Prolactin coordinates with the ovarian steroids to orchestrate mammary development and lactation, culminating in nourishment and an increasingly appreciated array of other benefits for neonates. Its central activities in mammary epithelial growth and differentiation suggest that it plays a role(s) in breast cancer, but it has been challenging to identify its contributions, essential for incorporation into prevention and treatment approaches. Large prospective epidemiologic studies have linked higher prolactin exposure to increased risk, particularly for ER+ breast cancer in postmenopausal women. However, it has been more difficult to determine its actions and clinical consequences in established tumors. Here we review experimental data implicating multiple mechanisms by which prolactin may increase the risk of breast cancer. We then consider the evidence for role(s) of prolactin and its downstream signaling cascades in disease progression and treatment responses, and discuss how new approaches are beginning to illuminate the biology behind the seemingly conflicting epidemiologic and experimental studies of prolactin actions across diverse breast cancers. Factors that regulate cell-specific proliferation and differentiation repeatedly have been shown to be significant actors in oncogenesis and potential therapeutic targets in established cancers. Prolactin (PRL) cooperates with the ovarian steroids, estrogen and progesterone, to orchestrate the cycles of mammary development and differentiation that lead to successful lactation, providing nourishment for the offspring. PRL-initiated signals that expand alveolar cells during pregnancy and coordinate their differentiation at the time of birth have been mechanistically defined [ –4 and reWhile control of PRL expression by pituitary lactotrophs during pregnancy and lactation is well understood [reviewed in ], its exHere we review the epidemiologic evidence linking PRL to oncogenesis in the breast, and recent experimental studies implicating multiple underlying mechanisms. We then address the more controversial role(s) for PRL in established breast cancers. PRLR is highly expressed in many breast cancers across all different subtypes, and epidemiologic analyses and experimental studies are revealing that PRL can elicit both pro-differentiation and pro-aggression outcomes. We discuss how new approaches are illuminating the factors that determine the responses to PRL, including intrinsic tumor cell properties and the microenvironment, and point to directions for future studies that will integrate our understanding of this hormone in breast cancer progression and therapeutic responses.Multiple epidemiologic studies have examined the relationship between levels of circulating PRL and development of breast cancer . LargeThe ability of PRL to stimulate mammary tumorigenesis in rodent models has been recognized for some time. Many early studies manipulated pituitary PRL –7, especStat1 secondary to somatic truncating mutations in Prlr, resulting in an alternatively spliced protein resembling the human “intermediate” isoform  by both epithelial and non-epithelial target cells may complement mitogenic effects of PRL on mammary epithelium to augment mammographic density, suggesting another mechanism by which PRL may raise risk . In thisPrl or Prlr slowed growth of lesions induced by viral oncogenes . Interestingly, however, Clevenger and colleagues reported that the “intermediate” isoform of the PRLR, which would not have been detected with the antibody used in the study above, was most highly expressed in TNBC  found no significant effect on disease progression . In anotin vivo. However, Rui and his colleagues have developed a mouse in which the mouse Prl gene has been replaced with the human PRL gene (NSG-Pro), resulting in physiologic regulation of hPRL expression . Some investigators have examined PRL responses in murine recipients of transplanted cell lines by providing another source of hPRL, with conflicting results. Primary tumors of transplanted MDA-MB-468 breast cancer cells that expressed hPRL grew more rapidly than tumors that did not in nu/nu (nude) mice (Well-characterized breast cancer cell lines modeling different breast cancer subtypes have been extensively studied de) mice . Reductide) mice . In contde) mice , and grode) mice .ESR1 by PRL, and promoted less differentiated, more aggressive tumors upon transplantation to NOD/SCID mice. In HER2+ SKBR3 cells, loss of PRLR increased HER2 expression, and ability to colonize lungs of NSG recipients. These findings, together with associated in vitro analyses, indicate beneficial actions of PRLR in these models (See review by Ali and colleagues elsewhere in this series).In a different approach, Ali and her colleagues used CRISPR/Cas9 to reduce PRLR expression in the ER+ MCF7 and HER2+ SKBR3 breast cancer cell lines . Loss ofe models . , the PRL antagonist, G129R-hPRL, reduced metastases after removal of the primary tumor . In a suAlthough PRL/PRLR inhibitors have not shown robust promise as monotherapies, there has been long term interest in their interaction with other treatment modalities, especially with anti-estrogens in ER+ breast cancers. As noted in Section 2.2.5, PRL cooperates with estrogen by multiple mechanisms, which have been dissected primarily in the well-differentiated ER+ breast cancer cell line, MCF7 –103. Notin vitro and in a murine MMTV-neu-derived tumor ex vivo . Transgenic overexpression of STAT5A leads to mammary carcinomas in the absence of other oncogenes , PRL was less able to activate JAK2/STAT5, but more strongly activated FAK-SRC-ERK1/2, associated with increased localization of PRLR in focal adhesions  of PRL in the biology of established clinical breast cancers remains unclear. Although PRLR is highly expressed by many tumors across breast cancer subtypes, data from small clinical trials inhibiting PRL action are difficult to interpret, and studies of xenografts, particularly of breast cancer cell lines, are conflicting. Responses of phenotypically diverse heterogeneous cancers are complicated by different levels of PRLR isoforms with distinct signaling capabilities, selection and genomic evolution as tumors progress and respond to initial therapies, and environmental context, including site-specific responses of the metastatic niche , ECM prin vivo environment , and More for Stage IV.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Obesity and nutrition-related non-communicable diseases (NR-NCDs) are increasing throughout Africa, driven by urbanisation and changing food environments. Policy action has been limited - and influenced by high income countries. Socio-economic/political environments of African food systems must be considered in order to understand what policy might work to prevent NR-NCDs, for whom, and under what circumstances.Methods: A realist synthesis of five policy areas to support healthier food consumption in urban Africa: regulating trade/foreign investment; regulating health/nutrition claims/labels; setting composition standards for processed foods; restricting unhealthy food marketing; and school food policy. We drew upon Ghana and Kenya to contextualise the evidence base. Programme theories were generated by stakeholders in Ghana/Kenya. A two-stage search interrogated MEDLINE, Web of Science and Scopus. Programme theories were tested and refined to produce a synthesised model.Results: The five policies operate through complex, inter-connected pathways moderated by global-, national- and local contexts. Consumers and the food environment interact to enable/disable food accessibility, affordability and availability. Consumer relationships with each other and retailers are important contextual influences, along with political/ economic interests, stakeholder alliances and globalized trade. Coherent laws/regulatory frameworks and government capacities are fundamental across all policies. The increasing importance of convenience is shaped by demographic and sociocultural drivers. Awareness of healthy diets mediates food consumption through comprehension, education, literacy and beliefs. Contextualised data  and inter-sectoral collaboration are critical to policy implementation.Conclusion: Evidence indicates that coherent action across the five policy areas could positively influence the healthiness of food environments and consumption in urban Africa. However, drivers of (un)healthy food environments and consumption reflect the complex interplay of socio-economic and political drivers acting at diverse geographical levels. Stakeholders at local, national, and global levels have important, yet differing, roles to play in ensuring healthy food environments and consumption in urban Africa. Consequently, obesity and nutrition-related non-communicable diseases (NR-NCDs) are rapidly increasing; becoming an important public health challenge. Ghana and Kenya exemplify trends in rapid urbanisation and shifting dietary patterns. Rising overweight and obesity sit alongside persistent and significant burdens of under-nutrition and multiple micronutrient deficiencies in many African countries; and thus this “multiple burden of malnutrition” presents a considerable challenge within African food systems, particularly within urban food environments. The food environment merits particular attention as the interface that mediates people’s food acquisition and consumption within the wider food system, that is influenced by policy and governance. Preventive policy action to address obesity and NR-NCDs within African urban food systems has been limited. High-level political commitments and strategies have been made on hunger and food security , food trade, food retail, food prices, food provision (except schools). At the same time, evidence to inform action within food systems tends to be drawn from potentially less-relevant higher-income settings, partly due to data poverty in many African contexts. What works to enable healthy food consumption in one context need not indicate what could work in another. This lack of contextually-sensitive evidence on what could work in urban Africa undermines the transformative changes that will be required to create healthier food environments and healthier consumption.Many African countries are experiencing a nutrition transition with shifting dietary patterns, related to rapid urbanisation and changing food systems. In practice, this means that evidence to inform strategies to support healthier food consumption, particularly in urban Africa, needs to account for the diverse socio-economic and political environments that drive unhealthy food consumption.Transforming food systems to address the rising challenge of NR-NCDs, particularly in urban Africa, needs to be driven by well-governed policy that considers whether decisions would support or undermine changes elsewhere in the food system. Policy-makers need evidence on what might work, and for whom, in specific urban contexts, if it is to support their decision-making and to inform the political trade-offs that are inevitably needed to secure transformational food system change. and their governments have recognised NR-NCDs as a pressing public health concern through the development of national policies to prevent NR-NCDs. We focused on evidence relating to five ‘good preventative policy’ actions from those identified internationally and locally to improve the healthiness of food systems: regulation of trade in goods/services and foreign direct investment (FDI); regulation of food health/nutrition claims; setting food composition standards/targets for processed foods; restricting the promotion of unhealthy food; and clear/consistent healthy food promotion policy in schools.We carried out a realist synthesis against this backdrop, to contextualise what policy might work to prevent NR-NCDs in urban Africa, for whom, and under what circumstances. Given that food systems are diverse culturally, socio-economically and politically, we drew upon two African countries: – Ghana and Kenya – to contextualise the evidence base. We selected these two countries because they typify dietary and epidemiological transition seen in urban Africa, By extension, realist analysis offers insights on whether similar action might work in other contexts. Policy actions are complex interventions that operate across multiple levels of interconnected food systems. A realist synthesis approach thus aligned with our research objectives in exploring ‘context’ at diverse levels; for example, community environments, cultures, and organisational structures, as well as wider socio-economic and political conditions.Realist synthesis is an “approach to reviewing research evidence on complex social interventions, which provides an explanatory analysis of how and why they work (or do not work) in particular contexts or settings” . Our realist synthesis followed a ‘realist template,’ involving 4 main steps . Each country team used insights from these stakeholder discussions, alongside evidence identified in the Food-EPI policy review, to independently short list seven policies (one from each Food-EPI policy domain) for possible investigation in the realist review. The country teams then compared their short lists, identifying five focal policies for the realist review where there was complete overlap:As a starting point we focused on the seven policy domains  and associated good practice indicators identified in the Healthy Food Environment Policy Index (Food-EPI): a tool developed by INFORMAS  for monitoring government action to improve food environments.Regulate trade in goods/services and FDI (PT1) Regulate food health/nutrition claims (PT2) Set food composition standards/targets for processed foods (PT3) Restrict promotion/marketing of unhealthy food (PT4) Provide clear/consistent healthy food promotion policy in schools (PT5) The importance of these policies to Ghana and Kenya was later confirmed when future government action was prioritised by Food-EPI expert panels (n = 34 across Ghana/Kenya), who discussed evidence collected in the respective countries and independently ranked all five areas as priorities for implementation.Programme theories for the five policy areas are articulated in PT1: Regulate Trade in Goods/Services and FDI IF Governments introduce policies that seek to regulate trade in goods, trade in services and/or FDI THEN industry/businesses will make strategic choices that negate the import, production, processing, retail and/or marketing of unhealthy foodsi) Reduced consumption of unhealthy foods and (ii) Increased consumption of healthy foodsLEADING TO  Reduced consumption of unhealthy food and (ii) Increased consumption of healthy foodsLEADING TO  Reduced consumption of unhealthy foods and (ii) Increased consumption of healthy foodsLEADING TO  will not purchase unhealthy foodsi) Reduced consumption of unhealthy food and (ii) Increased consumption of healthy foodsLEADING TO , which will have a positive impact on their food choicesi) Reduced consumption of unhealthy food and (ii) Increased consumption of healthy foodsLEADING TO .For inclusion, sources had to be published in the English language and focus (in whole/part) on one of the five prioritised policy actions within Ghana, Kenya, or comparable African low- and middle-income countries (LMICs); or, within review papers, in LMICs more generally or at a global level. In contrast to other review types, a realist synthesis includes diverse literature types (unless an item does not relate to any of the theory areas).A two-stage search was conducted using MEDLINE, Web of Science and Scopus databases. Exploratory literature scoping was conducted on PubMed MEDLINE (April-May 2018) to identify initial PTs 1-5 from within the included sources. The scoping search was used to refine subsequent searches. Follow up iterative, comprehensive searches were carried out to identify studies/reports from Kenya and Ghana, to test the identified theories . Follow- One reviewer initially screened titles to eliminate obviously irrelevant references. The remaining titles and abstracts were divided between five reviewers who, after assessing a test set, applied inclusion criteria to all potentially-relevant sources. Decisions on inclusion were recorded in an Excel spreadsheet. A second member of the team reviewed each final selection of sources across the five policy areas to ensure consistency. For each policy area, sub-teams of two-three members extracted text against the evaluative framework . The outcome of interest was change in food consumption, but intermediate outcomes were recorded if mentioned. Data extraction was checked by a second member of the review team. Given that the value of risk of bias assessments within realist syntheses is contested, particularly in examining how complex policies operate  we assessed the overall profile of study designs  supporting each policy action. This approach to assessing the whole evidence base, not individual included studies, proved most useful. In summary, the team considered three Rs, namely, the ‘relevance’ of each source ; its ‘rigour’ ; and its richness . Sets of these configurations are presented within a Microsoft Excel table to show individual components and their relationships and to help to explain how, and under what circumstances outcomes relating to food consumption are achieved. The previously agreed programme theories were continually revised. We drew on our multi-country  and multi-disciplinary  expertise to discuss and challenge the logic models, revised programme theories and supporting evidence at regular team meetings. This added analytical rigour. The logic model for each PT was considered in turn, before combining these into a summary conceptual map by consensus.Data analysis was iterative. Evidence for each policy area was mapped against the evaluative framework derived from our initial programme theories. Logic models for each policy action were generated from extracted data, illustrating how each might work within the socio-economic and political environments of Ghana and Kenya. Aligned to retroductive approaches, connected Context-Mechanism-Outcome elements are identified and coded within the data.Overall, 13317 references were retrieved. After removal of duplicates 6162 were assessed for eligibility. Of these, 5727 were excluded at first screening  . The remThis section highlights findings for the five focal policy actions; discussing how each could support policy action to improve healthier food consumption in urban Africa, drawing on evidence from Ghana and Kenya to contextualise the findings identified . This, in turn, is assumed to precipitate changes in urban consumer food environments, altering availability, accessibility, desirability, purchasing and consumption of ‘healthy’ or ‘unhealthy’ foods  exemplify trade policies that create ‘technical barriers’ to cross-border movement of packaged foods. Governments can also regulate trade in services and FDI; for example, introducing diet- or health-oriented conditionalities on FDI by transnational food companies within an integrated package that covers marketing and provision of information .Import barriers  exemplify regulatory actions that theoretically work by disincentivising trade in goods: reducing cross-border flows of ‘unhealthy’ foods to urban centres by preventing/limiting trade in these foods, raising trade costs and/or encouraging ‘healthier’ product reformulation. Included sources highlighted Ghana’s ‘innovative’ standards for meat products to address rising imports of low-quality fatty meat. Reducing import restrictions  can incentivise trade in raw materials and finished products, bringing cheaper international food imports  into domestic markets, increasing food availability, price competition, affordability, and healthy food consumption. Similarly, removing restrictions on market access for foreign investors in domestic service sectors  and/or reducing restrictions on foreign ownership of companies, can potentially deliver urban dietary gains: enabling food processing, expansion of food retailing, widening food choices and lowering prices. Many LMIC governments, including in Kenya/Ghana, implemented liberalisation measures from the mid-1980s: removing price controls on domestic products like maize and sugar in Kenya, removing import licensing, and loosening investment rules.Removing barriers to trade in goods/services or FDI can theoretically improve healthy food consumption. For trade/investment regulations to work, stakeholders must respond in anticipated ways to incentives/disincentives and urban consumers must adapt purchasing/consumption behaviours in anticipated ways to food environment changes. In practice, many factors affect ‘success’ at these two points and it is difficult to anticipate what will happen in urban Africa due to limited knowledge of the complex socio-material dynamics of supply, demand and local competition, and in contexts occupied by many small and medium enterprises. Many LMICs collate limited data to inform/evaluate policy action . Effects of trade regulation on urban food consumption via, for example, import barrier removal not only depend on how domestic producers respond to new/cheaper products competing in local markets, but also on associated shifts in food environments, food culture and behaviours. Domestic producers could be displaced and/or domestic staples production converted to export cash crops, leading to reduced availability of traditional crops and increasing people’s exposure to ‘unhealthy’ imported food. Impacts on ‘healthy’ consumption depend on types of foods imported , competitive market dynamics and shifts in consumer responses over time. Included sources offered limited consideration of who might be affected by government action, though emphasised differential impacts across urban population groups, with the most vulnerable often disproportionately affected in negative ways.The effects of trade policy on healthy food consumption and on the wider food system are strongly mediated by global, regional, national and local contexts. one review reported mixed results for ‘trade openness’ and undernutrition: with evidence of reductions in underweight and increases in nutrient supply, intakes, and proxies for dietary quality. Yet lower tariffs have increased imports of sugar-sweetened beveragesand may also coincide with increased availability (and likely consumption) of ‘hazardous products’ like sweeteners via a ‘hazard substitution’ pathway. Kenya’s 2015 removal of sugar protections surfaced fears of cheaper sugar imports competing domestically, making greater quantities of confectionary available. Growth of vegetable oil imports in Kenya coincided with liberalising reforms, with vegetable fats now commonly consumed by Kenyan women. FDI is the ‘preferred method’ for transnational companies to enter new markets for processed foods: allowing product marketing that creates demand while adapting to consumer preferences. Evidence highlighted FDI’s role in exposing populations to unhealthy products  in LMICs, and undermining food security in Ghana.While trade liberalisation measures potentially decrease food importation costs, increasing food diversity and availability/affordability of ‘healthy’ foods  In Ghana, standards for meat products reduced imports and availability of low-quality, high-fat meats; with standards more useful than product-specific bans because they apply to imported and domestic meats, thus meeting multilateral trade commitments, and automatically apply to new meat products without needing amendments to legislation. Efforts by Ghanaian producers to get government to impose an additional duty on frozen poultry imports were less successful; tied interests of domestic/international producers/importers and lack of strategic policy framing  undermined such action.Stricter trade regulation can lead to reduced imports of ‘unhealthy’ food products. Higher tariffs on sugar-sweetened beverages can significantly decrease per capita imports.The complexity of trade-nutrition-health links, makes collaborative capacity between ministries important: promoting understanding and policy coherence, so economic objectives do not undermine ‘preventative’ action on trade and FDI. Achieving this can be challenging in LMICs where liberal economic policies may be stated priorities of government and partner international financial institutions. In Ghana, trade and health ministries collaborated successfully to control fatty meat imports but found enforcement challenging due to limited local authority capacities. A key challenge to regulation of FDI is that action may undermine country sources of external financing. Another challenge is regional trading dynamics, with no evidence identified on how membership of regional trading areas affected action in Ghana and Kenya. However, sources illustrated that treaties can limit ‘policy space’ to intervene, given that regulation of food labelling/composition/marketing may infringe agreements  whilst also incentivising trade in ‘unhealthy’ products. For example, South Africa’s regional ‘hub’ role promoted import, investment in and access to soft drinks and processed foods in other Southern African Development Community countries.Effective implementation of trade regulation requires government infrastructural capacity, policy framing, coherent/organised alliances/interest groups and public support . Seven studies from Ghana examine how food labelling impacts upon consumer attitudes/behaviour, with three studies identified from Kenya , time (convenience), adverts, label information, expiry data, nutritional information, ingredients, taste and appearance. African studies report that price is paramount when selecting food products, irrespective of quality and nutritional value. In Kenya, geographical origin may be influential. Too much information can confuse consumers and too little may mislead them. Advertising and price, cultural expectations, the taste-nutrition trade-off, and availability of affordable and attractive alternatives contend with regulation of health claims to influence purchase and consumption behaviour. Limited comparison exists with other African countries; Kenya (25%) and Ghana (33.3%) demonstrate higher non-compliance in nutrition claims than South Africa (10%).For labelling to work, consumers must read, understand, and believe the information content of the label. Nutrition knowledge exerts a low/average impact on consumer food choices Although 75/100 consumers in Ghana reported reading labels prior to selecting food self-reporting is inaccurate. One Ghanaian study showed that consumers are not dissuaded by a label in a language they could not understand. A contemporaneous study found low levels of label reading. Even Ghanaian respondents with tertiary education experience difficulties explaining recommended dietary allowances per serving. Older individuals, white participants and those with higher education and income read nutrition information more frequently. Females were most likely to read and be influenced by food labels.Consumers must have time to read and understand health claims. Consumers may read information to assess nutritional value or health properties, to avoid allergens and to determine quality. Consumers reading front-of-pack claims may conclude a product is healthier, making them more likely to purchase, particularly if predisposed towards that product. Combining short front-of-pack health claims with full claims on the back of the package can lead consumers to better process and believe the claim.Commentators suggest that improving educational levels in urban centres can be exploited as the population becomes interested in food labelling. For this type of action to work, consumers need to be educated to improve nutritional knowledge and to utilize information on food labels. Food labels often require simplification; simple terminology, pictures/colours, a single health endorsement logo and bigger fonts. Presentation of health claims may favourably impact upon product, nutrition attitudes, and purchase intentions. However, price and ingrained purchasing habits exert greater influence than the label.Studies link reading a label that advances a health claim to consumption of that product. A meta-analysis found that perceptions of health claims varied based on the food involved and how it was perceived before the health claim.i) reduced intake of unhealthy food and (ii) increased consumption of healthy foods”  of Ghana enforces food labelling on all processed pre-packed foods.PT3 postulates that, “if governments set targets for composition of processed foods, in terms of healthy and unhealthy ingredients, consumers will reduce their purchase of unhealthy foods and this will ultimately lead to . This aims to ensure that consumers are provided with adequate useful nutritional, compositional and other information related to food products manufactured locally or imported and sold in South Africa. This has impacted positively on changes in consumers’ behaviours towards healthier food choices and preventing food waste.In high-income and LMICs, some evidence supports the important role of high quality food composition data in national food standards guidelines development and implementation to regulate processed unhealthy foods and sugar-sweetened beverages, and the impact of this in reducing diet-related NCDs burden. Lack of expertise and skills of African nutrition scientists, as well as capacities of governments to collect and undertake food/nutrient analysis, are identified as key factors influencing food composition data quality, and/or lack of food composition tables in setting targets. The West African Food Composition Table, including local ingredients from Ghana, provides an opportunity to regulate the sales of processed foods, and in Kenya, national food composition tables have recently been published. However, no such regulation has yet taken place in either country.In African countries that lack high quality food composition databases to provide accurate estimates of nutrients for local food items, such restrictions barely exist. For instance, in Uganda, the lack of a national food composition database is suggested as a potential reason for the lack of national dietary guidelines.Independent of other factors, exposure to unhealthy food marketing is also a modifiable risk factor for obesity. As a result, the World Health Organization (WHO) through World Health Assembly (WHA) Resolution 63.14 called for governments to ensure that “… settings where children gather are free from all forms of marketing of unhealthy foods .” Ghana and Kenya are both signatories.PT4 hypothesizes that if governments introduce policy measures that restrict marketing of unhealthy foods, their production, processing, importation, marketing by industry will reduce–leading to reduced consumption . Plentif Only Morocco has fully achieved implementation of NCD progress monitoring indicator #7C “marketing to children restrictions.” In Ghana and Kenya, government policies exist to restrict exposure and power of promotion of unhealthy foods to or for children across diverse settings. The Ghana FDA requires products to be registered, and advertisement scripts approved before they can be advertised. National NCD Policy priorities to achieve healthy diets for Ghana include regulating advertising of unhealthy foods and non-alcoholic beverages, particularly to children. In Kenya, the Code of Advertising Practice and Direct Marketing (2003) restricts advertisements addressed to or targeted at children with potential to harm them mentally, morally, physically or emotionally. The Kenyan National School Health Policy (2017) prohibits marketing foods and beverages within and around schools. Both Ghana and Kenya embed policies within the national NCD prevention and control strategies focusing on restriction of unhealthy food promotion to children in schools, however local policy-makers rated implementation as low.Globally, countries are actively implementing WHA Resolution 63.14, although few are from Africa. Unhealthy foods are sold to Ghanaian children in school by private/independent vendors and soft drinks are heavily advertised. Contextual evidence from beyond Ghana and Kenya demonstrates that incentives (positive or negative), and underlying factors such as food security, food cost, food/nutrition literacy, health literacy and the respective political economies are important.Effective implementation and enforcement of these interventions requires robust planning processes and resources. Additionally, all stakeholders (government and non-government) should respect the core values of the intervention. Also critical is the mode of regulation; governments favour approaches that encourage the food industry to self-regulate, notwithstanding accumulating evidence that industry policies have minimal impact on reducing children’s exposure to unhealthy food marketing. In Ghana and Kenya, political will is communicated in national policies, but with insufficient implementation. While systematic review evidence relating to the impact of food marketing on children is plentiful, it fails to address the effect of promoting healthy food on healthy food consumption.Recent research confirms inadequate government action in enforcing the policies. Food and beverages consumed at school are typically available through meals provided/sold by school/around school or brought to school. A whole school policy should therefore target all avenues that increase access to unhealthy foods as well as encouraging healthier food choices. School food policies can promote healthy food consumption through several means: nutrition guidelines- providing nutrition standards for menu planning based on food and/or nutrients and applied at school meal programs or at other meals sold in school environment; regulation of food and beverage availability- by controlling the type of food and beverages sold or provided at school; price intervention- free or subsidised provision of healthy foods, or controlling the price of foods or beverages sold in schools. A systematic review of school food policies globally showed that nutrition guidelines and price interventions focused on healthier foods are effective in improving the school food environment and dietary intake.Literature is sparse on school food environments in Ghana/Kenya . Evidenc Where schools lack a functional implementation committee, such as in some regions in Ghana, implementation is a challenge. Linking school feeding policies directly with agricultural development in communities surrounding schools, can lead to success in implementing policies, incentivising farmers to grow healthier foods and thus sustaining local food systems.Effective, enforced school food policies require school-level implementation; Ghana and Kenya have successfully used a decentralized approach. The Ghana school feeding programme relies on financial and technical support from government and development partners; partnerships with the Ministry of Agriculture are needed to ensure that local products are used. In Kenya, a national policy framework for school feeding programs exists, although national school feeding guidelines are not established. School feeding in Kenya lacks stable funding and the institutional and implementation arrangements necessary for sustainability and efficiency. Success of school policies also depends on appropriate monitoring. For example, in Kenya, an innovative approach was the development of a computer-based monitoring system jointly run by the Ministry of Education and World Food Programme, identifying poor management practices and assistance needs in vulnerable areas. Overall, school feeding programme targeting and implementation are best undertaken at school, rather than, national level. Most LMICs do not have standalone policies promoting healthy food consumption in schools for preventing NR-NCDs and instead are established to increase school attendance and address food security of vulnerable populations; thus, emphasizing food supply (quantity) rather than healthy food consumption . For example, Ghana piloted and scaled-up the Ghana School feeding programme to encourage educational participation and improve nutrient intake; while Kenya targeted socially-disadvantaged and nutritionally-vulnerable children, used as an incentive to attract school-aged children to class and was largely supported by World Food Program funding but transitioned to a government funded program in 2009.To be successful, government at all levels need resources for implementing and evaluating policies. Programmes are planned at national level, with local authorities/councils and municipalities responsible for implementation. In some cases, parents take responsibility for preparing meals. Other programmes promote healthy diets to teachers and parents, regulate products sold in school canteens and by neighbourhood vendors and improve dietary knowledge, leading to higher nutrient intakes and dietary diversity, and increased vegetable consumption in families with recipient children. Consequently, overall health outcomes can be better among children in schools where school feeding programs are implemented.Positive elements in guidelines to provide healthy food consumption include micronutrient fortification of commonly-eaten foods and school education to increase children’s knowledge of healthy dietary behaviours. and private food vendors frequently sell unhealthy food adjacent to school exposing children to energy dense foods. In both Ghana and Kenya, children who take money or food to school often purchase energy-dense nutrient poor foods. In Ghana, the national policy for prevention and control of NCDs recommends restriction of fizzy drinks, however this has not served to curb vendors from selling unhealthy foods and drinks to schoolchildren. Evidence suggests that school food policies may support changes to the order of foods presented in buffets which may, in turn, encourage shifts towards healthier consumption. However, in African contexts, school staff and economic dynamics influence behaviours.Although the main school-based food policies in Ghana and Kenya involve school feeding programs, unhealthy foods are frequently available. More than half of Ghanaian schools sell food, most of which is unhealthyThis review examined policy actions to support healthier food consumption in urban Africa, with Ghana and Kenya as focal examples to contextualise what policy actions might work to prevent NR-NCDs, for whom, and under what circumstances. Taken together, findings provide a novel and comprehensive evaluation of how five types of government action, identified as particularly important for our selected African countries, may exert effects. We found evidence that implementing these five policies could positively influence the healthiness of food environments and consumption in urban Africa. However, the drivers of (un)healthy food environments and consumption are clearly shaped by a complex interplay of economic, social and political drivers acting at a range of geographical scales. Stakeholders at all levels  have differing roles to play in ensuring healthy food environments and consumption in urban Africa. Our contribution adds to existing academic work that seeks to understand how food policies work, by more fully considering the context of urban Africa and the “nutrition transition.” the contextual reality of urban Africa sees Governments influenced by competing actors, including industry actors, leading to poor enactment and enforcement. Where policies exist and are enforced, outcomes can be suboptimal due to a myriad of contextual and sociocultural influences. For instance, where textual labelling is in English, functional literacy and health literacy are required, yet these are low in urban Africa. Additionally, literate, but busy urban consumers lack time to read and process information. Similarly, school food policies (PT5) seek to “overcome barriers to meeting healthy preferences caused by lack of financial or physical access…and poor information skills.” Elsewhere, evidence suggests that school food policies may encourage children to reassess their preferences at point-of-purchase. However, in African contexts, school staff and economic dynamics influence behaviours.The five focal PTs suggest relatively simple causal pathways between intervention and effects; with success predicated on assumed predictable and rational choices of stakeholders to differing forms of state intervention. Yet our findings illustrate the socio-cultural, economic and political complexity of policy action in practice, revealing whole system impediments to compliance and illustrate the interdependence of policies seeking to effect transformational preventative action within African food systems. For example, trade and investment regulations (PT1) are inevitably enacted (or not) against a politically and economically contingent backdrop. Intersecting with trade, nutrition labelling, as explored through regulation of food claims (PT2) and setting targets for food composition (PT3), is similarly complex. While in theory the aim is to “induce a food-systems response; overcome barriers to meeting healthy preferences caused by inadequate information,”Integration of the different programme theories  highligh while Kenya has recently launched its own locally developed food composition tables. Significantly, all programme theories lacked other evidence directly relating to urban African contexts. Our findings thus highlight a need for better data systems to inform and monitor transformational policy action. Research, monitoring and evaluation systems are critical to government policy infrastructure for LMICs, including Ghana and Kenya, for effective action within food systems to address NCDs.Contextualised Food Composition Data are key to how policies operate, for example in controlling marketing to children or food labelling, and could explain contextual differences between Ghana and Kenya. Yet, such data are either unavailable, poor in quality of or dated. Food Composition Tables specific to Ghana have not been updated since 1975 with Ghana relying, instead, on regional West African Food Composition Tables An additional challenge is the persistence of food insecurity impacting on government policy decisions on the promotion of healthy foods consumption in Africa.Therefore, there is the need to highlight the potential double duty actions of the policies being discussed. These issues are highlighted in relation to trade policy actions, and substantiate studies that find limited institutional checks and balances on commercial policy influence in Ghana and Kenya, which can drive resistance to policy implementation. As evidenced in other LMICs, policy action to regulate private sector activity relating to food and nutrition can quickly be revoked following extensive industry lobbying at government-level. For example, Morocco repealed its Sugar Sweetened Beverage tax in 2018 prior to implementation in 2019 in response to pressures from the agri-food industry. A year later, an attempt was made to introduce a significantly ‘watered-down’ bill. Only South Africa within the African Region has been able to introduce such a tax with concerted efforts, resources, and alliances of civil society, academia, and government to defeat resistance from food companies. Implementation of WHA Resolution 63.14 has faced similar challenges.Interdependencies between policy actions , and the critical role of contextualised food composition data, highlight that transformational policy action for urban contexts in Africa depends on the complex interplay of economic, political and socio-cultural forces. These interdependencies illustrate the need for policy coherence; particularly challenging in African contexts where ‘policy space’ to address healthy food consumption is limited by the prioritisation of economic growth by governments and partner IFIs, the role/interests of economic stakeholders in external financing and policy decision-making, and limits in government infrastructural capacities. The political economy of implementing healthy food system actions within the African context is unique and complex. Studying the myriad of influencers , is required to fully appreciate the variegated political economies of the African food system, and to intervene effectively.Of 54 countries in Africa, only Morocco fully achieved implementation of WHO NCD progress monitoring indicator #7C “marketing to children restrictions.”Our findings highlight the importance of government institutional capacities here: to develop policy collaboratively across government, to develop alliances with non-government actors, and to implement and enforce decisions at multiple levels. Capacity for enforcing regulatory action was highlighted as important to trade policy action, yet limited by what authorities can achieve within existing resources. Controls on food labelling and food composition similarly require capacity for enforcement as highlighted in previous studies.One such actor is The Scaling Up Nutrition Network . This Network may have a potential role to play; in supporting cross-sectoral alliances, in better understanding local contexts and in framing strategic action to prioritise NR-NCDs. Key strengths of this realist review include an integrated approach that draws on systematic reviews, primary data and the involvement of high-income and LMIC stakeholders. However, such a synthesis is limited by the evidence available for inclusion. Much of the data was indirect; ie, the research questions of sources did not always directly correspond with the focal programme theories. Findings from the two focus countries may not be applicable internationally. Additionally, many studies employed weak research designs potentially impacting on the results. Lastly, in synthesising the current evidence base, the realist review highlights the dearth of country-specific information on how and why policy actions might ‘work’ (or not). Results, although indicative of the available evidence, should be viewed cautiously until directly-relevant studies become more plentiful. Not applicable. AB holds an honorary contract with the WHO. MH holds an honorary contract with the University of Sheffield. No other relationships or activities have been disclosed. AB and MH are both responsible for the study design. AB performed the searches and led data extraction and analysis. All Authors selected items for inclusion, extracted data, checked data extraction and contributed to data analysis. All authors contributed to writing the manuscript. The views presented in this paper are those of the author(s) and do not necessarily represent the views of the funder or of their employing organizations. Sources of support: Review undertaken as part of the Dietary Transitions in African Cities (TACLED), involving a collaboration between the University of Ghana, African Health and Population Centre, Kenya; and the Universities of Sheffield, Liverpool, Loughborough in the UK. The TACLED project was supported by a Global Challenges Research Fund Foundation Award from the UK MRC [grant number MR/P025153/1], and supported by AHRC, BBSRC, ESRC and NERC. The funders played no role in the design of the study, data collection, data analysis, interpretation of the data or writing of the publication.1School of Health and Related Research (ScHARR), University of Sheffield, Sheffield, UK. 2School of Public Health, University of Ghana, Accra, Ghana. 3Institute for Health and Society, Newcastle University, Newcastle upon Tyne, UK. 4African Population and Health Research Center (APHRC), Nairobi, Kenya. 5French National Research Institute for Sustainable Development (IRD), NUTRIPASS Unit, IRD-Univ Montpellier, Montpellier, France.Supplementary file 1. Search Strategy.Click here for additional data file.Supplementary file 2. Pathway From Trade/Investment Policy to Food Consumption.Click here for additional data file.Supplementary file 3. Pathway From Nutritional/Health Information to Healthy Food Consumption.Click here for additional data file.Supplementary file 4. Pathway From Food Composition Standards to Healthy Food Consumption.Click here for additional data file.Supplementary file 5. Pathway From Restricting Promotion/Marketing of Unhealthy Foods to Reduced Consumption of Unhealthy Foods.Click here for additional data file.Supplementary file 6. Pathway From Food Promotion/Regulation in Schools to Reduced Consumption of Unhealthy Foods.Click here for additional data file.


Trypanosoma parasites, the causative agents of Human and Animal African Trypanosomiases. Like many animals subsisting on a limited diet, microbial symbiosis is key to supplementing nutrient deficiencies necessary for metabolic, reproductive, and immune functions. Extensive studies on the microbiota in parallel to tsetse biology have unraveled the many dependencies partners have for one another. But far less is known mechanistically on how products are swapped between partners and how these metabolic exchanges are regulated, especially to address changing physiological needs. More specifically, how do metabolites contributed by one partner get to the right place at the right time and in the right amounts to the other partner? Epigenetics is the study of molecules and mechanisms that regulate the inheritance, gene activity and expression of traits that are not due to DNA sequence alone. The roles that epigenetics provide as a mechanistic link between host phenotype, metabolism and microbiota (both in composition and activity) is relatively unknown and represents a frontier of exploration. Here, we take a closer look at blood feeding insects with emphasis on the tsetse fly, to specifically propose roles for microRNAs (miRNA) and DNA methylation, in maintaining insect-microbiota functional homeostasis. We provide empirical details to addressing these hypotheses and advancing these studies. Deciphering how microbiota and host activity are harmonized may foster multiple applications toward manipulating host health, including identifying novel targets for innovative vector control strategies to counter insidious pests such as tsetse.Tsetse flies have socioeconomic significance as the obligate vector of multiple  Glossina, and are divided into four groups: morsitans, fusca, palpalis, and austeni  varying in their occurrence and ranging in their impact toward host biology from parasitism to mutualism. The obligate mutualist, Wigglesworthia, is a focal point of this review and will be further discussed below. A commensal Sodalis (W. pipientis (supergroup A) may be harbored by tsetse typically within reproductive tissues , a debilitating disease caused by the parasitic invasion of the central nervous system which is lethal if left untreated. The disease is endemic to 36 countries in sub-Saharan Africa. Animal African Trypanosomiasis  is a wasting disease caused by trypanosome infections of domestic animals, contributing to food insecurity within impacted areas. Trypanosome infections of tsetse impose a reproductive burden on females , riboflavin (B2), nicotinamide (B3), pantothenic acid (B5), pyridoxine (B6), and folate  noncoding RNAs with a primary function in sequence-specific post-transcriptional gene regulation . Gene rekaryotes  with seqkaryotes . For exa genomes . High coosophila . Despiteosophila , consequosophila . While mosophila , their ploquacious protein  forming a ~22 nt miRNA:miRNA* duplex  phenocopies the compromised peritrophic matrix and offers an exciting  technological advancement toward studying the regulatory roles of other miRNAs. Lastly, tsetse with symptomatic Salivary Gland Hypertrophy Virus (SGHV) infections exhibit different tsetse miRNA and SGHV miRNA expression profiles upon comparison to asymptomatic flies. With symptomatic flies, the most highly expressed miRNAs are predicted to target immune-related mRNAs, including those encoded by fibrillin-1 (FBN1) and Ras-related protein-27 (Rab27), and others involved in reproduction such as apolipoprotein lipid transfer particle (Apoltp) and vitellogenin receptor  have differential expression in aposymbiotic compared to wild-type flies, which may indicate a similar regulatory role toward these genes could be played by tsetse miRNAs  which facilitates Wolbachia association with microtubules enabling its transfer during mosquito oocyte or embryonic development  groups to cytosine residues (5mC) typically within 5′-cytosine-phosphate-guanine-3′ (CpG) dinucleotides  is particularly deficient within blood . SAM donates the methyl group during DNA methylation via the action of DNA methyltransferases , needed for the formation of methionine from homocysteine . Once mesferases . Folate ymbionts .D. melanogaster and the characterization of DNA methylation in the fruit fly genome . Figures were created through BioRender.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exposure to environmental toxins, such as cigarette smoke, polluted air, and pesticides, can negatively impact brain health, leading to cognitive decline and an increased risk of neurodegenerative diseases, such as Alzheimer's Disease. Additionally, exposure to certain toxins during critical periods of brain development can lead to developmental disorders and birth defects. The precise mechanisms by which these toxins affect brain health are yet to be elucidated. Some proposed mechanisms include oxidative stress, inflammation, and changes to neurotransmitters. In this Research Topic, which focuses on the impacts of environmental toxin exposure on brain health, five peer-reviewed papers tackled such research questions from different perspectives addressing toxins coming from natural sources (air pollutants) or human activities . These findings provide new insight into novel potential therapeutic targets and preventative measures to mitigate the detrimental effects on those exposed and their offspring.Li et al. reviewed existing evidence to identify the mechanisms of by which common air pollutants (PM2.5) induce brain damage. PM2.5 refers to the fine particles in polluted air with a diameter of 2.5 micrometers or smaller. These fine particles can enter the bloodstream and damage the brain via various actions, including cerebrovascular damage, brain nerve damage and brain tumor formation. Furthermore, they suggested the underlying mechanisms of action, including autophagy, oxidative stress, neuroinflammation, and autonomic dysfunction. Questions remain as to which brain circuits are involved and whether epigenetic mechanisms play a significant role.Chronic exposure to tobacco cigarette smoke is a significant risk factor for chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide -1 was decreased in cigarette smoke exposed mice compared to sham controls. This supports existing evidence of a compromised blood brain barrier initiating brain damage in those with smoking-induced COPD. They also demonstrated the development of microglia-dependent neuroinflammation in the hippocampus due to chronic cigarette smoke exposure. This could provide a framework for future clinical interventions to protect the brain from neuroinflammatory sequalae in patients with COPD.Using an elegant series of behavioral and molecular measures, Wang et al. explored the association of cigarette smoking with post-operative pain by examining cerebrospinal fluid (CSF) levels of the neuropeptides beta-endorphin and substance P in a Chinese cohort. Compared to non-smoking controls, active smokers exhibited higher subjective pain intensity and longer post-surgical recovery time accompanied by lower beta-endorphin and higher substance P levels in the CSF. The findings suggest that interventions to increase beta-endorphin and reduce substance P levels in the CSF of active smokers may be effective in reducing post-operative pain intensity. Substance P is a sensory neurotransmitter important in the perception of pain  receptor is abundant in the developing human brain. Although buprenorphine was found to convey inhibitory signals via the NOP receptor and alter gene expression and interneuron migration into the cortex, neural activities were not changed. In rats, the authors demonstrated that prenatal exposure to buprenorphine alters interneuron migration in the adult cortex. These findings may implicate buprenorphine exposure in utero in the etiology of neurological diseases arising from disrupted interneuron function in the cortex , especially given that buprenorphine has modest NOP receptor agonist activity. Future investigation of the pharmacological interactions between NOP receptor antagonists or NOP receptor knock out animals, in the context of prenatal buprenorphine exposure, will continue to give essential information about the influence of buprenorphine on neural circuits involved in psychiatric illness.Like smoking, maternal exposure to opioids can also have adverse and long-lasting outcomes on the developing human brain. Due to the opioid epidemic, the Schedule III opioid analgesic buprenorphine is now preferentially used for treating pregnant patients with opioid use disorder. Buprenorphine is clinically indicated as a first line treatment over other opioids, such as oxycodone and fentanyl, due to its safer therapeutic index, effectiveness, and favorable outcomes for both mother and offspring (Maciag et al., The articles published in this Research Topic highlight cigarette smoking, maternal cigarette smoking, opioids use during pregnancy, and PM2.5 as environmental toxins contributing to the pathogenesis and etiology of brain dysfunction. These studies advance our understanding of the molecular mechanisms involved in acute and long-term exposure to environmental toxins.NK prepared an initial draft of this editorial. It was carefully revised by HC. All authors approved the final version for submission.


Precision medicine has put forward the proposition of "precision targeting" for modern drug delivery systems. Inspired by techniques from biology, pharmaceutical sciences, and nanoengineering, numerous targeted drug delivery systems have been developed in recent decades. But the large-scale applications of these systems are limited due to unsatisfactory targeting efficiency, cytotoxicity, easy removability, and instability. As such, the natural endogenous cargo delivery vehicle—extracellular vesicles (EVs)—have sparked significant interest for its unique inherent targeting properties, biocompatibility, transmembrane ability, and circulatory stability. The membranes of EVs are enriched for receptors or ligands that interact with target cells, which endows them with inherent targeting mission. However, most of the natural therapeutic EVs face the fate of being cleared by macrophages, resulting in off-target. Therefore, the specificity of natural EVs delivery systems urgently needs to be further improved. In this review, we comprehensively summarize the inherent homing mechanisms of EVs and the effects of the donor cell source and administration route on targeting specificity. We then go over nanoengineering techniques that modify EVs for improving specific targeting, such as source cell alteration and modification of EVs surface. We also highlight the auxiliary strategies to enhance specificity by changing the external environment, such as magnetic and photothermal. Furthermore, contemporary issues such as the lack of a gold standard for assessing targeting efficiency are discussed. This review will provide new insights into the development of precision medicine delivery systems. Tracing the full range of factors affecting EVs targeting mission in terms of EV components, cell sources, and delivery methods.Strategies for EVs surface display and post-isolation methods facilitate the targeted delivery efficiency of the loading therapeutic cargo.EVs complemented by non-invasive strategies such as magnetic fields, ultrasound, and photothermal further enhance targeting efficacy. Precision medicine poses new challenges for modern drug delivery systems, the most important of which is targeted precision delivery . AlthougThe main equipment for EVs to accomplish targeted missions is structures enriched on the surface of EVs, such as receptor-ligand proteins. The surface enrichment of EVs with numerous receptors or ligands that have a natural affinity for receptors heavily expressed on the surface of target cells is the method by which they are targeted . TherapeUnfortunately, the surface shape, cellular origin, and mode of administration targeting missions provided to EVs are insufficient to contend with the complicated in vivo microenvironment. Structural fragments such as proteins on the surface of EVs are expected to customize switches for their targeting capabilities. Nano-engineered EVs are those created by changing EVs’ parent cells or directly on EVs , 23. ForIn this review, we provide a comprehensive description of the natural homing mechanism of EVs, starting from the enhanced permeability and retention (EPR) effects of EVs in targeting tumor cells and the specific targeting molecules on the surface of EVs. Furthermore, we discussed the effects of various cell sources and administration routes on EVs reaching the target cells. We highlighted and analyzed numerous methodologies for genetic alteration and direct modification of EVs in the realm of nanoengineered EVs targeting technology. The use of magnetic fields, ultrasound, photothermal and PH-sensing peptides as targeting aids to facilitate the effective accumulation of EVs at the site of injury. Finally, we describe some difficulties associated with EV-targeted drug delivery design systems.Tetraspanins are often viewed as surface markers of EVs and are involved in processes such as cell activation and adhesion , 31. ForThe phospholipid bilayer of EVs, composed of phosphatidylserine (PS), cholesterol and sphingolipids, is also responsible for the process of EVs targeting to specific cells. For example, PS can target immune cells by binding to the phosphatidylserine receptor (PSR) that causes immunosuppression, but more often, PS is recognized by macrophages and leads to EV clearance , 38. FlaDue to the removal of macrophages and the reticuloendothelial system (RES) , EVs areWhen focusing on tumor cells in receptor cells, we found that natural EVs have been extensively studied for targeting tumors, which is largely attributed to the EPR effect , 46. SpeThe above targeting effect is mainly determined by the molecules on the surface of EVs, and EVs inherit some proteins from parental cells when they are secreted , so the Due to the organ tropism of extracellular vesicles, most tumor-derived EVs will home to the tumor cells themselves. Based on this feature, EVs can be used as a powerful tool for targeting cancer therapy. Cancer cell membrane antigens E-cadherin is expressed on the surface of urine-derived EVs from prostate cancer patients, which allows EVs to preferentially target homologous tumors , 57. Li However, tumor-derived EVs also target other sites, such as immune cells, and endothelial cells. Molecules on tumor-derived EVs that achieve natural targeting include tumor antigens expressed on their surface, which are presented to immune cells as specific stimuli and mediate anti-tumor effects. Chen et al. demonstrated that B lymphoma cell-derived EVs containing heat shock proteins (HSP60 and HSP90) and major histocompatibility complex (MHC) molecules enabled them to act as antigen presentation vehicles, targeting T cells and exhibiting more potent immunostimulatory activity . The preDendritic cell (DC) -derived EVs express MHC on their surface and trigger immune effects . It was LFA-1 is a part of integrins and is involved in cell adhesion and T-cell activation . LFA-1 iMesenchymal stem cells (MSCs) have become an effective treatment for various diseases. After systemic injection, because of the large size of bone marrow mesenchymal stem cells (BMSCs), they quickly became trapped in the pulmonary vascular bed and typically less than 1% of BMSCs could be homed to the target site . MSC-derIn addition to the abovementioned common EVs-producing cells, many other cells could be suitable sources for EVs-targeted therapy, including endothelial cells, blood, and milk. The lack of bone targeting strategies has been a bottleneck in the clinical management of degenerative bone lesions . EndotheDifferent routes of administration lead to different rates of drug arrival at the target organ and eventual accumulation in different organs. For example, when nitroglycerin is used to treat angina pectoris, sublingual administration is often chosen to reach the treatment site more rapidly. The EVs serve as vehicles for drug delivery, and the way they are injected is also critical for targeted therapy. In this section, we will focus on the effect of the mode of administration on the destination of the EVs Fig. .The most common mode of administration is intravenous, considering that intravenous injections allow direct access to the body circulation. Wiklander et al. evaluated the effect of the injection route on EVs distribution and found that intravenous injection accumulated more in the liver compared to other methods (60% ± 3.9) . The resIntraperitoneal injection is also an effective method for EVs to deliver therapeutic molecules to target cells. Heidari et al. injected mesenchymal stem cell-derived EVs into the intraperitoneal cavity for treating acute colitis caused by dextran sulfate sodium . Nojehde−/− mice and collagen-induced arthritic mice [With the development of milk as one of the promising sources of EVs, oral EVs have become a more convenient and safer way to administer drugs without the need for a medical professional. Oral administration of milk-derived EVs can be used to observe improvements in joint inflammation. The data conclude that EVs in the treatment group do affect T-cell immune gut microbiota directly or indirectly through inflammation and are important for autoimmune arthritis in IL-1Ratic mice . Nazmek tic mice . One restic mice .+) EVs in cholesterol liposomes resulted in a significant reduction in tumor growth in mice (58%), which depended on the intratumoral EVs administration [In addition to the abovementioned injection methods, local delivery, particularly intratumoral delivery in cancer treatment, has received a lot of attention. This is because it allows the drug to concentrate on a specific site. Taking advantage of this feature, local injection of the drug facilitates its accumulation in the injured tissue. In the prostate cancer mouse model, intratumor-injected EVs were more bound to tumor tissue, and TNF-related apoptosis-inducing ligand  and lysosome-associated membrane protein-2b (Lamp-2b), tetraspanins , which can be combined with specific targeting ligands for enhancing their targeting ability. Lamp-2b, often found on dendritic cell-derived EVs, is a commonly used membrane protein that contributes to EVs homing . AlvarezIn addition to Lamp-2b, other membrane proteins on the surface of EVs can serve as anchor points for targeting motifs, for example, cluster of differentiation 9 (CD9) can bind to human antigen R (HuR), an RNA binding protein ; the traThe above methods for modifying the parental cells of EVs suffer from low transfection efficiency, and researchers are currently working on further modifications of the secreted EVs to improve targeting efficiency. A nanoengineered EVs membrane targeting platform that also enables EVs to carry targeting peptides has been established, called cloaking Fig. , in whicResearchers have discovered an advanced method for improving the targeting function of EVs: chemically attaching some functional groups to the surface of EV membranes. Copper-catalyzed azide-alkyne cycloaddition (click chemistry) is an efficient and promising technique for introducing various functional azide groups into EVs for subsequent bioorthogonal reactions. Smith et al. applied this click chemistry approach and demonstrated that the method not only allows EVs to be more easily targeted to recipient cells by chemical bonding, but also does not alter the size of the EVs or affect the endocytosis of the target cells . Jia et Recently, a more effective, simpler and newer technology for actively targeted EV delivery has emerged: nanoengineering of aptamer-targeted EV delivery. Compared with the previously used antibodies and peptides, aptamers have some significant advantages, including higher stability, stronger specificity, less toxicity, and superb tissue penetration . ConsideKV11, which could efficiently deliver an anti-angiogenic peptide and suppresses neovascularization of the retina [For precisely targeted drugs to treat disease, CP05 peptide was turned out. CP05 peptide can bind to CD63, a marker on the surface of EVs, with high affinity to the second extracellular loop  in hepatocytes or HepG2 cells. The disadvantage of targeting EVs based on electrostatic interactions is the cytotoxicity of pullulan . After tThe surface of EVs can be modified with the receptors of some targeting ligands to enhance their targeting capability. It is well known that folate (FA) receptor is overexpressed on the surface of the majority of cancer cells. Based on this, Zheng et al. constructed FA/exosome to deliver siRNA for effective cancer inhibition . Yang etAlthough nanoengineered EVs have been extensively tested for targeted therapies, there are limitations to their various approaches. The typical approach to nanoengineered EVs is to transfect genes into the parental cells of the EVs so that the secreted EVs membrane expresses the target protein. The EVs subsequently bind specifically to receptors that are highly expressed at the site of the lesion. However, this gene modification method has some problems, such as safety issues, complex and expensive gene transfection, and unstable transfection, targeting peptides may affect the structure of the EVs surface . One limSurface modification of EVs has already yielded many remarkable results in improving targeting specificity, and surprisingly, researchers have found that targeting effects will be aided by synergistic external stimulation. Several researchers have considered the use of external magnetic fields to enhance the targeting of EVs. For example, magnetically functionalized blood EVs can be effectively targeted to treat cancer by preparing superparamagnetic iron oxide nanoparticles (SPIONs). The surface of EVs produced by reticulocytes within blood contains membrane proteins including TfR that can bind to superparamagnetic nanoparticles coupled to Tf.  HarvestiTo achieve precise treatment of cancer, Wang et al. provided a revolutionary strategy, which can achieve nanoengineered EVs active targeting and controlled drug release: using RGD and FA as double ligand modification, combined gold nanorods (AuNRs) to selectively eliminate tumor cells under the irradiation of near-infrared light without affecting the surrounding normal tissues . An emerUltrasound can enhance cell membrane permeability and promote the uptake of drugs by cancer cells and is commonly employed as an auxiliary tool for tumor treatment . Liu et Current studies of EVs-targeted therapies typically use EVs as nanocarriers loading proteins or therapeutic RNAs that bind specifically to target cells, but little attention has been paid to the evaluation of various nanoengineered EVs-targeted therapeutic approaches. Commonly used methods for observing the distribution patterns of EVs include lipophilic fluorescently labeled EVs, X-ray, and positron emission tomography (PET) , 156. FlAn ongoing clinical trial for Alzheimer's disease has shown that MSCs-Exos are nasally dropped in amounts of 5–20 μg and is required twice a week for 12 weeks. These exosomes take several weeks to extract . The selection of a suitable source of EVs for the experimental purpose was mentioned above as an effective way to address the low EV production. There is no clear method for meeting the volume of EVs required for large-scale clinical trials, and every process for producing therapeutically targeted EVs requires a safe protocol that reduces batch-to-batch variability. Alternatively, EVs mimics that are structurally and functionally similar to EVs but with higher yields are gaining interest in the field of cancer therapy . HeterolThe environment to which EVs are exposed (pH and temperature) can significantly affect their uptake by the cells. Although low pH decreases the stability of EVs to some extent, it increases the uptake of EVs . It has Although many studies have demonstrated that EVs are promising therapeutic vehicles for targeted drug delivery, the EVs that are currently isolated often contain "impurities" such as apoptotic vesicles or microvesicles . Most peSome studies have also shown that the environment in which cells are cultured, such as hypoxia and low PH, can also enhance the EV production  and evenEVs, as cell-released vesicles, are emerging as promising nanoplatforms for drug delivery. Natural EVs themselves abundantly express unique membrane surface molecules including proteins, lipids, and glycans that facilitate EV targeting mission. Moreover, EVs injection modalities and parent cells determine the fate of EVs after release into the extracellular matrix. In order to upgrade natural EV-targeted drug delivery capabilities, researchers explored a range of nanoengineering approaches, including surface display and post-isolation methods. We further compared different nanoengineered EVs strategies. Several non-invasive external stimuli can assist natural and nanoengineering EVs in targeted drug delivery. Aggregation of therapeutic EVs to the injured tissue by magnetism, ultrasound or photothermal can reduce off-target effects of EVs and avoid damage to normal tissues. Despite these encouraging achievements, there are still some obstacles to the application of EVs in precision delivery platforms, such as the lack of a gold standard for measuring EVs targeting efficiency and the need to overcome the problem of insufficient EVs yield. Thus, more advanced approaches are still needed to expand the potential of EVs in targeted therapies. In the future, we expect a multidisciplinary effort to achieve a scale-up production of quality-controlled targeted EVs and break the therapeutic bottleneck of EVs in targeted therapy.


Guided bone regeneration (GBR) has become a clinically standard modality for the treatment of localized jawbone defects. Barrier membranes play an important role in this process by preventing soft tissue invasion outgoing from the mucosa and creating an underlying space to support bone growth. Different membrane types provide different biological mechanisms due to their different origins, preparation methods and structures. Among them, collagen membranes have attracted great interest due to their excellent biological properties and desired bone regeneration results to non-absorbable membranes even without a second surgery for removal. This work provides a comparative summary of common barrier membranes used in GBR, focusing on recent advances in collagen membranes and their biological mechanisms. In conclusion, the review article highlights the biological and regenerative properties of currently available barrier membranes with a particular focus on bioresorbable collagen-based materials. In addition, the advantages and disadvantages of these biomaterials are highlighted, and possible improvements for future material developments are summarized. Guided bone regeneration (GBR), as one of the most common strategies for alveolar ridge preservation/augmentation, is regarded as a standard treatment modality. Barrier membranes play a key role in GBR by forming a barrier between soft tissue and the bone defect area, thus facilitating the proliferation of osteoprogenitor cells and supporting new bone tissue formation. In addition to the space maintenance function, a successful material design of the “ideal” GBR membrane should take into account the following properties: 1) biocompatibility: does not damage the surrounding tissue and the healing process; 2) cellular occlusion: prevents the invasion of non-osteogenic cells into bone defect from the mucosa; 3) easy handling: not too rigid without sacrificing space maintenance function; (4) bioactivation properties: promotes wound healing and tissue integration . It. It77]. The main factors currently limiting collagen membranes are their low stiffness and rapid degradation in vivo. After implantation, collagen membranes can be absorbed by enzymatic degradation mediated by collagenases, bacterial proteases, and macrophage-derived enzymes  80,81].,81.80,81®Plus, for example, is a very densely structured ribose crosslinked membrane that is absorbed after eight months in vivo , a collagen membrane derived from the equine Achilles tendon, has also not shown a satisfactory degradation pattern, with a standing time of only 4–6 weeks [The collagen of tendon accounts for up to 85% of its dry weight and is composed almost entirely of type I collagen ,89. Ther–6 weeks , which sCollagen from the pericardium is also attractive in terms of mechanical properties because it retains the biomechanical properties of the pericardial tissue . It ofteMoreover, it was shown in a preclinical study by Alkildani et al. that a pericardium-based barrier membrane was completely ossified over a period of 16 weeks and thus contributes to bone tissue regeneration in combination with a bone substitute material (manuscript in preparation) .per capita [Among mammals, bovine and porcine are the most important sources of collagen extraction because they are the most consumed meat r capita . Howeverr capita . Collager capita . Equine r capita . Equine r capita ,97. Thisr capita . Similarr capita . Equine ®-3D scaffolds, Jellagen® Ltd., Cardiff, UK) induced an overall weaker immune response than porcine pericardial collagen scaffolds. It induced long-term M2 cell responses and optimal vascularization patterns within the implantation bed [In this context, non-mammalian marine organisms become a very attractive alternative source . Jellyfition bed .Poor mechanical properties and high degradation rates of native collagen-based barrier membranes are still dominant limitations in clinical applications. To enhance durability and mechanical strength, crosslinking as an efficient method has attracted extensive studies. In principle, crosslinking inhibits sliding between collagen molecules under pressure by introducing intramolecular and intermolecular covalent or non-covalent bonds, which increases the stiffness, tensile strength, compressive modulus, and reduced extensibility of collagen fibers . At the Physical crosslinking methods of collagen mainly comprise dehydrothermal (DHT) and ultraviolet (UV) irradiation. Both processes do not require the incorporation of any chemical agents avoiding the threat of biological toxicity.® as one of the most common commercial collagen membranes. Altogether, the UV crosslinking membrane is mainly limited by the difficulty of achieving satisfactory mechanical properties compared to chemical crosslinking. It is therefore often used as an auxiliary crosslinking method, in combination with other crosslinking methods. In addition, UV crosslinking is more suitable for fabricating thin or transparent membrane considering the penetration of UV irradiation.UV induces the formation of highly reactive free radicals, which mediates the formation of intra- and extra-fibrillar carbonyl-based covalent bonds at aromatic amino acid residues . This me® besides exhibiting well tissue integration in vivo [Dehydrothermal (DHT) is another common physical method for collagen crosslinking that involves exposure to high temperatures in a vacuum condition to cause the formation of intermolecular amide and ester bonds through dehydration . As earl in vivo . Thus, DGlutaraldehyde (GA) is the most widely used traditional crosslinking agent for collagen, due to its low cost, high reactivity, and high solubility in aqueous solutions. However, it has been rarely used for tissue engineering studies in recent years due to its local cytotoxicity and induction of calcification as well as inflammatory responses  (Shi 202®) in a rat subcutaneous model [In contrast, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC-NHS) is a “zero-length” crosslinker that chemically activates the carboxylic acid groups of aspartic acid and glutamic acid on collagen to conjugate with hydroxylysine and amines of lysine residues through direct covalent bonds without any linkers or spacers ,106. Allus model ,121. Theus model .Gardenia jasminoides L. or Gardenia jasminoides [Enterococcus faecalis, Porphyromonas gingivalis, and Clostridium difficile) [Natural crosslinkers have significant advantages over traditional chemical crosslinkers in terms of biocompatibility. Genipin is the best-known natural crosslinking agent mainly extracted from the fruits of minoides . It is ominoides . Nair etminoides . Howeverminoides . This stminoides . With inminoides . Naturalminoides . Proanthminoides ,128,129.minoides  and micrfficile) ,132. In fficile) . The colfficile) . This isfficile) . Epigallfficile) . The incfficile) . EGCG crfficile) ,138. Notfficile) .The crosslinking and stability of collagen in vivo largely depend on enzymatic reactions. The most representative one is the transglutaminase that catalyzes the formation of ε-(γ-glutaminyl)-lysine isopeptide bonds to assemble various proteins related to mineralized tissue formation  into polymeric forms involved in matrix stabilization, chondrocyte and osteoblast differentiation, and matrix mineralization . NaturalThe binding of multiple bioactive molecules to membranes has received the most attention because of their multifunctional role in osteogenesis, particularly cell recruitment, proliferation, and differentiation . CommonlThe binding of active molecules to the membrane is based on the hypothesis that exposure of the treated area to multiple different growth factors can trigger the development of a favorable microenvironment and promote bone regeneration ,155. PlaAlthough all these studies demonstrate the feasibility of delivering active molecules through membranes, the instability  and early burst release of protein factors greatly hinder their clinical application. The supraphysiological dose of active molecules to compensate for their instability also raises toxicity and cost concerns . DeliverMany studies have been conducted to demonstrate that the doping of trace elements, such as zinc (Zn), magnesium (Mg), cobalt (Co), and strontium (Sr), can further enhance the biological activity of collagen. In addition, the accompanying release of trace elements can regulate the local biological environment. The interest in the osteogenic properties of Sr stems from the therapeutic effects of strontium ranelate in osteoporosis. Sr supports osteoblast differentiation and activates the expression of one of the osteoblast markers, osteopontin, a marker of late osteoblasts . Sr alsoStaphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) without obvious cytotoxicity. The AgNP-coated membrane also has effective anti-inflammatory effects by inhibiting the expression and release of anti-inflammatory cytokines such as IL-6 and TNF-α. In addition, the resultant membrane was able to induce osteogenic differentiation of mesenchymal stem cells, demonstrating its osteogenic potential [Antibacterial agents and antibiotics, such as tetracyclines , metroniotential . Amoxiciotential . Polysacotential . In partotential . The asyotential . In contotential . PeptideBarrier membranes have been shown to provide more than just barrier functionality in GBR procedures . For exaAlthough the current wealth of histological evidence relating to bone healing and regeneration beneath barrier membranes is insufficient to explain the clear role of barrier membranes in defect healing involving inflammation, cell recruitment, and bone remodeling, it has been shown that the immune response elicited by different materials is specific, depending on the physicochemical properties of the material . This “fThis raises the key question of whether the foreign body response triggered by the membrane is necessary and beneficial for tissue regeneration at the defect. This is based on the fact that the immune response appears to mediate the degradation of the material and transmembrane vascularization.The inflammatory tissue response is a cascade effect. Inflammatory cells such as monocytes, macrophages, and neutrophils direct the pattern of the immune response cascade through their interaction with proteins and the release of cytokines ,205. WitAlthough the relationship between the different material properties and the activation and expression patterns of MNGCs is unclear, it has been established that the immune response is crucial in the bone healing process. Modification of material properties can optimize the immune response of biomaterials to better support bone healing.Angiogenesis is an important part of the microenvironment that facilitates bone healing. The concept of “transmembrane vascularization” has been proposed for collagen barrier membranes . PreviouBarbeck et al. evaluated two porcine dermal-derived collagen membranes (Mucoderm and Collprotect) and noted that although neither membrane showed successful transmembrane vascularization, the Mucoderm membrane containing the vascular backbone allowed for microvascular penetration associated with the inward growth of connective tissue . This prIn conclusion, understanding the biological mechanisms of membranes and the related integration pattern in vivo is essential for material preparation. Once the relationship between different material properties and mechanisms related to bone remodeling is clearly understood, material properties can be used to regulate key biological events to support better tissue regeneration.The use of barrier membranes to block the inward growth of soft tissue is a standard strategy that has been used successfully in clinical practice. Resorbable and non-resorbable membranes differ in terms of clinical procedures, complication rates, and long-term outcomes. Despite the need for secondary surgery, non-resorbable membranes are noted to be irreplaceable in clinical situations dealing with large or vertical bone defects because of their excellent mechanical and barrier properties. The most common complications of non-resorbable membranes are early membrane exposure and subsequent bacterial infections. Titanium mesh demonstrates superior clinical performance to PTFE membranes in this regard. Not only is the clinical exposure rate significantly lower than that of PTFE, but it also supports successful tissue regeneration after exposure has occurred. In recent years, the development of emerging technologies, such as digitally planed and patient-specific titanium meshes, has made non-resorbable membranes highly tailorable in clinical settings. Of the many resorbable membranes, collagen membranes are dominant in clinical practice due to their well-established scientific background and extensive clinical validation. The unique biological properties of collagen membranes and the absence of secondary surgery are considered potential candidates for the ideal barrier membrane.The source of collagen has been shown to be an important factor influencing cellular responses and membrane degradation patterns. Mammals remain the primary source of extracted collagen. Collagen membranes from the pericardium show greater resistance to tearing and longer degradation cycles. However, marine sources of collagen  have gained much attention in recent years due to the absence of infectious disease and religious factors involved, showing favorable bone regeneration immune response and vascularization patterns.Modification of collagen membranes is necessary and effective for achieving better clinical tissue regeneration, generally by crosslinking and carrying bioactive molecules. The initial hypothesis of crosslinking is to improve the mechanical strength and degradation cycle of collagen membranes and thus influence the clinical outcome of collagen membranes. Various chemical/physical/enzymatic crosslinking methods have been developed to successfully prepare crosslinked collagen membranes. However, the high degree of crosslinking of collagen fibers is associated with a higher exposure rate and sometimes affects the foreign body reaction during resorption. The loading of bioactive molecules is mainly based on the hypothesis of constructing an optimal microenvironment to increase bone remodeling. Most of the loaded active molecules undergo two phases: explosive release and slow release. Their effect on inducing tissue regeneration is usually concentration-dependent, and therefore, more clinical evidence is needed to verify the optimal loading concentration.The biological mechanisms of collagen membranes are not yet fully defined. However, the importance of membrane bioactivity has been repeatedly highlighted. Increasing histological evidence showed that the foreign body response of collagen membranes in vivo is closely associated with macrophages and multinucleated giant cells. A comprehensive understanding of the molecular mechanisms and cellular responses associated with tissue healing of different membranes has important implications for regulating bone regeneration through material properties. On the other hand, it is unclear whether different membranes have similar cellular responses and molecular mechanisms in different hosts. Collectively, this review summarizes the basics of barrier membranes for GBR, focusing on advances in collagen membrane modification and their biological mechanisms. The summarization and synthesis of this information are essential to guide the development of the next generation of barrier membranes.