance.Frequently, primary health-care planning took place at the district level and was consolidated at regional and national levels – all countries had a national planning and budgeting process. In many cases, planning was a top-down process and there was little participation by low-level workers or communities. Consequently, primary health-care policy was not responsive to local needs or capacities. The main primary health-care policy issues were: adopting an integrated or sector-wide approach; decentralization; accountability for performance ; and socBox 7In Ethiopia, health plans were embedded in five-year strategic development plans for growth and transformation. These plans were developed through extensive consultations that included communities and multiple stakeholders. Districts had autonomy, which allowed for both bottom-up and top-down planning processes. Districts could argue for resources during the budgetary process and district committees could develop health plans to suit the local context.In 2017, the number of nurses and doctors combined ranged from 0.40 per 1000 population in the United Republic of Tanzania to 6.94 per 1000 in South Africa. In all 20 countries, the distribution of the health workforce, especially doctors and nurses, was uneven. In particular, rural areas were underserved: poor working conditions in rural areas was a disincentive in 35% of countries (7/20), whereas centralized decision-making and financial control were bottlenecks to recruitment and deployment in others. Absenteeism was common and was linked to poor working conditions, the absence of incentives and nonfunctioning regulatory and accountability mechanisms, particularly for doctors. Five countries  had more doctors than nurses, whereas nurses predominated in all African countries studied. Professionals frequently worked simultaneously in both private and public sectors, which contributed to inequitable coverage. Traditional health practitioners were generally unregulated but Ghana, South Africa, Sri Lanka and Thailand were addressing regulation.Two types of structure for community participation were identified: (i) elected representatives at national, regional, district and local government levels; and (ii) appointed community participation bodies that play an advisory role. Community bodies  existed in 55% of countries (11/20) but wielded little power or influence. In countries where health services had largely been privatized , local government and community bodies had limited oversight of primary health-care services.Our multicountry assessment found commonalities in the performance of primary health-care systems in low- and middle-income countries, derived lessons for improving performance in these countries and identified strategies for enhancing primary health-care policy-making and systems. Substantial improvements in primary health-care systems had been made in the 20 countries studied, including better community-based services. Overall, however, the effort and investment devoted to strengthening health promotion and prevention was not commensurate with the swiftly increasing morbidity and mortality associated with noncommunicable diseases. Frequently, a primary focus on curative services undermined the comprehensiveness and continuity of primary health care. ,Although primary health care was given a high priority in policy-making and agenda-setting in the countries studied, in practice it was limited by inadequate human and financial resources, by the limited availability and suboptimal quality of commodities, and by weak support systems. The predominant primary health-care delivery approach remained the provision of an essential package of health services and the principal focus was on high-impact, efficient and equity-enhancing interventions and services. Yet, there were weaknesses with the comprehensiveness, integration and quality of essential primary health-care interventions: for example, the inclusion of essential interventions in planned packages of primary health-care services was uneven and coverage was limited. Moreover, inequitable resource allocation and a lack of regulation and accountability also impeded the implementation and scaling up of high-quality primary health-care services. A UHC intervention compendium is being developed by WHO to inform the development and redesign of health benefits packages, while recognizing that the implementation of essential primary health-care services can vary across countries according to context, priorities, resources and capacities.Our analysis also highlights the importance of primary health-care systems being adaptive so that services can be scaled up and funding can be increased when more domestic resources become available. Moreover, the inclusion of essential primary health-care interventions in UHC benefits packages should be appropriate to the context, should be informed by evidence from local, disaggregated data and real-world knowledge, and should consider the views of primary health-care stakeholders .,In addition, our study identified gaps in the measurement and reporting of primary health-care-sensitive indicators that can weaken the monitoring, development and accountability essential for improving primary health care. Our findings corroborate previous evidence that more sensitive metrics of primary health care, including financial indicators, are required and confirm the importance of strengthening health information and surveillance systems.Support for CHWs could be strengthened by providing adequate resources and effective referral systems, by ensuring proper recognition, training and supervision, and by enforcing regulations. Although guidance is now available on how health policy and systems can help improve community health worker programmes,Regulation and accountability are important for mixed health systems that involve fragmented, heterogeneous, private-sector provision, such as systems where the private sector is dominant , systems with private-sector hospitals and clinics for better-off people and systems with extensive use of private drug shops .Maintaining inclusive, universal mechanisms that protect against high out-of-pocket spending remains a challenge, for example, in countries with good service coverage but a high level of financial hardship.Applying a complex, adaptive, health-systems approach to primary health care can also illuminate the interdependency of primary health-care domains and the system-level effects of primary health-care interventions.Our study’s findings were limited by the skewed distribution of the PRIMASYS countries towards middle-income countries. Moreover, although the case studies addressed policy- and systems-level determinants of primary health care, the nature and complexity of the evidence made it difficult to associate the performance of a primary health-care system or an improvement in primary health-care policy or implementation with a specific factor or initiative. In addition, countries used different definitions of primary health care: for example, whether or not district hospitals are included in primary health care. The analysis was also limited by the absence, under-reporting and suboptimal quality of data on primary health-care performance. Finally, we acknowledge that primary health-care systems and policies might have evolved since we collected the data.We have identified several priorities to improve primary health-care systems in low- and middle-income countries. In particular, communities should be engaged in a meaningful manner and primary health care should better adapt to evolving population needs. These actions will improve the people-centred nature and responsiveness of health systems. As public resources increase, primary health-care systems are well positioned to support the development of schemes that provide essential packages of health services and UHC benefits. In fact, strengthening primary health-care interventions and existing models for providing essential packages of health services are critical for achieving UHC and other health-related sustainable development goals between 2020 and 2030. Greater transparency and public accountability are also vital for setting priorities for essential benefits packages and primary health-care policy-making and for ensuring equity and human rights. For example, some low- and middle-income countries have embedded a rights-based approach to strengthening primary health-care systems by including sexual and reproductive health and rights interventions in primary health-care and UHC policies.The COVID-19 pandemic demonstrates that strong primary health care is the cornerstone of preparedness and response planning, particularly for infection prevention and control. In addition, primary health care can act as a strategic entry point for multisectoral initiatives and for addressing issues around the political, commercial and legal determinants of health. For instance, the rapid rise in noncommunicable disease is expected to impede poverty reduction initiatives in low-income countries because household expenditure on health care will increase. By promoting the integration of health and social services, multisectoral collaboration, continuity of care and equity-enhancing schemes, primary health care can help address these complex challenges and accelerate progress towards UHC and the sustainable development goals.


There is a recognized increase in vulnerability to psychosis in autistic people (AP). However, the construct of psychosis (particularly schizophrenia) contains several distinct factors, making understanding the relationship between autism and psychosis complex. Previous research has suggested that affective lability may be particularly related to psychotic experiences for AP who have experienced psychosis (AP-P). There is also a suggestion that psychosis might be a state of extreme (over)empathizing, perhaps related to emotional processes.We recruited three groups: AP-P (N = 23), a group of AP who had not experienced psychosis  and a neurotypical control group . Participants completed measures of autistic traits, schizotypal traits (as a proxy for psychosis-proneness), emotional processes, and perspective taking . As well as comparisons between groups, regression analyses were used to understand the influence of dependent variables on schizotypal traits.We found that AP-P had significantly higher rates of schizotypy (positive and disorganized), as well as higher rates of emotional difficulties. Across all groups, affective lability had a positive and significant association with positive and disorganized schizotypal traits. Differences in perspective taking between groups were small and generally non-significant, particularly in adjusted comparisons; additionally, its impact on schizotypy was small and non-significant.Our findings suggest that positive and disorganized schizotypy, in particular, have a relationship with affective lability. This, in turn, supports the idea of emotional processes as related to the development of schizotypal traits and psychosis across all individuals, regardless of autism diagnostic status. We found no evidence of empathy relating to any subscale of schizotypy, or the total schizotypy score. We contend that emotional processes should be considered in exploration of the relationship between autism and schizotypy in future. This may help to explain some of the findings of overlap between these constructs in previous research. Factors known to affect neurodevelopment of emotion systems such as history of early trauma, challenges during pregnancy and birth, and early childhood experiences of adversity during critical windows of development need further consideration in future research. Autism spectrum disorders (ASDs) are life-long neurodevelopmental conditions affecting an individual’s perception of, and interaction with, the world . ASD refChisholm and colleagues  reviewedIn order to understand the relationship between ASD and psychosis, researchers have attempted to map ASD traits and psychotic traits into the same conceptual ‘space’. A personality construct called schizotypy has been used as a proxy for ‘psychosis-proneness’ . SchizotUsing factor analysis, two of the largest studies in this area drew differing conclusions about the relationship between autistic and schizotypal traits. Dinsdale et al.  favored A factor that may be involved in the relationship between schizotypal and autistic traits is empathy. Empathy is a complex skill that involves predicting and reacting to how you believe another person will feel. It has been conceptualized as having broadly two factors: cognitive empathy ; and affective empathy . The diametric model of ASD and schizophrenia suggests that increased empathy may be linked to increased risk of psychosis, through a mechanism of overly empathizing with the perceived contents of others’ minds , 20. EmpIn order to contribute to better understanding of this area, and the potential interactions between emotion regulation difficulties, affective lability, schizotypy, and autistic traits, we have attempted to investigate these concepts in the same theoretical space. The following hypotheses were tested:H1. AP-P will use less effective emotion regulation strategies and report more affective lability than AP-NPH2. AP-P will be better at perspective taking than AP-NPH3. Schizotypal traits will be higher in AP with a history of psychosis (AP-P) compared with AP who have no history of psychosis (AP-NP)H4. Emotion regulation difficulties and affective lability will be associated with higher schizotypal scores across participant groupsEthical approval for the study was given by the North of Scotland NHS Research Ethics Committee in January 2016. The study was conducted between January 2016 and April 2017.via participation in previous research or via social media advertising, and were incentivized with the opportunity to participate in a prize draw.An observational study comparing self-reported measures of autistic traits, schizotypal traits, and emotional processes between participant groups. Participants were recruited either Participants were all adults (aged 18 or older), and were required to have English as their first language. They were recruited to three groups.via the Autism Research Centre’s (ARC’s) database (https://www.autismresearchcentre.net/), they were asked to confirm they had no significant mental health history. ASD diagnosis was not confirmed, but the database is maintained by a respected research group who check participant eligibility: thus, we considered this group representative of AP. A total of 59 participants were recruited.Recruited Consisted of:participants invited from previous research ;new participants self-identified through the ARC database.Participants from the ARC database were screened using the Diagnostic Interview for Psychosis (DIP-DM) , which gA total of 23 participants were recruited from both sources.Participants were recruited through social media advertising. Participants were not formally screened, but were asked to confirm they had neither history of ASD diagnosis nor any significant mental health history. A total of 41 participants were recruited.The following self-report measures were used:Autism Spectrum Quotient (AQ) (ent (AQ) : A 50-itent (AQ) , 34, altSchizotypal Personality Questionnaire—Brief Revised (SPQ-BR) ((SPQ-BR) : A 32-it(SPQ-BR) ; however(SPQ-BR) .Affective Lability Scale-18 (ALS-18) ((ALS-18) : This 18(ALS-18) .Emotion Regulation Questionnaire-9 (ERQ-9) ( (ERQ-9) : This is (ERQ-9) , and hasQuestionnaire of Cognitive and Affective Empathy (QCAE) , perspecAll participants were asked their age and gender. No other demographics were collected.Data were analyzed using R (R Core Team).Measures section. Univariate comparisons between AP-NP and AP-P were made using Fisher’s Exact test and t-tests. Univariate comparisons across all three groups were made using Fisher’s Exact test and one-way analysis of variance (ANOVA). Non-parametric versions  of these comparisons, along with appropriate descriptive statistics, are reported in the Supplementary Materials. These sets of analyses are compared to determine robustness of conclusions.Initially, the AP-NP, AP-P and NC groups were compared in terms of gender, age and the measures introduced in Descriptive Statistics and Univariate Comparisons section. Adjusted group comparisons were made using linear regression, with a separate regression for each scale. Independent variables  included: Participant Group; Gender; Age; ALS-18 Total; ERQ-9 Cognition Reappraisal; ERQ-9 Emotion Suppression; QCAE Perspective Taking.To begin examining our hypotheses, unadjusted comparisons of affective and empathy measures between groups were conducted as outlined in Linear regression models were used to look for adjusted relationships with SPQ-BR scales . In these models, the following independent variables were included: Participant Group; Gender; Age; AQ Total; ALS-18 Total; ERQ-9 Cognition Reappraisal; ERQ-9 Emotion Suppression; QCAE Perspective Taking. In each regression, multicollinearity was examined using variation inflation factors (VIFs).For each regression, a key area of interest was the potential interaction between Participant Group and each of the dependent variables. To explore these interactions, we fitted a series of models that extended each base regression with all possible combinations of (first order) interactions between Group and other dependent variables. Fitted models were compared using the second-order Akaike Information Criterion (AICc) , 43, wheWhere interactions were fitted, the Effects  package Descriptive Statistics and Univariate Comparisons, Group Comparisons of ALS-18, ERQ-9 and QCAE Perspective Taking and Schizotypy Regressions sections, replacing ALS-18 Total with its subscales.Having conducted the analysis outlined in the above sections, affective lability was highlighted as a key factor. Thus, we repeated the analyses described in Pearson’s correlation coefficient (r) was used as the primary measure of effect size. Generally, Pearson’s r was calculated from t-statistics , using aGroup sizes, descriptive statistics and tests of differences between groups  are reported in Gender split was similar between AP-NP and NC groups; however, there were significantly (proportionately) fewer women in AP-P compared to AP-NP. The AP-NP group was significantly older than the AP-P and NC groups. A large and significant difference in AQ scores was found between AP-NP and AP-P. As would be expected, the NC group had the lowest AQ score. Between AP-NP and AP-P groups, across SPQ-BR scales, the only significant difference was found on the Positive scale. NC had the lowest means across all SPQ-BR scales. The same differences between groups were found in the equivalent non-parametric analysis reported in Detailed unadjusted comparisons of these measures are given in Detailed adjusted comparisons of these measures are given in not have the lowest AICc; however, its fit is only negligibly worse than the best fitting model (765.52 versus 764.38)—given this fit, and the lack of consistent interactions included across the other models, we chose the model with no interactions for reasons of parsimony. Residual plots indicate no substantial problems with model fit. Except for one variable, all VIFs are below four; the remaining variable has a VIF of just over five.Regression models within two of the best AICc for each SPQ-BR scale  are shown in The fit of the final models is given in Between groups, schizotypy differences are minimal. For total schizotypy, there was a significant difference between NC and AP-P, with AP-P scoring approximately 11 units higher. On positive schizotypy, AP-P scored significantly higher than both AP-NP and NC. Other group differences were small and non-significant.Across Positive, Negative and Disorganized scales, AQTotal was only significantly associated with negative schizotypy. This was the second largest effect on the Negative scale, and likely drives the significant relationship with AQTotal on the Total scale.ALS-18 Total had a significant and the largest impact within each of the Positive and Disorganized scales. Accordingly, ALS-total significantly impacts on SPQ-BR Total and has the largest impact thereon. ERQ-9 Emotion Suppression had a significant effect on the Positive and Negative scales, and had the third and largest impact on these respectively. These relationships likely drive the significant impact of ERQ-9 Emotion Suppression on SPQ-BR Total.There were no significant relationships between the SPQ-BR subscales and gender, age, ERQ-9 Cognitive Reappraisal, or QCAE Perspective Taking respectively.not proof for the contrary).With no interactions with Participant Group, we had no evidence for effects noted above differing by Participant Group  than NC, although not all of these comparisons were statistically significant.The premise of this hypothesis is that emotional processes are involved with or impacted by schizotypy, regardless of other factors. This hypothesis was supported. Affective lability has a significant and positive association with overall schizotypy ; further, this is also the strongest association with schizotypy among the variables considered here . This suggests an important relationship between these constructs. Exploratory analysis of the subscales of the ALS-18 suggests that different affective processes may be related to different schizotypal traits. For example, we found that switches between anxiety and depression (ALS-18 anxiety/depression) were more strongly associated with Positive and Negative schizotypy. Switches between depression and elation associated more with Disorganized schizotypy. We also found that, as predicted, Emotional Suppression is significantly associated with greater overall schizotypy, and specifically Positive and Negative schizotypy.It is important to note that the relationship between emotional processes and negative schizotypy is complex and appears to be affected by the presence of an ASD diagnosis. The two group by measure interactions that we found in the analyses both related to negative schizotypy, and in both cases, the AP groups showed a different relationship between affective lability, emotion regulation, and negative schizotypy, when compared to NC. This suggests that there may be something fundamentally different in the way that AP report experiencing and managing anger. It is also interesting to consider how AP, who show more negative schizotypal traits, report better use of an emotion regulation strategy that relies on logically appraising the situation . The finding may indicate a specific relevance of or different understanding of the descriptions of Cognitive Reappraisal between AP and NC, for example. This requires further investigation.Potentially impacting on generalizability, this study’s sample sizes were small. This was partially a by-product of selecting a rare dual diagnostic group such as AP-P, and also the nature of the study as exploratory investigation of a novel hypothesis. The sample size particularly impacts on the statistical power to detect interactions. Conclusions, particularly based on interactions, should be interpreted with caution given the small sample size. It may be helpful to consider our results as preliminary in light of these limitations, and further research is clearly required to confirm and expand on them.Recruitment methods varied between groups, potentially introducing bias: for example, mental status relating to autism and psychosis were retrospectively determined, which may produce bias and impact on the results of the study. Additionally, further screening of individuals may have been useful: AP-NP and NC groups were not screened, and so may have mis-represented their diagnostic status. Psychopathology screening would have helped rule out other confounding relationships. However, additional screening would have added to participant burden and so may have not been as useful as hoped as we are not aware of evidence to suggest research participants have been found to under-represent their mental health status.Given the limitations of the small sample and limited screening, but the interesting relationships highlighted, future research replicating these results would be valuable. Increased screening of participants to confirm group diagnoses and rule out general psychopathology would help reduce bias from unmeasured confounders. Controlling for additional demographic variables may also further reduce bias. Other designs—such as matching—might also be considered.As found in previous research, there appears to be a complex relationship between negative schizotypy, disorganized schizotypy, and autistic traits, and our results have suggested that these traits correlate with emotional processing differences. Future studies would benefit from comparing AP-P to other populations with psychosis to further understanding in this area.It seems plausible to us that emotional processes, particularly affective lability, add to the model of relationship between autistic and schizotypal constructs. To our knowledge, these factors have not previously been considered in this research field. Lability involving anxious emotional states is associated with positive and negative schizotypy, while lability involving elated emotional states is associated with disorganized schizotypy, suggesting that different emotional experiences may give rise to or be caused by different patterns of thought or behavior. AP as a whole in our study reported significantly higher negative schizotypal traits than NC, replicating previous findings. However, this difference was complicated by interactions with euthymia/anger lability and use of cognitive reappraisal as an emotion regulation strategy, which require further research to replicate and explore further.Clinically, we would hypothesize that individuals with emotion regulation difficulties and affective lability are likely to be those with either underlying neurobiological differences and/or histories of traumatic experiences such as difficulties during pregnancy/birth [e.g. ], insecuWe believe that our finding could be considered a development of the stress-vulnerability theory of psychosis , and thaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involved human participants and was reviewed and approved by North of Scotland NHS Research Ethics Committee. All patients/participants provided informed consent to participate in this study, either written or electronic.FL led design of the study, with contributions from AW, SW, RR, and KC. FL gathered the data. AW conducted data analysis in collaboration with FL. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Multivariable logistic and negative binomial regression models tested the association between adults recruited pre- or post-California lockdown and past month cigarette use, past month electronic cigarette use, past month cigarette consumption, and past month e-cigarette consumption among current users, controlling for demographic differences. Adults pre- and post-lockdown had equal odds of using cigarettes during the past month. Cigarette users who responded post-lockdown had higher cigarette consumption rates compared to cigarette users who responded pre-lockdown . Adults who responded post-lockdown had lower odds of using electronic cigarettes during the past month compared to participants surveyed before the order . Cigarette users may be using more cigarettes during the state mandated lockdown. Possible causes for this increase in cigarette use may include increased stress, the change in workplace smokefree protections coverage, and increased opportunities for smoking or vaping.To determine if cigarette smoking, electronic cigarette use, and rate of consumption of these products differed before and after a pandemic lockdown order, two convenience samples of adults in Central California were recruited and surveyed before (March 2020) and after (May 2020) COVID-19 lockdown orders were implemented in California ( Tobacco users are at higher risk for having more severe COVID-19 outcomes ,4,5,6,7.Research on the impact of COVID-19 on adult tobacco-related behaviors is emerging ,11,12,13We examined two ways in which pandemic-related lockdown orders may have impacted the use of tobacco products among a sample of adults living in Central California before and after the state mandated lockdown order was issued on 19 March 2020: (1) changes in past month use of traditional cigarettes and e-cigarettes, and (2) changes in cigarette and e-cigarette consumption rates. Past month use is an indicator of the number of current users in the community. Consumption rates are an indicator of whether current users are increasing or decreasing their usage of cigarettes or e-cigarettes. In this regard, our study seeks to understand lockdown impacts at the community level (number of users), as well as at the individual user level (intake amount). Central California represents a vast area that includes urban, suburban, and rural communities. Moreover, this region is home to a diverse cross-section of racial/ethnic and socioeconomic status groups. Like many regions across the U.S., Central California has historically faced economic challenges that have translated to a weaker health infrastructure, including mental health and tobacco cessation services, translating to higher tobacco use and potential for greater susceptibility and severity of the COVID-19 virus ,16,17. SThe convenience sample consisted of 2571 adults (1510 surveyed in early March 2020 and 1061 in May 2020). Participants resided within an 11-county region in Central California. All participants were recruited via focused advertisements on social media that targeted any resident of the 11-county area. Interested participants were directed to an online screening survey deployed via Qualtrics, where participants were screened for age and county of residence. Eligibility criteria included being 18 years or older, living in the 11-county area, and being English literate. If eligible, participants were directed to the full survey; participants were compensated a gift certificate of $5 for completing the survey. To assure validity of responses, participants whose survey response time did not fall within two standard deviations of the average survey completion time were excluded from analysis. While there have been many cutoff points used in the literature [Participants were classified as a past-30-day  cigarette user if they had used a cigarette at least once during the last 30 days. Past-30-day e-cigarette users were classified as participants who reported that they had used an e-cigarette product at least once during the past 30 days. In order to compute a respondent’s cigarette consumption rate that accounted for both number of days smoked (0–30) and number of cigarettes smoked per day (0–100 plus), we modified a measure used by Johnson and colleagues , where aParticipants were categorized as responding pre-lockdown if they responded to the survey in early March 2020 and post-lockdown if they responded in May 2020. Participants were asked basic demographic questions  and whether they were living with a partner (yes/no). In order to categorize racial/ethnic affiliation, participants were first asked if they identified as Hispanic/Latino. Participants were then asked to select which other racial/ethnic groups they identified with African American/Black, American Indian/Alaska Native, Caucasian/White, East Asian , Middle Eastern , Pacific Islander/Native Hawaiian, Southeast Asian , South Asian , or Other. Respondents’ race/ethnicity was classified as Hispanic/Latino (of any race), non-Hispanic (NH) White (no other race/ethnicity), NH African American/Black (no other race/ethnicity), and NH Other . Two measures were used for socioeconomic status (SES): participants were asked about their highest level of education attained (high school diploma or GED or less/some college/college degree or higher) and household income level which was divided into four-levels .p ≤ 0.05), indicated that individuals who responded pre/post-COVID varied demographically on the basis of age, sex, race/ethnicity, living with a partner, education, and income. As a result, the final models pertaining to cigarette use were adjusted for all these factors. An analysis of e-cigarette users  indicated pre- and post-COVID respondents varied demographically on the basis of sex, age, race/ethnicity, living with a partner, and education. As a result, the models pertaining to e-cigarette use were adjusted for these factors, and for income, as income has been shown to be correlated with e-cigarette use [Two multivariable binary logistic regressions were run to explore the relationship between participants surveyed before and after the lockdown order and (1) past 30-day use of cigarettes and (2) past 30-day use of e-cigarettes. To test whether or not pre- and post-lockdown respondents varied demographically, we used a chi-square test see . The finette use . Cases wParticipants were primarily female 64.72%, , betweenParticipants surveyed pre- and post-lockdown had equal odds of smoking cigarettes  during the past month compared to participants surveyed pre-lockdown (holding other variables constant in the model). There was no significant difference in the incidence rate for e-cigarette consumption over the last 30 days  for participants surveyed post-lockdown versus pre-lockdown (holding other variables constant in the model). Models controlled for sex, age, race/ethnicity, living with a partner, income, and education.The COVID-19 pandemic lockdown in California was not associated with an increase in the number of individuals who used cigarettes or e-cigarettes. Indeed, the odds of being an e-cigarette user was lower post-lockdown than pre-lockdown. However, among current cigarette users, the lockdown was associated with an increase in cigarette consumption.Many public health and healthcare professionals have worried that stress and isolation during the COVID-19 pandemic may lead to, or exacerbate, addictive behaviors ,23,24. TWhen considering this increase in cigarette consumption, it should be noted that stress and other mental health issues may not be the only mechanism at play. Another factor potentially contributing to the increase in cigarette consumption concerns the impact of workplace smokefree policies. Most indoor workplaces in the U.S. have banned indoor smoking, and these smokefree workplace laws are associated with reduced smoking among workers ,30,31. MUnlike previous adult studies that demonstrated an increase in respondents using e-cigarettes , the curWhile literature on tobacco use during COVID-19 is increasing, it is important to clearly delineate community-level effects vs. individual-level effects. Our study indicates that while the number of users may be down, the pandemic does have an effect on tobacco usage, particularly as our study points to current cigarette users increasing their consumption rates.Similar to other research, we found in our whole sample (pre- and post-pandemic respondents combined) that women and Hispanic/Latinos had lower odds of using a cigarette in the last 30 days, while adults 18–25 years old were more likely to use e-cigarettes than individuals over 25 years old ,37,38,39This study uses a convenience sample of participants recruited from social media and not a random sample, which impacts generalizability of study findings. However, the counties surveyed in the current study are akin to regions across the U.S., given their higher smoking rates and mix of rural, suburban, and urban populations. Additionally, this sample contains significantly more tobacco users than the general population of the region. However, this inadvertent oversample allows for a better understanding of tobacco user behavior. The oversampling of tobacco users may have occurred in response to the fact that our online ads had the word “nicotine” in the title, which may have made the study more attractive to smokers. The data are cross-sectional, and thus findings do not represent changes in a particular participant’s behavior, but rather differences at the community level; no causal inference can be made. Furthermore, the data cannot make inferences on the mechanism by which these pre-post differences occurred. The data are also subject to the limitations of self-reported data and the biases of recall. Respondents pre- and post-lockdown differed on some demographic factors, which was potentially due to the fact that participants who would not otherwise participate in surveys were more available during lockdown given confinement to home. While the analyses controlled for demographic variables that differed between respondents collected in March and May, there may be other underlying factors, such as perceived stress, social support, or other unobserved pandemic-related factors that could also possibly explain the differences in tobacco use between pre- and post-lockdown respondents. However, our primary goal was to understand how one major event in time might be related to community-level behavior and health.Our findings indicate a shift in tobacco consumption at the community level in Central California post-COVID-19-related lockdown. While public health attention has been focused on a possible increase in addictive behaviors due to pandemic-related social and economic stress, another possible contributing factor could be that cigarette users were removed from protective indoor smokefree workplace laws and have increased time for cigarette smoking due to changes in working schedules. Interventions for quitting or reducing consumption need to take this into account to address the needs of tobacco users during the pandemic.


Human-based computational modelling and simulation are powerful tools to accelerate the mechanistic understanding of cardiac patho-physiology, and to develop and evaluate therapeutic interventions. The aim of this study is to calibrate and evaluate human ventricular electro-mechanical models for investigations on the effect of the electro-mechanical coupling and pharmacological action on human ventricular electrophysiology, calcium dynamics, and active contraction.The most recent models of human ventricular electrophysiology, excitation-contraction coupling, and active contraction were integrated, and the coupled models were calibrated using human experimental data. Simulations were then conducted using the coupled models to quantify the effects of electro-mechanical coupling and drug exposure on electrophysiology and force generation in virtual human ventricular cardiomyocytes and tissue. The resulting calibrated human electro-mechanical models yielded active tension, action potential, and calcium transient metrics that are in agreement with experiments for endocardial, epicardial, and mid-myocardial human samples. Simulation results correctly predicted the inotropic response of different multichannel action reference compounds and demonstrated that the electro-mechanical coupling improves the robustness of repolarisation under drug exposure compared to electrophysiology-only models. They also generated additional evidence to explain the partial mismatch between in-silico and in-vitro experiments on drug-induced electrophysiology changes.The human calibrated and evaluated modelling and simulation framework constructed in this study opens new avenues for future investigations into the complex interplay between the electrical and mechanical cardiac substrates, its modulation by pharmacological action, and its translation to tissue and organ models of cardiac patho-physiology. They provide virtual tools, embedding our knowledge on human physiology, to investigate disease, and develop and evaluate therapeutic interventions . Recent Over the last six decades, computational modelling and simulation have played a key role in cardiac science. They have accelerated the mechanistic understanding of cardiac patho-physiology, usually through separate paths for electrophysiology and contractility. Iterations between experimental and modelling and simulation science have enabled the development and validation of biophysical models . TechnicBuilding on previous models , Tomek eMoreover, The aim of this study is to investigate the effects of electro-mechanical coupling in human ventricular electrophysiology, calcium dynamics and active contraction, and their modulation by pharmacological action using multiscale modelling and simulation. To address this goal, the most recent models of human ventricular electrophysiology and active contraction are coupled and calibrated using experimental data. For electrophysiology, we consider both the ToR-ORd and its predecessor, the ORd model, and we hypothesise that the improvements in the excitation-contraction coupling in the ToR-ORd (with respect to the ORd model) will yield a better agreement between simulations and experimental data.Simulations are conducted to quantify the effect of electro-mechanical coupling on the action potential, calcium dynamics, and active contraction in healthy virtual human ventricular cardiomyocytes and tissue and under drug exposure. This study provides a human-based integrated pipeline for the simultaneous simulation of electrophysiology, calcium dynamics and contractility in ventricular cardiomyocytes. The proposed modelling and simulation framework offers the opportunity to extend human-based investigations into the mechanisms of disease and drug action to incorporate heterogeneous electrical and mechanical features of cardiac function and their strong coupling.22.12.2Previously-published human electrophysiology, calcium dynamics and active tension models were coupled through forward (excitation-contraction coupling) and reverse (mechano-electric feedback) processes. Specifically, the new ToR-ORd model published in The key improvements of the ToR-ORd compared to the ORd models are an in-depth re-evaluation of the ICaL formulation, the re-assessment of the IKr, the replacement of the INa and IK1, as well as the incorporation of additional currents . This allowed an improved representation of the AP plateau, action potential duration (APD) adaptation, and responses to sodium current block .The Land model  was adopThe bidirectional electro-mechanical coupling was achieved as previously reported in 2.3The coupled electro-mechanical models (from now on referred to as ToR-ORd+Land and ORd+Land) were calibrated against human experimental recordings to achieve physiological active tension. This was necessary because the CaT from the ToR-ORd and ORd models was in close agreement with human recordings  but was As in Land, isometric twitch data in intact human preparations from the literature  were use2.4Firstly, we compared the simulated APs and CaTs using the ToR-ORd+Land and ORd+Land models with human experimental data , as in T2.5We simulated the drug-induced effects on cellular and tissue electro-mechanical function and compared the results with reported effects on human preparations. We focused on the following multichannel action reference compounds: Dofetilide (antiarrhythmic drug class III), Verapamil (antiarrhythmic drug class IV) and Quinidine (antiarrhythmic drug class Ia). Compounds selection was based on preclinical  and clinDrug effects were incorporated in the electro-mechanical models using IC50 and Hill coefficient data , by mean2.6ode15s. Stimulus currents of −53 Cellular simulations were conducted using MatLab  using the ordinary differential equation solver 2.7Alya , fixing it in all directions. A stimulus current of −100 33.1Further details on how the calibration affects the contractility model and the steady-state calcium-force relationship can be found in the supplementary material (S5). The supplementary material (S6) also includes information on the preserved force-frequency and length-dependence properties of the calibrated ToR-ORd+Land and ORd+Land models.We next analysed how the coupling with the contractility model affects cellular electrophysiology. The AP morphology and kinetics of the electro-mechanically coupled models perfectly resemble those of the purely electrophysiological models -A, C. HoThe differences between the electrophysiology-only and the electro-mechanical models stem from the change in buffering scheme adopted: in the coupled formulation, the combined troponin-calmodulin buffering scheme of the ToR-ORd and ORd models is split up into its two components and the Land dynamic formulation for troponin C is adopted. As a consequence, the coupled models now predict that less calcium is bound to these buffers  selectively blocks IKr. It induces a dose-dependent increase in the QT interval, and TdP is the most dangerous side effect of this therapy. In this study, Dofetilide concentrations were explored in the range of 0.02–0.1 μM, in correspondence with experimental investigations (igations .Simulations conducted with the cellular ToR-ORd+Land model at the lowest drug concentration (0.02 μM) show that Dofetilide prolongs the AP, which in turn induces an increase in intracellular calcium, and therefore of active tension  and their respective electrophysiology-only (ToR-ORd and ORd) models (for both 0.25 and 1 Hz pacings) is reported in Verapamil. Verapamil blocks both IKr and ICaL, with minor effects on APD and QT interval. Verapamil has a TdP risk of 0 (not classified).Despite the very similar inotropic response, mediated by a similar effect on CaT, the ToR-ORd+Land and the ORd+Land models provide different predictions in terms of APD prolongation -C2. The Additional analyses with the ToR-ORd+Land model show that, in order to reproduce experimental data on APD, IC50 for IKr has to be one order of magnitude bigger  we observe a more robust electrical repolarisation in the presence of the electro-mechanical coupling, compared to electrophysiology-only models. While consistent for different pacing frequencies, this is more evident at slow pacing. We are able to explain such behaviour by detailed analyses of the time course of the simulated AP and CaT. Coupling-induced changes on the CaT allow for calcium to decay almost completely within the duration of the AP. Under such circumstances, the APD prolongation induced by drug exposure has less opportunity to translate into calcium abnormalities. This can be also seen in terms of the electro-mechanical window (EMw), defined as the difference between the duration of electrical and mechanical systole and computed in previous studies as the difference between the APD and CaT duration at 90% repolarisation/decay . In base4.5While the models presented in this study have been able to recapitulate some of the transmurally varying electro-mechanics features of the healthy human myocardium, additional sources of heterogeneity have been reported in the literature. Inclusion of these aspects of heterogeneity in the models would provide a more comprehensive description of the heterogeneous cardiac substrates. As suggested in previous studies  myosin eAnother key limitation of this study is the lack of after-load simulations. Contractility response is critically affected by pre-load and after-load conditions and their simulations, together with isometric ones, would provide a more exhaustive description of contraction modes. Further model development is needed to include a mathematical representation of the dynamic changes in sarcomere length. Evaluation of such simulation results would also require human-relevant data currently unavailable.Finally, a key factor to consider for the comparisons reported in this study between experimental and simulation data is the differences between setups. In particular, experiments are often conducted at a fixed muscle length, which would allow some sarcomere shortening even in isometric contractions , whereas5In this study, we integrated the most recent models of human ventricular electrophysiology and active contraction and calibrated them using human experimental data. This process yielded physiological simulated active tension, AP, and CaT in agreement with experiments obtained in human preparations. Furthermore, the simulated electro-mechanical function reproduced the reported transmurally heterogeneous properties of human myocardium.We quantified the effect of the electro-mechanical coupling on electrophysiology and force generation in virtual human ventricular cardiomyocytes and myocardial tissue, both in control conditions and under drug exposure. Specifically, we correctly predicted the inotropic response of different multichannel action reference compounds. Furthermore, we generated additional evidence to explain the partial mismatch between in-silico and in-vitro predictions of drug-induced APD prolongation. Simulations demonstrated that cardiac electro-mechanical coupling provides a more robust repolarisation under drug exposure compared to electrophysiology-only models.The human-based mechanistic framework presented in this study will enable future investigations into the complex interactions between electrophysiology and contractility, also allowing the simultaneous assessment of pro-arrhythmia and inotropic liabilities of pharmacological therapies.Francesca Margara: Conceptualization, Methodology, Formal analysis, Software, Writing - Original Draft, Writing - Review & Editing. Zhinuo J. Wang: Conceptualization, Methodology, Software, Writing - Original Draft, Writing - Review & Editing. Francesc Levrero-Florencio: Methodology, Software, Writing - Review & Editing. Alfonso Santiago: Software, Writing - Review & Editing. Mariano Vázquez: Software, Writing - Review & Editing. Alfonso Bueno-Orovio: Conceptualization, Methodology, Writing - Original Draft, Writing - Review & Editing, Supervision. Blanca Rodriguez: Conceptualization, Methodology, Writing - Original Draft, Writing - Review & Editing, Funding Acquisition, Resources, Supervision.Personalised In-Silico Cardiology (PIC) project (10.13039/100010661European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 764738); a 10.13039/100010269Wellcome Trust Fellowship in Basic Biomedical Sciences to B.R. (214290/Z/18/Z), a 10.13039/501100000274British Heart Foundation (BHF) Intermediate Basic Science Fellowship to A.B. (FS/17/22/32644), the CompBioMed 1 and 2 Centre of Excellence in Computational Biomedicine , an NC3Rs Infrastructure for Impact Award (NC/P001076/1), the TransQST project , and the Oxford BHF Centre of Research Excellence (RE/13/1/30181). The authors gratefully acknowledge computational resources provided by a PRACE project (2017174226) and an Amazon Web Services .This work was funded by the Code and supporting research data are available under request.None.


Mycobacterium tuberculosis infection, being at risk of developing active tuberculosis (TB) during their lifetime. Given the inefficacy of prophylactic measures and the increase of drug-resistant M. tuberculosis strains, there is a clear and urgent need for the development of new and more efficient chemotherapeutic agents, with selective toxicity, to be implemented on patient treatment. The component enzymes of the shikimate pathway, which is essential in mycobacteria and absent in humans, stand as attractive and potential targets for the development of new drugs to treat TB. This review gives an update on published work on the enzymes of the shikimate pathway and some insight on what can be potentially explored towards selective drug development.Roughly a third of the world’s population is estimated to have latent  Mycobacterium tuberculosis is the main causative agent of human tuberculosis (TB). In 2018, approximately 10 million people developed TB, which resulted in 1.3 million deaths in HIV-negative and 300,000 deaths in HIV-positive patients. This recent report from the World Health Organization (WHO) indicated that, worldwide, approximately 400,000 people developed multidrug-resistant TB (MDR-TB), where M. tuberculosis strains are resistant to isoniazid and rifampicin, two of the most effective TB first-line drugs -ones and the 2′-hydroxy-4′-pentoxy, which inhibited the activity of bifunctional CS enzymes, such as N. crassa, Plasmodium falciparum, Salmonella typhimurium and Streptococcus pneumoniae pyridine-3-carboxylic acid, which inhibited hypoxia-induced tolerance to isoniazid (INH) in M. tuberculosis, prevented selection of drug-resistant strains and reversed INH resistance in katG mutants. However, further efforts should be pursued to translate this chemical compound into a chemotherapeutic agent to treat TB infection in human hosts infected with INH-resistant strains of M. tuberculosis harboring katG mutations [Extracellular olerance . A modifolerance  identifiutations . AlthougM. tuberculosis.Host-directed therapies have emerged as a novel and promising approach to treat TB, whose modulation of host response is likely to avoid the development of bacterial resistance ,166. Unlhttps://broad.io/cgtb) to, in their words, accelerate the discovery of new inhibitor chemotypes and their targets.A new strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets) was developed in which large chemical libraries  are screened against pools of mutant strains depleted in essential bacterial targets (100-150 hypomorphs) and their hypersensitivity are exploited to generate large-scale chemical-genetic interaction profiles (CGIPs). The PROSPECT strategy simultaneously identifies whole-cell active compounds and predicts their mode of action from the primary screening data, which provides putative target information that can help guide hit prioritization . MoreoveIn this section we have attempted to give a broad view of expanding experimental strategies to devise new therapies to treat TB. There is also an increased need for expanding the variety and complexity of cell-based assays for biological research and drug discovery. Stem cell-derived cells and tissues have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The Japanese Global Health Innovative Technology Fund proposed a set of criteria for hits and leads identified in screening campaigns of chemical compounds to treat neglected tropical diseases, including TB . These c


Introduction: Arterial brain vessel assessment is crucial for the diagnostic process in patients with cerebrovascular disease. Non-invasive neuroimaging techniques, such as time-of-flight (TOF) magnetic resonance angiography (MRA) imaging are applied in the clinical routine to depict arteries. They are, however, only visually assessed. Fully automated vessel segmentation integrated into the clinical routine could facilitate the time-critical diagnosis of vessel abnormalities and might facilitate the identification of valuable biomarkers for cerebrovascular events. In the present work, we developed and validated a new deep learning model for vessel segmentation, coined BRAVE-NET, on a large aggregated dataset of patients with cerebrovascular diseases.Methods: BRAVE-NET is a multiscale 3-D convolutional neural network (CNN) model developed on a dataset of 264 patients from three different studies enrolling patients with cerebrovascular diseases. A context path, dually capturing high- and low-resolution volumes, and deep supervision were implemented. The BRAVE-NET model was compared to a baseline Unet model and variants with only context paths and deep supervision, respectively. The models were developed and validated using high-quality manual labels as ground truth. Next to precision and recall, the performance was assessed quantitatively by Dice coefficient (DSC); average Hausdorff distance (AVD); 95-percentile Hausdorff distance (95HD); and via visual qualitative rating.Results: The BRAVE-NET performance surpassed the other models for arterial brain vessel segmentation with a DSC = 0.931, AVD = 0.165, and 95HD = 29.153. The BRAVE-NET model was also the most resistant toward false labelings as revealed by the visual analysis. The performance improvement is primarily attributed to the integration of the multiscaling context path into the 3-D Unet and to a lesser extent to the deep supervision architectural component.Discussion: We present a new state-of-the-art of arterial brain vessel segmentation tailored to cerebrovascular pathology. We provide an extensive experimental validation of the model using a large aggregated dataset encompassing a large variability of cerebrovascular disease and an external set of healthy volunteers. The framework provides the technological foundation for improving the clinical workflow and can serve as a biomarker extraction tool in cerebrovascular diseases. Stroke is one of the leading causes of death worldwide and 15 million people each year suffer a stroke  time-of-flight (TOF) imaging. It is fast, has no ionizing radiation exposure and can depict the arterial vasculature in high detail. Clinical reading of vessel imaging is based on visual judgment alone, as there is a lack of fully automated vessel segmentation methods. Fully automated segmentation means in the clinical context that the segmentation results would be rapidly available on the scanner console without the need of extra post-processing. Extensive, time-consuming and non-standardized image post-processing is an important obstacle to widespread clinical application. Fully automated analysis of TOF images could facilitate the diagnosis of vessel abnormalities and allow the quantification of the cerebrovascular status, e.g., arterial vessel density or arterial vessel diameters, to make potential biomarkers for diagnostic stratification easily available —a type of artificial neural networks (ANN) tailored for image analysis—have become the method of choice for vessel segmentation including the brain vasculature and showed promising results in recent applications  with cerebrovascular disease. This approach ensured the high performance and high generalization of BRAVE-NET.Recognizing this challenge, this work presents a high performance fully automated framework for vessel segmentation in patients with cerebrovascular disease addressing all the above-mentioned limitations, coined BRAVE-NET (BRAinVEssel-NETwork). BRAVE-NET is a multiscale 3-D CNN model designed to dually capture high- and low-resolution volumes thus enabling enhanced distinction of brain vessels from other structures, such as skull areas and arteries not feeding the brain, making masking and other post-processing steps obsolete. The model was developed and validated using high-quality labeled data derived from multiple datasets of patients  Magnetic Resonance Angiography (MRA) images were used to train all benchmarked models. TOF relies on the fact that, within an imaged volume, inflowing blood has high magnetization compared to stationary tissue that becomes magnetically saturated by multiple radiofrequency pulses. TOF-MRA is one of the most important methods for non-contrast neurovascular and peripheral MRA. In the PEGASUS and 1000Plus studies, TOF imaging was performed on a Magnetom Trio 3T whole-body system  using a 12-channel receive radiofrequency (RF) coil  tailored for head imaging. In the 7UP study, TOF imaging was performed on a Magnetom Verio 3T whole body system  using a 12-channel receive radiofrequency (RF) coil  tailored for head imaging. Parameters of the TOF imaging of each dataset are shown in regiongrowingmacro module implemented in MeVisLab . To tackle inter-rater variability in label generation, these pre-labeled data were thoroughly manually corrected by either OUA and EA (both junior raters) and then cross-checked by the other rater. Junior raters were trained for image segmentation and were only allowed to independently label images once their performance met ground truth standards. These labels were then checked subsequently by VIM (9 years experience in stroke imaging). Thus, each ground-truth was eventually checked by three independent raters, including 1 senior rater. The total labeling time with this framework amounted to 60–80 min per patient. For 1000Plus data, the MeVisLab-based pre-segmentation step was replaced by using a 2D Unet segmentation model, developed in earlier work  The images were smoothed with a 16 × 16 × 16 averaging filter in a shifting window fashion. (2) A threshold of intensity values at 10 was applied (intensity > 10 implies ROI). This provides an automatically generated mask for the training phase which contains all the brain tissue but also a thick border area around the skull. The inclusion of patches from these border areas proved to be crucial for the learning process, particularly for the distinction of the skull and the ability to capture the distinctive neighborhood of small vessels in that area. We note that this step serves the purpose of pre-selection of training patches only and is fully optional for the training phase. Inference with any of the models is possible on any raw image without a selection mask, consequently no masks were utilized in the testing phase. Similarly, instead of prior normalization of input data, an initial Batch-normalization layer was introduced to each architecture before other layers to learn and adjust to moving mean and variance statistics of training samples. This approach eliminates data pre-processing and the need of stashing statistics as well as enables quick adaptation when necessary through “fine-tuning” of Batch-normalization layers.Images from each dataset were used for training, model selection and evaluation. A 4-fold cross-validation methodology was employed to ensure the robustness of all models toward different training and test sets. Therefore, data was split randomly into 4 distinct training and test sets making sure that each set contained the same number of patients from each source dataset . Furthermore, 15% of images were separated randomly without overlaps from each training set for validation and used for parameter selection. The task of brain vessel segmentation on 3D MRI images challenges deep learning techniques from several aspects. First, processing of whole brain volumes at once requires significant resources in terms of GPU memory. Second, the distribution of vessels compared to brain tissue is very sparse. The physiological arterial vascular volume fraction of the brain is 1.5%. TOF-detectable arterial vessel voxels can be as low as on average 0.3% of all voxels within the brain. Finally, sufficient training of deep neural networks requires numerous images, while our dataset is—like most medical imaging datasets—limited in size. To solve these challenges, we redesigned the segmentation task as voxel-wise classification and trained our models on patches that represent an arbitrary neighborhood of the center voxel. Accordingly, 2,000 locations per TOF image  were randomly sampled within the heuristical mask, ensuring 50% of the samples were vessel-centric. Patches of sizes [64 × 64 × 8] and [128 × 128 × 16] were extracted around each location, and comprised the input patches of our proposed framework.In recent years, the medical AI research field showed a tendency to develop an overwhelming focus for performance in homogeneous samples in contrast to the requirements for a medical imaging product  = 1/(1+exp(f(x))), where f(x) denotes the output value of the last [1 × 1 × 1] convolutional layer at the voxel position x ∈ Ω with Ω ∈ Z3and p(x) is the approximated probability of a voxel x to be a vessel. The same activation is applied to each output of the deep supervision method. DSC loss was used to update the model parameters and to monitor training convergence, throughout all experiments. DSC between two binary volumes, i.e., prediction and ground truth—is defined as DSC = 2TP/(2TP+FP+FN), where TP is the number of true positive voxels, FP is the number of false-positive voxels and FN is the number of false-negative voxels. Further derived:The designated input  and ground truth patches ([64 × 64 × 8]) were used to train the models. All models were implemented in Python using Tensorflow Deep learning framework , false positives (FP), and false negatives (FN) were assigned different voxel values (True negatives (TN) remained labeled with 0). The images were then visualized by overlaying these new masks with the original scans using ITK-Snap software  as well as small vessels . Large vessel error was dominated by false positives , whereas small vessel error was dominated by balanced error .Besides excellent vessel segmentation performance, the visual inspection revealed a high rate of falsely labeled rare pathologies and other tissue classes. The false-labeling-rate was lowest for the BRAVE-NET model (40%), followed by Context-Unet (43%), DS-Unet (49%), and Unet (50%) with the highest false-labeling-rate. The results were consistent in case of our external validation set. BRAVE-NET achieved highest performance on all measures but Recall, where Context-Unet was superior. Details can be found in Visual inspection of the results revealed similarly high performance in terms of granularity and completeness of segmentations. The lower overall performance originated from the inclusion of large sinuses in the segmentations yielding a high number of false positives. As Precision values also indicate, the BRAVE-NET architecture was superior in reduction of false positives compared to other architectures. We present the BRAVE-NET model for fully automated arterial vessel segmentation from TOF-MRA images in cerebrovascular disease. The model combines multiscaling and deep-supervision as extensions of the standard Unet approach and yielded a new state-of-the-art performance. This was achieved by robust validation using the largest presented dataset in the literature so far. BRAVE-NET is suitable for clinical application, as it can be applied on raw images without the necessity for additional image processing. The superiority of the BRAVE-NET was demonstrated quantitatively and was confirmed via visual assessment. It outperformed the other tested models especially in cases of visible pathologies and false labeling. The performance was further validated on an external set of healthy volunteers, where BRAVE-NET proved superior in quantitative measures as well as in exclusion of non-relevant brain structures . BRAVE-NET marks a significant step toward increased applicability of deep learning based segmentations in the clinical setting and the continued development of quantitative vascular biomarkers in cerebrovascular disease.Reported models in the literature for brain vessel segmentation are usually limited to a DSC of 0.90  similar to our study.First, the majority of MRA-based vessel segmentation frameworks presented so far in the literature require image pre-processing, such as downsampling, brain masking, intensity correction, image normalization, and various other methods before they can be applied  pathologies. Exceptions are the work of Meijs et al.  who deveSixth, we included an external validation set to demonstrate the applicability of our framework with regards to no need of pre-processing steps, and test the generalization of our models on a public, out-of-sample dataset. Here, we made the contribution to generate 20 high quality ground truth labels for voxel-wise segmentation and make them publicly available (Hilbert et al., To summarize, the presented framework add to the literature through the unique combination of high performance, full automation and robust validation. It thus has the largest applicability for the clinical setting so far.The excellent performance of BRAVE-NET can also be attributed to architecture design choices. CNNs and encoder-decoder Unet variations, in particular, were proven in recent years as the state-of-the-art method for semantic segmentation including medical imaging applications (Milletari et al., Our study has several recognized limitations. First, while the dataset in this study was acquired from multiple scanners and patients exhibited a large spectrum of steno-occlusive cerebrovascular disease, the data heterogeneity was still limited. It is highly warranted to specifically include in future work datasets with other pathologies, such as aneurysms, other MR scanner- and MR imaging sequence types and other imaging modalities, such as CT. Here, transfer learning approaches seem beneficial. Heterogeneity is not only essential in training sets but also in testing for thorough and robust assessment of performance. Even though our external validation set yielded consistent ranking of our models, segmentation performance was rather limited due to a high false positive rate throughout all architectures. From a development perspective, we expect this to be easily overcome by minor fine-tuning on a few samples from the dataset, and this highlights that all variations of input data need to be used for tools with planned clinical applications. Second, it was beyond the scope of this work to integrate all potentially beneficial architecture modifications. Further future modifications could utilize densely connected convolutional blocks to enhance feature propagation through encoding levels (Zhang et al., We present a fully automated deep learning framework for TOF-MRA brain vessel segmentation. We utilize multiscaling for better spatial information integration and deep supervision for improving model convergence. We employ a multi-scanner dataset of yet unmatched size and range of pathologies in the published literature. We also ensure a high degree of robustness of our framework through extensive quantitative and qualitative evaluation. We explicitly analyze and communicate model performance in presence of relevant pathologies to further increase applicability in the clinical setting. Our results thus constitute a new state-of-the-art for fully automated frameworks for brain vessel segmentation. Our work strongly facilitates and promotes the usability of automated vessel segmentation in the clinical setting and can be swiftly translated to the development of cerebrovascular biomarkers for clinical applications.ethikkommission@charite.de. The imaging data used for external validation and the generated labels are available under https://www.insight-journal.org/midas/community/view/21 and http://doi.org/10.5281/zenodo.3968844 respectively.The datasets used in this article for model training are not readily available because data protection laws prohibit sharing the 1000Plus, PEGASUS and 7UP datasets at the current time point. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by Ethics committee of Charité University Medicine Berlin and Berlin State Ethics Board. The patients/participants provided their written informed consent to participate in this study.AH, VM, AT, JF, DF, and ML: conceptualization. JS, JW, PD, TN, JF, and DF: funding acquisition and project administration. EA, OA, JB, VM, AK, IG, and JF: data curation. AH, VM, JS, IG, AK, AT, JW, PD, TN, JF, DF, and ML: methodology. AH, VM, DF, and ML: model development and validation. JS, JW, TN, JF, and DF: resources. AH, VM, DF, and ML: writing—original draft. AH, VM, EA, OA, JB, JS, IG, AK, AT, JW, PD, TN, JF, DF, and ML: writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, its prognostic value for BC patients remains controversial. To address this issue, the present systematic review aims to summarize the available evidence and give a picture of a prognostic significance of miR-155 in BC pathology. All eligible studies were searched on PubMed and EMBASE databases through various search strategies. Starting from 289 potential eligible records, data were examined from 28 studies, comparing tissue and circulating miR-155 expression levels with clinicopathological features and survival rates in BC patients. We discuss the pitfalls and challenges that need to be assessed to understand the power of miR-155 to respond to real clinical needs, highlighting the consistency, robustness or lack of results obtained to sate in translating this molecule to clinical practice. Our paper suggests that the prognostic role of miR-155 in the management of BC needs to be further verified.There is an unmet need for novel non-invasive prognostic molecular tumour markers for breast cancer (BC). Accumulating evidence shows that miR-155 plays a pivotal role in tumorigenesis. Generally, miR-155 is considered an oncogenic miRNA promoting tumour growth, angiogenesis and aggressiveness of BC. Therefore, many researchers have focused on its  Breast cancer (BC) represents one of the most frequent malignant neoplasms in women worldwide, and its mortality rate is still increasing, due to population growth and ageing . WorldwiMicroRNAs (miRNAs) are a class of nucleic acid-based molecules approximately 19 to 24 nucleotides long and regulate gene expression at the post-transcriptional level . MiRNA iFollowing our search strategy, a total of 289 potential eligible records related to the prognostic role of miR-155 in BC were extracted from PubMed and EMBASE public databases. The main characteristics of each study are detailed in Among the 12 selected studies assessing the prognostic value of tissue miR-155 in BC patients , three oMost of the selected studies agree with the positive correlation between miR-155 overexpression and tumour grade ,11, TumoMost of the selected studies associated miR-155 level with poor prognosis in BC patients. Chen et al. proposed miR-155 as a negative prognostic marker using a risk model of DFS and OS . In detaRaychaudhuri et al.  evaluateIn two of the 12 selected studies, miR-155 expression was correlated with oncogenic markers. Hafez et al.  reportedMost of the eligible studies selected  assessedThe studies focusing on the deregulation of miR-155 in blood samples of BC patients with prognostic factors provide heterogeneous results. Regarding tumour size, Answar et al.  assessedFinally, Fortis and colleagues  evaluatePrognostic rates, such as OS and DFS, were poorly investigated in association with circulating miR-155 expression level. Song et al.  reportedWe interrogated the Human miRNA disease database (HMDD 3.2v) , experimHere, we have analysed the potential prognostic value of miR-155 in BC. MiRNAs are useful tools for diagnostic and prognostic purposes in several diseases . In receMany researchers have focused on the possibility of utilizing miR-155 as a biomarker for diagnostic and prognostic purposes in BC. Nevertheless, the benefits of using this miRNA as a prognosis marker remain unclear. We have carried out a systematic review of all studies in which miR-155 seems to provide promising results as a prognostic molecule. In this qualitative analysis, starting from 289 identified records, we selected a total of 28 studies that revealed the potential role of miR-155 for prognostic purposes in BC. Because these studies were highly heterogeneous and only a few of them performed survival analysis, the data have been somewhat difficult to aggregate. In most of them, authors performed correlation analyses of miR-155 expression with prognostic patient-related factors  and with prognostic tumour-related factors . Regrettably, however, not in every study was OS or DFS calculated, maybe due to a lack of follow-up data, making the clinical application of this miR doubtful. However, about 90% of these studies correlated increasing tissue and/or circulating miR-155 with poor prognosis, whereas only in three of them was miR-155 up-regulation linked to better prognosis ,17; authMore concordant results have correlated miR-155 expression with therapeutic response ,29,37. Ihttps://clinicaltrials.gov/), started on 2015, involves the miR-155 as a biomarker of cardiotoxicity induced by RAdioTherapy in BC, but results are not yet available. Unlike what was expected, the selected studies did not provide sufficient information to give a consistent answer to our research question. In this framework, the differences in sample size, cohort demographics, inhomogeneous subtype distributions and follow-up time-table could account for the general lack of strength of results. Thus, further, large-scale studies with better design and more comprehensive follow-up data support will need to be conducted to clarify the prognostic value of miR-155 in BC. Overall, this work aims to highlight the weaknesses that need to be overcome to validate the prognostic role of miR-155 in BC, as its use in clinical practice appears still premature. Although miRNA application in clinical practice is lacking to date, experimental results could make it possible to use these molecules for prognostic purposes. However, despite the interesting intrinsic characteristics of miRNAs, several challenges for their clinical application in the management of oncological diseases still needs to be overcome.Summarizing, our systematic review has elucidated that miR-155, although promising it is still far from being an effective biomarker for predicting prognosis of BC patients. Nevertheless, to date, only one on-going clinical trial  on PubMed and EMBASE databases. We used the following search strategies:  and breast cancer and (prognosis or prognostic or tissue or circulating or serum or plasma)  independently, after having screened identified studies for titles and abstracts, included or excluded articles basing on the following criteria. Inclusion criteria: (1) prognostic role of miR-155 in breast tissues and/or blood samples of BC patients; (2) original articles; (3) English language. Exclusion criteria: (1) letters, case reports, reviews, conference abstracts; (2) non-English papers; (3) studies performed on immortalized BC cell lines; (4) methodological studies; (5) studies not focused on BC. The discrepancies between the three reviewers were resolved by consensus or by discussion with a fourth researcher (GC).After the selection procedure, data that met the inclusion criteria were summarized into a customized Excel spreadsheet database by one investigator (AMG) and verified by a second and third (SI and MI). For each study, the following characteristics were collected: publication year; country of the population enrolled; cohort study; sample; method; miRNAs; miR-155 expression; prognosis; significantly associated parameters; survival analysis; reference.The articles were classified based on the sample type used in studies: tissue and/or plasma/serum samples. This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  (See TabWe integrated our systematic review data with the open web-based resource Human miRNA disease database (HMDD v3.2) . We asse