To evaluate how self-measured blood pressure (SMBP) monitoring interventions impact hypertension equity.While a growing number of studies have recruited participants from safety-net settings, racial/ethnic minority groups, rural areas, or lower socio-economic backgrounds, few have reported on clinical outcomes with many choosing to evaluate only patient-reported outcomes . The studies with clinical outcomes demonstrate that SMBP monitoring (a) can be successfully adopted by historically excluded patient populations and safety-net settings and (b) improves outcomes when paired with clinical support. There are few studies that explicitly evaluate how SMBP monitoring impacts hypertension disparities and among rural, low-income, and some racial/ethnic minority populations.Researchers need to design SMBP monitoring studies that include disparity reduction outcomes and recruit from broader populations that experience worse hypertension outcomes. In addition to assessing effectiveness, studies must also evaluate how to mitigate multi-level barriers to real-world implementation of SMBP monitoring programs. Disparities in hypertension continue to be a major challenge in public health contributing significantly to mortality worldwide , 2. WithFactors contributing to hypertension disparities occur at the level of the community/neighborhood, healthcare system, and individual. Community factors such as access to healthy food or underinvestment in rural areas or neighborhoods with predominantly non-white populations are associated with higher blood pressure in individuals who live in those neighborhoods –16. ThesAlthough these socio-economic factors contribute to poor outcomes, hypertension disparities are by no means inevitable or static. They can be made better or worse by the way we deliver care. Healthcare process factors that are controlled or influenced by the health system, such as infrequent follow-up and suboptimal prescribing of medication treatment for hypertension, are major barriers to achieving BP control –22. For Several scientific societies and professional organizations have recommended out-of-office BP (OOBP) measurement to confirm the diagnosis of and manage hypertension –35. OOBPOOBP include ambulatory blood pressure monitoring (ABPM), which is an automated technique of recording BP, often over a 24-hour period. Although ABPM is strongly associated with cardiovascular disease (CVD) related events, this OOBP technique is poorly-tolerated by patients due to interference with daily activities and sleep . MoreovePublished meta-analyses have demonstrated that SMBP monitoring in combination with co-interventions including team-based care, regular support from trained clinicians, antihypertensive medication titration, lifestyle counseling, and/or patient education is associated with significant improvement in BP and reduction of BP , 45–49. During the COVID-19 pandemic, there has been a rapid expansion in SMBP monitoring and the integration of SMBP monitoring into telemedicine. Telemedicine use increased rapidly across health systems to address gaps in clinical care and mitigate the spread of COVID-19. Although telemedicine has been highlighted as a potential strategy to address disparities in health care access, there is also an acknowledgment that digital literacy challenges and disparities in device and broadband access may further exacerbate health disparities . HoweverAs noted above, many factors contribute to disparities in hypertension control. SMBP monitoring programs are particularly well-positioned to address inequities in hypertension outcomes that result from disparities in access to in-person clinical care or patient engagement.Historically excluded populations disproportionately face time or transportation barriers to accessing brick and mortar clinical settings that are only open during work hours. For example, low-income low-wage workers may be employed at multiple jobs without the ability to take time off for a healthcare visit; patients who rely on public transit or reside in rural areas may spend hours commuting to and from an office visit. These barriers result in fewer clinical visits and therefore fewer blood pressure measurements and opportunities to identify uncontrolled hypertension , 58; misSMBP monitoring programs also provide opportunities to address disparities in patient activation. By engaging in SMBP, patients are empowered to understand and recognize how individual behaviors  impact blood pressure control. Though these individual behaviors play only a small role in equity, SMBP facilitates increased patient engagement and may facilitate improved medication adherence, disease understanding, and lifestyle behaviors as well as nudge clinicians into providing more timely treatment .Below, we discuss several issues related to SMBP monitoring and equity. We first start off with a narrative review of the literature to describe what we know and do not know about how SMBP monitoring interventions can improve hypertension equity. Due to differences in healthcare system infrastructure in high-income countries versus LMICs, we focus on studies conducted within high-income countries. Based on these studies, we then describe the issues related to implementing SMBP monitoring interventions in a way that ensure equity. We conclude with recommendations to advance the utility of SMBP monitoring interventions to mitigate hypertension disparities.Despite the potential for SMBP programs to address hypertension equity, there is limited literature on whether it improves equity. A 2021 guide from the United States Centers for Disease Control and Prevention  notes thTo better understand how SMBP impacts equity, we reviewed studies that met one of the following criteria: (a) ensured adequate inclusion (or target recruitment of) patient populations that experience worse hypertension outcome or (b) explicitly reported outcomes focused on disparity reduction in key clinical or process outcomes. Within Table Most SMBP-related studies that have focused on hypertension equity have done so by either prioritizing recruitment of disadvantaged populations or conducting the study in health centers or settings that disproportionately care for patient populations that experience worse health outcomes. Many pilot studies have specifically focused on racial/ethnic minority patient populations, with the greatest number of studies focused on improving hypertension control among Black patients , 70•, 71We found that many equity-focused SMBP monitoring studies reported engagement in monitoring programs or adherence to monitoring activities. Consistent with other digital health interventions for hypertension management , equity-Along these lines, many studies that have explicitly centered equity have paired self-monitoring interventions with other intervention components . While these types of multi-component interventions are often more successful in improving disease outcomes, it makes it difficult to ascertain how much improved clinical or process outcomes can be attributed to SMBP monitoring versus other components of the intervention.Although some studies have conducted practice-level analyses or support for bringing SMBP into routine clinical care •, 77•, sWe identified few studies that explicitly measured the impact of monitoring programs on disparities. Hirshberg et al. • describWhile most studies reported the impact of these programs on clinical or process outcomes, there was rarely specific evaluation of disparity reduction. Although improvement in clinical/process outcomes is important, if the difference in these outcomes is not measured between the advantaged and disadvantaged population, it is possible that the improvement in the population experiencing disparities is smaller than the advantaged population, thereby widening the disparity. This phenomenon has been called an intervention-generated inequity and is a common occurrence for digital health interventions, particularly since digital health tools may be less accessible to populations that experience disparities in health outcomes . Many ofLastly, studies focused on SMBP used a variety of communication modalities to return the blood pressure values back to the clinical team, including telephone phone calls, short-messaging system (SMS)/text messaging, mobile phone applications, and patient portals . FrequenGiven the growing interest in SMBP monitoring, we also wanted to understand the research questions being explored in yet to be published studies. In Table Specifically, while there continues to be a focus on Black patients, other patient populations  that experience hypertension disparities have poor representation (or unknown representation if this information is not collected). Moreover, no studies have proposed measuring a reduction in disparity as a primary or secondary outcome for clinical or process outcomes. All these interventions included SMBP monitoring with several other components, and it is unclear if studies will evaluate which components of each intervention are most responsible for any changes in outcomes. For example, are improved clinical outcomes driven primarily by digital communication, or is human-coaching and intensive in-person support a crucial aspect to improved outcomes?Fortunately, there are upcoming studies focused on how to implement SMBP in real-world clinical care. The importance of this evaluation has been brought to light by the COVID-19 pandemic and the well-documented challenges safety-net systems faced in implementing remote patient monitoring tools . For exaGiven the early literature in this space and still limited number of future studies in this space, there are many areas that require further investigation to understand the impact of SMBP monitoring programs on hypertension equity. We outline three broad areas for future study.There is a need for larger, high-quality studies that include a greater diversity of participants that experience worse hypertension outcomes. While there are a growing number of studies focused on Black/African–American participants, there are fewer focused on other racial or ethnic groups that experience worse hypertension outcomes, including Latinx, Pacific Island, Indigenous, and some Asian ethnicities. Furthermore, more studies should evaluate interventions in populations with low income (or Medicaid insured as a proxy) and limited educational attainment. Unfortunately, this information is not always collected by researchers during clinical trials. Despite the nearly 10% disparity in hypertension control between rural and urban populations, studies are more frequently conducted in urban areas where most research centers are based.As noted above, while it is crucial that equity studies focus on BP outcomes or key process outcomes  that result in BP change, it is also necessary to explicitly measure the disparities between populations. Without measurement of the disparity between populations, it is difficult to know if interventions will advance equity. We acknowledge that it can be challenging  to power a primary outcome based on disparity reduction; however, at the very least, researchers with an eye towards equity should at least plan to evaluate and measure the impact of interventions on disparities. This therefore requires researchers to recruit from both the advantaged and disadvantaged populations; moreover, to power a clinical trial on a primary outcome of disparity reduction, research trials will need to include a large enough sample size from both populations.It is also necessary to advance understanding about how best to implement remote monitoring interventions to advance equity. Using implementation science tools in SMBP monitoring studies would improve understanding of (1) which intervention components work for which patients, and (2) how best to implement remote monitoring programs in under-resourced clinical settings that disproportionately care for patients that experience hypertension disparities. Due to the multicomponent nature of many SMBP monitoring interventions , researchers need to closely evaluate which component of these interventions drive change in outcomes. Moreover, evaluation of clinic or community-level levers to support implementation is needed. Although some trials are beginning to conduct pragmatic evaluations of SMBP monitoring as part of clinical care, there is wide variation in clinical settings and SMBP monitoring implementation •, 85. MoAs implementation scientists, we want to highlight this last point about evaluating implementation outcomes to ensure equity. While it is necessary to evaluate the efficacy of SMBP monitoring on reducing disparities, it is also crucial to explore ways to facilitate effective and equitable implementation of SMBP monitoring interventions. Findings in real world settings are frequently different from clinical trial results and conducting trials in diverse settings can be helpful for providing insights on the effectiveness (rather than efficacy) of interventions. For example, the study team involved with the Hirshberg et al. study on SMBP for postpartum patients found that when they implemented their intervention at a second site, the percentage of their study population that identified as Black decreased from 70% to 40%. Fortunately, the authors still found that in real-world implementation, there was similar improvement in process and clinical outcomes between their Black and non-Black participants . HoweverWe are particularly concerned about unintended consequences because as with other digital health tools, there are multiple levels of barriers to implementation that need to be considered to ensure equity , 90. FigWithin this multi-step process, there are possibilities for drop-off or gaps along the entire spectrum, from onboarding to routine clinical management incorporating home BP data. These barriers to adoption of this digital health intervention can occur at multiple levels –91. For It is well known that patients from racial/ethnic minority backgrounds and those with low-income are more likely to receive healthcare from public healthcare or safety net healthcare settings in the USA, and therefore barriers to healthcare systems must also be considered . Safety-Moreover, both patients/families and clinical care teams need digital tools that will meet their needs and preferences, rather than having to continuously adapt tools not designed for their use for their setting . At a coIn addition, as with other digital health tools, many organizations have failed to develop SMBP programs that support patients who speak languages other than English and are available in digital formats that are easy to use  . InvestiLastly, as is clear from the discussion above, the available evidence about SMBP is not often centered on these patients or settings. For example, in addition to randomized controlled trials, it is critical to collect real-world evidence on engagement and adherence to SMBP across patient groups and in multiple settings to advance our understanding of how best to implement these programs. Similarly, all studies should routinely document key characteristics of their sample related to equity, such as use and effectiveness of SMBP by race/ethnicity, gender, age, language, socioeconomic status, and health and digital literacy .Given the promise of SMBP monitoring programs to improve hypertension control, but the real challenges to equitable implementation, we provide the following recommendations to key stakeholders to ensure that SMBP implementation can improve equity.Researchers and research funders have a clear role to play to advance equity in SMBP implementation. As noted in the section on what we still need to learn, funders and researchers need to (a) both better define the populations that experience hypertension disparities and broaden evaluations to include more of those populations; (b) explicitly conduct subgroup analyses to evaluate the impact of intervention implementation on disparities; (c) conduct implementation-focused studies that increase understanding of how to implement these programs in real-world settings and which components of multi-component interventions are most important for specific populations. Although specific study designs and methodologies are beyond the scope of this review, we encourage funders and researchers to utilize pragmatic hybrid effectiveness-implementation clinical trials  to addreAnother key aspect to facilitating development of a useful evidence-based is ensuring that researchers collect all relevant sociodemographic traits  to better understand for which patients these programs work. While some funders require reporting of the age, gender, and race/ethnicity of anticipated research participants, there is little enforcement of these planned targets during the recruitment process. Also, funders should provide a larger budget and longer timeline that acknowledges the additional time, effort, and resources to recruit historically excluded populations . Moreover, there should be consideration of expanding what sociodemographic traits  are collected from participants to understand the applicability of research findings to marginalized populations.While there are areas for future research, there are key steps that other stakeholders can take now to increase equitable implementation of SMBP.Policy makers and payors need to acknowledge the innumerable barriers that patients and healthcare systems face to implement a successful SMBP programs. At a basic level, increasing access to healthcare and health insurance will improve equitable access to SMBP monitoring programs across the USA. Specific to SMBP monitoring, payors should not return to pre-pandemic policies that required SMBP values to be transmitted digitally  for clinicians to receive “credit” for pay-for-performance metrics or reimbursement. Given both the patient-facing challenges of using telecommunication tools for remote patient monitioring and health system challenges  of integrating these data into electronic health records, it would be inequitable to force use of only telemonitoring to improve hypertension outcomes, especially since studies have not demonstrated the superiority of telemonitoring.Despite no proven superiority of telemonitoring, we recognize that many healthcare systems are moving towards digital communication of BP values. Many patients face structural barriers to accessing the devices or high-quality Internet access to utilize these telemonitoring tools. Policy makers should pursue policies that increase access to low-cost digital devices and internet access and increase investment in infrastructure that makes high-quality internet accessible to all communities. Similarly, if SMBP programs rely on apps or other digital health tools, regulatory agencies can build in baseline accessibility requirements into their approval processes to address equity. For example, as the U.S. Federal Drug Administration (and similar agencies in other countries) begin approving digital therapeutic tools, there could be requirements related to digital platform usability and language access.For all SMBP programs, payors can also address cost-related barriers for patients by reimbursing for BP monitoring devices. Harmonization of policies from all payors  would facilitate equity. At this time, not all payors have the same policies which is confusing for both patients and clinical teams; continued reimbursement for telemedicine visits must be paired with reimbursement for patient monitoring tools (such as BP monitors) that support high quality telemedicine visits. Moreover, with an eye on equity, payors should strongly consider reimbursing for a wider variety of BP monitors, including BP monitors with extra lurge cuffs or BP monitors that ease communication of values back to clinical teams . Reimbursing for monitors that meet all patients’ needs may help ensure SMBP monitoring produces equitable improvements in clinical outcomes.To address the disproportionate burden that safety-net systems face in supporting marginalized patients to use digital tools, payors need to consider how to provide reimbursement or compensation for the additional work conducted by the clinical team to support patient monitoring at home. For example, US payors should continue moving away from fee-for-service payment models and towards value-based payment, alternative payment models, and other approaches where clinical teams can choose the best method to care for a patient that may include more or less in-person care based on the patients’ needs. Payors can also center equity by prioritizing pay-for-performance metrics that are focused on disparity reduction.Digital tool developers need to begin to specifically innovate for historically excluded populations. This involves including these patient populations in design conversations to ensure that tools are usable by patients with limited health literacy, limited digital literacy, or with communication barriers (such as a non-dominant language preference). Tools should also be validated in diverse populations, as studies have shown that ignoring diverse populations clearly leads to disparities and inaccuracy in clinical decisions , 105.While conversations about digital design equity have often focused on patient-facing barriers, we want to highlight that digital tools should also consider challenges faced by clinicians in underresourced safety-net settings. Given the importance of both patients and clinicians in successful implementation of SMBP monitoring, any digital tool used to facilitate SMBP monitoring should be designed to work well for safety-net systems that have fewer financial resources to pay for expensive software or hardware, are more likely to use electronic health records with fewer advanced features, and are more likely to be understaffed. Therefore, it is crucial to design tools that can be easily integrated into clinical team workflows and structure in safety net systems.Front-line clinicians and healthcare delivery systems also can take direct steps to ensure equitable access to SMBP monitoring programs. Clinicians should recognize that some patients may need additional support to adopt and use these tools and be prepared to provide training to patients as needed. If there is an opportunity to choose different BP monitors or data submission platforms, clinicians should choose products with greater usability. For example, by selecting a cellular-enabled blood pressure monitor that allows automatic uploading of BP values for clinician review rather than asking patients to sync via a Bluetooth device or manually entering values into a patient portal. Clinicians should also consider avoiding programs that require patients to have advanced digital literacy skills, which poses unnecessary additional barriers to equitable patient participation.Given the higher proportion of hypertension among patients with low-income, clinical teams should learn about payor coverage for BP monitors so they can help patients secure home BP monitors. While payors can make this easier for clinical teams by having similar coverage, in the meantime clinical teams should be prepared to support patients with acquiring BP monitors by developing explicit workflows for acquiring BP monitors and teaching patients how to use these devices.Healthcare system leaders must also recognize that collecting and responding to home blood pressure values requires significant clinical team time. Instead of expecting clinical teams to do this work between office visits, healthcare systems that adopt these programs should create a team-based infrastructure that will optimize the chances of successful implementation. Investing in hardware, software, personnel, or culture change that makes this work sustainable for clinical teams will help ensure accessibility and equal treatment for higher-needs patients. These investments may include: selecting electronic health records and other software with advanced features to facilitate data integration and usable digitally connected care; reallocating personnel to robustly support population health panel management; and optimizing performance reporting and billing processes to capture reimbursement available with current policies to secure funding that sustains the infrastructure required to provide equitable SMBP programs.SMBP monitoring is a well-known approach to improve hypertension control, but its impact on equity is understudied and largely unknown. Early studies are promising and suggest that SMBP can be successfully adopted by historically excluded patient populations and implemented in community health centers and safety-net settings. Future research needs to include better representation from more populations that experience worse hypertension outcomes; explicitly evaluate the impact of SMBP monitoring programs on disparities between patient populations; and evaluate both effectiveness and implementation outcomes to inform real-world implementation of SMBP monitoring. There is a role for all stakeholders — including funders, researchers, policy makers, payors, digital tool developers, and healthcare teams — to advance implementation of SMBP monitoring to improve hypertension equity.


Increased reliance on the use of alternative ingredients in the formulations of diets for farmed fish has long been a priority to sustain the unparalleled growth of aquaculture. This need which has led the feed industry to explore many alternative protein and oil sources, has also led to many physiological impacts on those species being fed those alternatives. The intensification of aquaculture under a global climate change scenario also contributes as another major challenge to feed the blue revolution, since there is a need to develop diets that enhance fish robustness. Recent research on farmed fish species has resulted in the development of a range of innovative novel ingredients that are able to sustain fish growth performance, improve fish welfare and resilience, and still assure a safe, nutritious and tasty product for human consumption. Since the suitability and sustainability of vegetable ingredients in diets for carnivorous fish has increasingly being questioned, other resources need to be explored and increasingly we need to identify and develop alternatives that do not compete directly with human food supply. Insects and agrofood byproducts have great nutritional value and may contribute towards a circular economy concept. Biotechnology also offers a wide range of possibilities, including microbial biomass and single cell products, but scale-up is still required for many of these initiatives to realize any serious benefit. Micro and macroalge are also receiving attention, not only as sources of macronutrients, but also for their richness in bioactive compounds. However, the potential of each new feed ingredient has to be thoroughly evaluated before their wide acceptance by the feed industry. There is little to gain by simply transferring risk from one ingredient to another, when ideally what the feed sector needs is greater security of supply, quality and on a cost-effective basis.This Research Topic on “Feeding the blue revolution” represents a Research Topic of 14 original research articles, highlighting the latest findings related to the evaluation of the physiological consequence of novel ingredients on farmed fish. In the following collection of papers, the readers will note that such an evaluation relies on classical methodological approaches, but also on some cutting edge tools associated with the use of omics to unravel the impact of nutritional clues on the physiology of farmed fish species that are emerging.Basto et al. have explored the impact of partial and total replacement of fish meal by defatted Tenebrio molitor larvae in a comprehensive approach focused on European sea bass (Dicentrarchus labrax) growth performance and nutrient utilization, but also further addressed the underlying physiological mechanisms involved in nutrient metabolism. In this carnivorous species, the authors concluded it was feasible to replace up to 80% fish meal without growth impairment, and still assuring fillet nutritional value for consumers. Another important insect species, black soldier fly, Hermetia illucens, was assessed by Li et al. as protein source for tongue sole (Cynoglossus semilaevis). The replacement of 25% of the fish meal by defatted black soldier fly larvae meal had positive effects on growth, but with some drawback effects on feed conversion. They also noticed intestinal structural damage and abnormal liver function at higher replacement levels. These studies highlighted the potential of insect meals to be included in aquafeeds, but identify that there are maximal inclusion limits depending on the fish species considered. It is also clear that further studies are warranted to fully validate such nutritional approaches at farm scale considering the full production cycle.Growing interest in the use of insect protein since the inclusion of this ingredient in aquafeed was authorized by the European Union (EU) in 2017 presents new opportunities. In the present volume, Kousoulaki et al. considered a series of low trophic ingredients  in fishmeal and fish oil free diets when fed to Atlantic salmon . Extensive raw material and dietary chemical characterisation was undertaken to identify constraints and opportunities for using such novel ingredients. Overall, each of the studied ingredients were well accepted by the fish, resulting in high performance and efficiency. It was highlighted that the relevance and impact of the quality of fishmeal and fish oil on comparative studies with such novel ingredients is an important consideration.Naya-Català et al. who evaluated processed animal proteins (PAPs), insect meal, yeast, and microbial biomasses in diets for gilthead sea bream (Sparus aurata) juveniles. This study focused on the interaction between intestinal microbiota and host transcriptomics to aid the identification of fish diets promoting gut health and metabolic homeostasis, and ultimately, the overall health of farmed fish. Using a circular economy concept, Terova et al. assessed crude glycerol, a primary by-product of biodiesel production, for heterotrophic cultivation of Schizochytrium limacinum. The resulting biomass, a rich source of docosahexaenoic acid, was further shown to be a useful alternative to marine-derived raw materials for European sea bass feeds, providing a much needed additional source of essential omega-3 fatty acids for indirect human consumption. However, the feed cost-effectiveness for that resource still needs to be improved.Insights in fish feeds for increased circularity and resource utilization were provided by García-Márquez et al. demonstrated the capacity of the microalga Chlorella fusca (15% inclusion level) to promote growth, metabolism and digestive functionality in a low trophic omnivorous fish species, the thick-lipped grey mullet (Chelon labrosus). In another paper, Fang et al., showed that a newly isolated strain of Haematococcus pluvialis GXU-A23 improved the growth performance, antioxidant and anti-inflammatory status, metabolic capacity and mid-intestine morphology of juvenile Litopenaeus vannamei. A 50 g/kg Haematococcus pluvialis was proposed to be used as a green additive in aquafeeds due to its rich astaxanthin and polyunsaturated fatty acid content. Ferreira et al. also explored a range of natural highly nutritious alternatives to replace fishmeal in gilthead sea bream diets. They found that microalgae were a good source of protein and lipids, able to promote the accumulation of LC-PUFAs in muscle, whilst selenised yeast was a good vehicle to fortify selenium levels in fish. However, the use of Laminaria digitata as strategy to increase iodine muscle content still requires further evaluation.Algae were examined in several papers in this Research Topic. Debaryomyces hansenii yeasts were shown to modulate physiological responses in plasma and immune organs of Atlantic salmon  counteracting possible consequences of hypoxic stress. Balbuena-Pecino et al. used an in vivo and in vitro approach to evaluate hydroxytyrosol-rich extract from olive juice (major phenolic compounds found in olives) as an additive in gilthead sea bream juveniles fed a high-fat diet. That study highlighted the beneficial use of hydroxytyrosol as a way to improve fish muscle-skeletal condition. The metabolic effects of flavonoids included in diets for hybrid grouper (Epinephelus fuscoguttatus♀×Epinephelus polyphekadion♂) was evaluated by Luo et al. Classical feeding trials combined with extensive tissue metabolomics analysis, helped to shed light on metabolic adaptations induced by such functional feeds. The use of glycerol, a by-product of biodiesel, was also evaluated in diets for rainbow trout (Oncorhynchus mykiss) and European sea bass. Viegas et al. suggested that these fish are able to catabolize glycerol and incorporate it into carbohydrates, but species metabolic differences were identified. An additional study by the same authors recommended a dietary glycerol supplementation of up to 2.5%, as it had no major effects on fish lipid metabolism or fat accumulation and therefore had some potential as a useful energy source . Finally, Selvam et al. provided new insights into intracellular trafficking of fatty acids using a rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model. This model provided comparable results to similar mammalian cell lines as well as in vivo studies in fish or mammals. It was concluded that carbon chain length and saturation level of fatty acids differently regulate their intracellular fate during fatty acid absorption.Functional diets continue to develop as strategies to boost fish health status. Hydrolyzed Overall, the “Research Topic” highlights a variety of current research in the area. This set of studies also shows how classical and novel research strategies are increasingly being used in tandem to underpin future directions to increase the range of alternative ingredient options for use in feeding farmed fish species. Beyond the theoretical aspects of these studies, increasing use of novel research strategies is providing an objective understanding of the physiological responses by fish to a growing range of alternative ingredients.


Conflict between humans and Asian elephants is a major conservation issue. Here we discuss common tools used to manage human-elephant conflict (HEC) in Asia and the potential of animal-borne satellite-linked shock collars or Aversive Geofencing Devices (AGDs) for managing problem elephants. Most current HEC mitigation tools lack the ability to be modified to accommodate needs of elephants and therefore are sometimes unsuccessful. AGDs currently used to manage livestock movement can be adapted to mitigate HEC to overcome this problem. AGDs can constantly monitor animal movements and be programmed to deliver sound warnings followed by electric shock whenever animals attempt to move across virtual boundaries demarcated by managers. Elephants fitted with AGDs are expected to learn to avoid the electric shock by associating it with the warning sound and move away from specified areas. Based on the potential shown by studies conducted using AGDs on other wild species, we suggest that experiments should be conducted with captive elephants to determine the efficacy and welfare impact of AGDs on elephants. Further, assessing public opinion on using AGDs on elephants will also be important. If elephants can learn to avoid virtual boundaries set by AGDs, it could help to significantly reduce HEC incidents.Asian elephants are a principal cause of human-wildlife conflict. This results in the death/injury of elephants and humans and large-scale crop and property damage. Most current human-elephant conflict (HEC) mitigation tools lack the flexibility to accommodate the ecological needs of elephants and are ineffective at reducing HEC in the long-term. Here we review common HEC mitigation tools used in Asia and the potential of Aversive Geofencing Devices (AGDs) to manage problem elephants. AGDs can be configured to monitor animal movements in real-time and deliver auditory warnings followed by electric stimuli whenever animals attempt to move across user-specified virtual boundaries. Thus, AGDs are expected to condition elephants to avoid receiving shocks and keep them away from virtually fenced areas, while providing alternative routes that can be modified if required. Studies conducted using AGDs with other species provide an overview of their potential in conditioning wild animals. We recommend that the efficacy and welfare impact of AGDs be evaluated using captive elephants along with public perception of using AGDs on elephants as a means of addressing the inherent deficiencies of common HEC mitigation tools. If elephants could be successfully conditioned to avoid virtual fences, then AGDs could resolve many HEC incidents throughout Asia. Elephas maximus (Linnaeus 1758) once inhabited areas between the Euphrates and Tigris Rivers in west Asia to the Yangtze-Kiang River in China [Asian elephants Linnaeus 758 once in China . The totin China . There iin China  and anotin China . Asian ein China ,7. AncieLinnaeus 758 once in China ,8,9,10. in China ,12,13. TDespite the elephant conservation legislation imposed, various anthropogenic activities have continued to threaten the survival of elephants. Legal and illegal capture and illicit trade of elephants to supplement captive populations occur in several nations, which contributes to the decline of elephant numbers in the wild ,15,16. HMany elephant and human lives are lost as a consequence of HEC with highest numbers recorded in India and Sri Lanka, where an average of 124 elephants and 571 humans in India  and 263 Various tools and strategies are used to mitigate HEC and keep damage-causing elephants away from crops and other human-dominated areas ,53. The Canis latrans, in 1976 where three out of the four shock-collared animals learnt to avoid black domestic rabbits and prey on white rabbits after 3–5 shocks [Satellite-linked electric shock collars or Aversive Geofencing Devices (AGDs) can automatically deliver a warning sound followed by an electric shock as an animal reaches a virtual boundary, and have been successfully used in managing livestock movement ,62,63. T5 shocks . AGDs ap5 shocks , and creHere we briefly review the use of common approaches to manage conflict between humans and elephants across Asia, highlighting their function and drawbacks. We then discuss the potential use of AGDs as a means to address these drawbacks and sustainably mitigate HEC. We further describe important research needs that require addressing to advance the use of AGDs on elephants. Our aim is to highlight the similarities and differences between AGDs and other HEC mitigation tools and outline a pathway forward for the trial and development of AGDs on elephants.A wide array of tools are used in Asia to mitigate HEC and several reviews have been published in the recent past on various aspects of HEC mitigation ,60,66,67Exclusion of elephants from conflict areas or restricting elephants to protected areas aims to keep elephants away from humans and their interests and is ostensibly intended to avoid the need for the direct killing of elephants in accordance with cultural and societal expectations. Exclusion is often achieved by aversive conditioning where animals learn to associate a particular behaviour with an unpleasant stimuli, and hence cease or modify that behaviour ,136,137.As an alternative to excluding elephants from human habitats, identified problem elephants may be physically removed from a population by either killing or translocating them. Large-scale culling of elephants is no longer sanctioned in Asian elephant range countries , but masVarious types of early warning systems are sometimes implemented to mitigate HEC, ranging from vigilance by farmers occupying traditional watchtowers to monitoring elephants using various remote sensing technologies . The useHuman centric methods are focused on encouraging human-elephant co-existence and developing tolerance towards elephants by providing financial relief or by educating stakeholders. The knowledge gap about HEC and the endangered status of elephants may intensify the conflict . Even thHabitat management through managing ecological corridors and enriching protected areas expects to reduce human-elephant interactions by reducing the need for elephants to venture into human-dominated habitats. Elephants are forest animals, but edge species, preferring habitats with intermediate disturbance rather than undisturbed forests ,145,146.Prevents HEC incidents before they occurKeeps elephants in or out of designated areasTargets specific individuals or small family groupsDoes not require the death of the animalProduces minimal harm to elephantsDoes not harm or impede non-target speciesDoes not require the construction of permanent or immovable structuresCan be altered, moved, or removed as neededIs long-lasting or sustainableIs automated, or does not require substantial human inputIs inexpensive or cost-effectiveIs culturally and socially acceptableOverall, many of the current mitigation efforts either address the symptoms of HEC or are not dynamic or flexible enough to be modified as needs change, and therefore are successful only in the short term or are not sustainable . Based oWith current mitigation tools each having only some of these characteristics , developCapra hircus) were also successfully contained in a designated area without a visible fence [Bos taurus [Ovis aries [AGDs have been used on domestic pets  and livestock for many decades . The firle fence . Since ts taurus ,151 and is aries ,153 as wTechnical development of AGDs has evolved since then and modern AGDs are now able to deliver stimuli automatically in conjunction with real-time GPS tracking, user alerts, and data logging capabilities similar to most standard GPS tracking devices. They have proven to successfully restrict livestock movement to large and dynamic user-specific areas without proximity-based sensors , overcomEven though responses of elephants to AGDs may vary from that of other animals, reviewing what is known from studies on other wild species may provide some insight into the potential and challenges that could be expected in conditioning elephants using AGDs. Scientific material published in the past 30 years (between 1993–2022) in the English language related to the use of AGDs on wild species were searched in Web of Science and Google Scholar using the following search string:  AND (“wildlife management” OR “wildlife conservation” OR “*wildlife conflict*” OR “predator management” OR “crop damage”). The initial search (last performed on 16th August 2022) resulted in 127 records. The titles and abstracts of each document was screened and eight empirical studies that involved direct experimentation with animal-borne electric shock collars on a wild species were extracted. References within articles were checked, and four articles missing from the initial list were added. The resulting list of articles (n = 12) are summarised in Canis lupus, dingoes Canis familiaris, island foxes Urocyon littoralis and black-tailed deer Odocoileus hemionus. The total number of wild animal studies conducted over the past 30 years are very few compared to the large number of studies available on livestock (see According to the search results, research using AGDs has been conducted with five wild species: coyotes, grey wolves tock see . Even thMany drawbacks and limitations were highlighted in these studies such as skin necrosis due to electrodes, irritation due to the collar belt material ,166,167,AGDs could help prevent HEC incidents before they arise if elephants learn to recognise the warning stimuli and predict the receipt of the electric shock and avoid it by moving away. This will minimise direct human interaction with elephants and prevent HEC incidents. AGDs may therefore be a good alternative when it is impractical to permanently erect electric fences in large areas  given thWhile the concept of testing AGDs on wild elephants to manage their movement may be attractive, elephants may not respond to the electric stimuli the same way livestock do and information available on other wild species may not be sufficient to foresee the potential of AGDs as an HEC mitigation tool. Virtual fences will also have to be established in much larger, heterogenous and complex landscapes than those that livestock are typically managed in. Fitting AGDs on wild elephants would also be a complex and costly process , so AGDsElephants appear to be good candidates for the use of AGDs, but elephant’s large size, strength, speed, and potentially dangerous behaviour poses a risk in testing AGDs on elephants. Individual variability in their capacity for learning and response to the electrical stimuli might also be expected ,168,181.The longevity of AGDs must be considered given that frequent replacement of collars on wild elephants is not possible. GPS collars have limited battery life and are typically scheduled to collect GPS points every few hours . HoweverAGDs typically expose animals to a high voltage electric shock with a very low amperage, delivered as pulses for a few milliseconds at a time ,183, theObtaining acceptance of all stakeholders, local communities, line agencies, local administration and government is required to mainstream the use of AGDs. All approaches to managing HEC cause some sort of pain, distress, or disruption to elephants, but public acceptance of AGDs depends on how these welfare impacts compare to or are perceived to be compared to other HEC mitigation tools . Use of Elephants are endangered and play a significant role in the ecosystem and culture. Conflict between humans and elephants is one of the most important environmental issues in Asian elephant range countries. A variety of approaches are used to mitigate HEC, although most have not been very successful given they are not flexible or dynamic enough to be modified according to elephants’ behavioural and ecological needs. AGDs may overcome many of these issues, but require further development. AGDs may safely prevent elephant movement into human habitations and help humans and elephants coexist if elephants successfully learn to associate the non-aversive auditory stimulus with the aversive electric shock. Use of AGDs may be a more ethical choice than elephant removal. However, AGDs first require field-testing with captive elephants under controlled conditions to refine their design and optimise their efficacy and welfare impacts. Understanding public perceptions about AGDs is also important. AGDs will not be a ‘silver bullet’ for HEC, but they do overcome many of the limitations of current tools and may therefore become a powerful new management tool for reducing HEC in the future.


Dry powder inhalers (DPIs) are drug–device combination products where the complexity of the formulation, its interaction with the device, and input from users play important roles in the drug delivery. As the landscape of DPI products advances with new powder formulations and novel device designs, understanding how these advancements impact performance can aid in developing generics that are therapeutically equivalent to the reference listed drug (RLD) products. This review details the current understanding of the formulation and device related principles driving DPI performance, past and present research efforts to characterize these performance factors, and the implications that advances in formulation and device design may present for evaluating bioequivalence (BE) for generic development. The challenges surrounding the use of metered dose inhalers (MDIs) became a major driver for the development of dry powder formulations and dry powder inhaler (DPI) technologies ,2. HowevDPIs are commonly used drug products for modern inhalation therapy of respiratory diseases that have specially designed delivery devices. These devices were developed to overcome certain limitations of MDIs, such as the complex coordination required for optimal lung delivery during times when patients may be undergoing bronchospasms , as wellMost DPIs contain three functional parts: powder drug formulation, drug dose measuring system, and a physical mechanism that allows dispersion of the powdered formulation. The following sections will examine each of these three aspects of a DPI, as well as the utility for patients and considerations for demonstrating BE.A DPI’s formulation is typically a complex dry powder mixture consisting of one or more active pharmaceutical ingredients (API) along with inactive, excipient ingredients. The use of excipients in the development of the formulation may not only enhance the chemical and physical stability of the API but can also impact the performance of the product. For example, this may include changing the dissolution of the API, the particle size, deposition to the lungs, and therapeutic efficacy. In general, excipients are usually “generally recognized as safe” (GRAS) substances that may improve the delivery and performance of a drug but cannot exert therapeutic effects by themselves . CompareWhen selecting which excipient(s) to include in a DPI’s formulation, understanding how the excipient properties will influence the formulation or affect its biocompatibility with the site of action in the lungs is critical. Polymers, such as methylcellulose that are usually used in oral formulations, cannot be used as excipients in DPI formulations because these are non-degradable and, therefore, should not be delivered to the lungs. Similarly, polysorbates or oleic acid, used as excipients in MDI formulations, may not be used in DPI formulations due to their semi-solid or liquid states and low melting points which are not suitable for dry powder phase-based formulation. While lactose is a well-known DPI excipient, its powder structure may impact the inhalation efficiency of the DPI formulation. For example, amorphous lactose can give rise to strong particle–particle interactions that may impede aerosolization of the formulation . To helpSugars, such as mannitol and trehalose, are other excipients commonly used in DPI formulation development. Mannitol is a sugar alcohol and being a non-reducing compound, it is compatible with APIs containing amines . Mannito® formulation, such as found in the recently approved Tyvaso® DPI ) form an aerodynamic structure that has shown improvements in aerosolization and phagocytic clearance from the lungs. The initial studies in rat models revealed that levodopa from porous particles was delivered to the alveolar membranes to be rapidly absorbed to the pulmonary capillary network and, thus, into the arterial circulation and to the brain, offering a more rapid and robust delivery [Porous particles with geometric sizes in the range of 5–30 μm (large porous particles) have been used in INBRIJAdelivery ,81.2), and a pore forming agent such as perfluorooctyl bromide (PFOB). The pore forming agent is almost completely evaporated from the final powder during the drying process. The drug in the phospholipid matrix of the particles obtained by emulsion-based spray drying may be amorphous in nature, which could pose challenges to the long-term physicochemical stability of these powders. To maintain crystallinity of API(s) with porous particle formulations, suspension-based spray drying has been used in preparing porous particles to minimize drug particle dissolution in the liquid feed [Various methods have been used to manufacture porous particle formulations. Emulsion-based spray drying is used in the manufacture of PulmoSphere™ particles to obtain phospholipid porous particles with a sponge-like morphology for high-dose delivery of tobramycin . The process of spray drying to achieve the specific particle morphology of tobramycin inhalation powder is described in uid feed . A numbeuid feed ,84,85,86® (developed by Nektar Therapeutics) or freeze drying . In these cases, insulin is preserved chemically and physically in the form of a powder that presents good dispersibility and aerodynamic properties suitable for pulmonary delivery to provide pulmonary bioavailability of 8–25%. Other types of engineered particles that are in early phase of development include liposomal dry powder formulations, where drug-encapsulated liposomes are converted into a dry powder form, particles coated with lipids, polymers, or leucine, and excipient-free nanoporous/nanoparticulate microparticles [Powder formulations containing PulmoSol™ technology used in EXUBERAarticles ,89.Research on porous particle preparation methods and their performance are summarized in Spray-drying technology is a developed technique that involves atomizing the drug formulation solution into liquid aerosol particles under a drying gas stream contact. The dry solid particles are produced through the evaporation process in the spray drying chamber and can be collected using a cyclonic powder collector or electrode separator . The phy2 is the most commonly used SCF technology in FDA approved DPI products, as it is recognized as safe and non-combustible. Supercritical CO2 can form the porous matrices due to its low viscosity, high diffusivity, and null surface tension [2, the precipitation of compressed CO2 antisolvent (PCA) and supercritical fluid antisolvent techniques provide the advantage to achieve the desired size for inhaled particles [Supercritical fluids (SCF) have been used for the formulation of DPIs as a cost-effective and non-toxic method to modify the solid-state form of the dry powder particles ,100. The tension ,97. Witharticles . A good articles .Recently, aerogels (which can be manufactured using SCF technology) have been explored for use in porous particle formulation development due to their diversity of textural properties and porosity . The higSpray freeze drying (SFD) is another well-developed technique for large porous particle (LPP) generation in the inhaled drug field. It has a high production rate and is suitable for thermally sensitive materials. The whole process consists of three sections which includes atomization, freezing, and lyophilization. The produced LPP have a desired aerodynamic deposition and improved solubility of the formulations. SFD has been successfully applied to generate voriconazole-loaded LPP for the treatment of pulmonary aspergillosis. Published reports have shown that dissolution studies with voriconazole can be rapidly released from the LPP based formulation, as compared with its release from solid voriconazole particles that required 2 h to dissolve .DPI microstructure including particle size, particle morphology, surface roughness, and interfacial chemistry have been shown to play a key role in governing particle–particle interactions. These properties also govern the relationship between the DPI device and the deagglomeration efficiency of the powder formulation . As suchIn addition to particle size and morphology, surface composition also influences cohesive forces and, ultimately, aerosol performance. X-ray photoelectron spectroscopy (XPS) as well as confocal Raman can be used for assessing the surface chemistry of powders ,107. RecTranslating properties of single particle (or sampled particles) obtained from SEM, AFM and MDRS to the bulk powder, particularly inside the capsule or blister, is challenging. In such cases, powder analytical techniques involving penetrating radiations such as X-ray microscopy (XRM) or X-ray computed tomography (XCT) could be employed for non-destructive analysis of the bulk powder to obtain size and shape (sphericity/aspect ratio) distributions . AlthougIn terms of in vivo based DPI performance characterization methods, recent FDA research studies conducted by University of Florida  have explored whether systemic PK measurements can detect differences in regional lung deposition of DPI drug products . Three cFinally, modeling and simulation techniques can be advantageous in identifying relationships between the formulation and device properties of a DPI and its performance. For example, computational fluid dynamics (CFD) can be used to understand the air flow and deagglomeration in the inhaler device by tracking single particle motion in the device. On the other hand, information about agglomerate break-up during impaction and interaction with the air turbulence may require modelling techniques such as the discrete element method (DEM) ,114. TheIn addition to the advancements in DPI formulation design, advancements are also continuing on the device front with the introduction of new designs and digital technologies. With the inclusion of digital technology, these digital DPI platforms have the ability to provide additional information on inhaler performance and/or use that may minimize device use errors. This has shown a positive impact on product use and performance, as well as patient satisfaction, compliance, and adherence, thus, overall clinical outcomes . A recen® used with ProAir®, AirDuo®, and Armonair® products, which were approved in 2018, 2019, and 2020, respectively [® contains an integrated digital sensor encompassed in an electronic module (eModule) on the top portion of the inhaler that can record usage (date and time), inhalation data relevant to the inhalation technique , and provide medication reminders [®, are beginning to emerge [® sensors designed for several DPI devices including Diskus® , Ellipta , and Neohaler  were approved by the FDA through the 510(k) pathway in 2015, 2016, and 2018, respectively [® sensors technology can record and monitor actuation activity (date and time) and utilizes Global Positioning System (GPS) technology to identify environmental asthma exacerbation triggers. Additionally, the Hailie® sensor (approved via the 510(k) pathway) was developed for MDIs as well as the Diskus® (SmartDisk™) and HandiHaler® (SmartHandy™), which is capable of being attached to the inhaler to record and track medication usage and set reminders.The currently FDA-approved digital DPIs include the Digihalerectively . The Digeminders ,118. Theo emerge ,119,120.ectively . The ProAs the inclusion of digital sensors to DPIs (either added on or fully integrated) become more commonplace, the real-world evidence of their potential benefits may begin to show, such as changes in patient adherence and clinical outcomes. As mentioned earlier in this review, other inhalation products such as MDIs and nebulizer-based products can present challenges to patient adherence and outcomes due to their need for better coordination with the patient, or the potential device bulkiness and long duration of administration. While DPIs can address these challenges due to their passive drug delivery approach and size, the cost for patients to use a DPI over other inhalation products may also be a consideration that impacts patient adherence and clinical outcomes. So, while the addition of digital versions of DPIs may offer benefits to patients, these would need to be considered along with any differences in costs for the patient when attempting to understand potential changes to patient behavior such as adherence to a prescribed treatment. Currently, there are no approved generic DPIs that incorporate a digital feature. These digital technologies present a new challenge for generic DPI development given the uncertainty with degree to which a generic DPI will be required to include digital technologies, or record and/or communicate this information to the patient, in order to match the RLD DPI. With that said, generic competition with these DPIs is expected to lead to reduced costs for the patient, which has been reported following the introduction of the first Advair Diskus generic . As thisLastly, the introduction of the Staccato inhalation platform provides an example of the continued device design advancements for inhalation powders. The Staccato device stands out among other inhalation devices due to its drug delivery mechanism. The device uses breath actuation, where an inhalation sensor triggers a heating element on the device that heats up a thin film of the formulation, causing it to sublimate and aerosolize the dose as dry particles . NotablyAs detailed earlier, the FDA has generally recommended an agglomeration weight of evidence approach to establish BE of a proposed generic DPI to its RLD. These recommendations are product-specific and include tests to address the complexities of these drug–device combination products and the challenges they present for evaluating equivalence. The advancements in DPI formulation and device design may necessitate new experimental techniques to sensitively detect differences between proposed generic and RLD DPI products that could impact performance.For example, study results on the role of the different types and amounts of agglomerates in a DPI formulation on its performance support the role that particle microstructure may play for these products. While the drug particle size is a well-known factor influencing regional deposition and subsequent drug release, the associated excipients in the depositing agglomerates may alter the surface area available for drug release of the API particles by preventing larger drug-drug agglomerates from forming. The speed at which these associated API particles become available for drug release may depend on the solubility of the excipient(s) in the agglomerates. Unlike lactose, which as a carrier particle has had its physical properties well studied, the various other excipient carrier particles  or surface modifiers  have limited information available for their potential influence on API drug release once formed into agglomerates.Moving away from the more well-studied carrier-based DPI formulations, the advancements in particle engineering have identified novel excipients, such as Bis-3,6(4-fumarylaminobutyl)-2,5-diketopiperazine (FDKP) and DSPC, as well as advanced manufacturing processes, such as those listed in With these novel formulation-based complexities, additional considerations for microstructural differences and their potential impacts on DPI performance may warrant additional characterization studies to support product quality and demonstration of BE. For microstructural characterization, this review has described a wide assortment of analytical approaches that DPI formulators may use, including ways for evaluating small sample or single particle/agglomerate associations and morphology , polymorphism , and inter-particle interactions . However, additional research is still needed to determine which of these methods can most sensitively discriminate between a proposed generic DPI and its RLD when clinically meaningful differences in performance are present that would affect BE.In relating the microstructural characteristics of a given DPI formulation to its performance, there are opportunities to utilize both in vitro and in vivo studies. For example, APSD studies using anatomical mouth-throat models and realistic breathing profiles, along with dissolution studies, are two potential in vitro approaches that may be useful for providing insight into how microstructural differences between DPI formulations may impact dose delivery, deposition, and drug release, provided discriminatory methods are developed and validated. For in vivo studies, PK studies may also provide a way to characterize microstructural impacts if differences are expected to affect regional deposition; however, more research is needed to better establish this method’s sensitivity to potential differences in the drug product microstructure. Lastly, given the various challenges and complexities presented by the different DPI formulations discussed in this review, the characterization methods selected to evaluate a proposed generic DPI and its RLD should be suitable to address the specific microstructural characteristics present in the DPI formulation. Therefore, characterization of DPI microstructure is likely to be product-specific and, so, should be appropriately justified.When considering the implications of new digital technologies and device designs for BE, there is considerably more uncertainty at the present time with what potential differences that may be permissible for a generic DPI. As a starting point, and to facilitate generic drug–device development, the FDA has provided its current thinking in published guidance on considerations and approaches for evaluating substitutability of a generic drug–device product. Given the ever-changing landscape for device designs and user interfaces, FDA encourages prospective generic applicants to utilize available communication mechanisms, such as the pre-abbreviated new drug application (pre-ANDA) meeting request process , to obtaThis review has provided a discussion of the FDA’s current understanding of the principles and contributions that a DPI’s formulation and device provides to its performance, as well as a brief overview of performance characterization methods that can be used during drug development and establishing BE. With the challenges that novel DPI formulations, manufacturing approaches, and device designs can present for evaluating potential generic DPIs, the FDA has funded research initiatives to better understand what factors are critical for ensuring BE with the RLD. This research has highlighted the role formulation microstructure can play in a DPI’s performance and identified novel in vitro and in vivo characterization methods that may be able to sensitively evaluate how microstructural differences impact performance. As DPI technology continues to advance, the FDA remains committed to building on the current understanding of DPI performance and encourages prospective generic applicants to communicate early in their development programs when challenges with evaluating DPI performance are encountered.


The measurement of circulating tumor DNA (ctDNA) has gained increasing prominence as a minimally invasive tool for the detection of cancer-specific markers in plasma. In adult cancers, ctDNA detection has shown value for disease-monitoring applications including tumor mutation profiling, risk stratification, relapse prediction, and treatment response evaluation. To date, there are ctDNA tests used as companion diagnostics for adult cancers and it is not understood why the same cannot be said about childhood cancer, despite the marked differences between adult and pediatric oncology. In this review, we discuss the current understanding of ctDNA as a disease monitoring biomarker in the context of pediatric malignancies, including the challenges associated with ctDNA detection in liquid biopsies. The data and conclusions from pediatric cancer studies of ctDNA are summarized, highlighting treatment response, disease monitoring and the detection of subclonal disease as applications of ctDNA. While the data from retrospective studies highlight the potential of ctDNA, large clinical trials are required for ctDNA analysis for routine clinical use in pediatric cancers. We outline the requirements for the standardization of ctDNA detection in pediatric cancers, including sample handling and reproducibility of results. With better understanding of the advantages and limitations of ctDNA and improved detection methods, ctDNA analysis may become the standard of care for patient monitoring in childhood cancers. Pediatric cancer is the leading cause of disease-related death in children worldwide . While aIn hematological malignancies including acute lymphoblastic leukemia (ALL), the most common childhood malignancy, MRD cells are maintained in the bone marrow throughout chemotherapy and the level of MRD at the end of induction therapy is proportional to mortality and incidence of relapse . The detIn recent years, considerable technological advances have allowed for the detection of tumor-specific biomarkers in biofluids, e.g., plasma, including circulating microRNA/RNA (ctmiRNA/RNA), circulating tumor cells (CTCs), exosomes and circulating tumor DNA (ctDNA) . In partThere are fundamental differences between childhood cancers and adult cancers  and thesThe differences in genetic mutations are an important issue to consider, as the mutation to be detected will depend on the purpose of ctDNA testing, e.g., detecting treatment resistance to tyrosine kinase inhibitors for Philadelphia chromosome-like ALL, a high-risk subtype of pediatric ALL . CurrentIn this review, we discuss the recent findings of ctDNA as a biomarker in the context of pediatric malignancies, including the challenges associated with detection in liquid biopsies. We examine ctDNA studies for risk stratification, treatment response and relapse prediction, highlighting the lack of fundamental knowledge of ctDNA in pediatric malignancies. We outline the requirements for the standardization of ctDNA detection in pediatric cancers, including sample handling and reproducibility of results. With better understanding of the advantages and limitations of ctDNA and the value of ctDNA for answering key clinical questions, ctDNA analysis may contribute to the standard of care for ongoing monitoring in childhood cancers.IKZF1 or translocations (BCR-ABL1 or KMT2A) (–5)  for the rapid detection of specific clonal rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes, microdeletions, i.e., r KMT2A) . Howeverr KMT2A) . This li2A) (–5) . Achievi2A) (–5) . Further2A) (–5) . AnotherUnlike hematological malignancies, MRD testing in solid tumors for risk stratification is not standardized, mostly due to the difficulties in sampling low concentrations of tumor cells in the blood . DetectiIt is important to note that MRD detection, i.e., the sampling of small numbers of cancer cells is inherently different from blood sampling and plasma-based ctDNA detection of disease. Therefore, while MRD analysis provides a standard model for liquid biopsy, these methods are measuring residual cancer cells from a single site versus circulating nucleotide biomarkers that represent an average of the total body tumor burden, depending on tumor type and phase of treatment. Given the challenges of diagnosis and detecting MRD in childhood cancer, liquid biopsy may provide significant advantages over conventional tissue biopsies including its use as an adjunct to disease detection and risk classification at diagnosis, assessing the quality of treatment response during treatment and providing an overall assessment of disease burden. Longitudinal monitoring of molecular composition and characterization of a patient’s tumor has further advocated for the use of liquid biopsy-derived markers in disease management, particularly ctDNA.Total cell-free DNA (cfDNA) from normal cells is found at low levels (around 10–15 ng/ml) in all healthy individuals . CtDNA ivia exosomes  single target PCR assays which are useful for disease monitoring; 2) small generic panels defined by the tumor subtype to allow for detecting subclonal markers which could potentially identify treatment resistance in addition to disease montoring; and 3) patient-specific panels using next generation sequencing (NGS) which allow for sensitive detection of many less common but clinically relevant mutations in multiple genes. The data obtained from sequencing the patient tumor can then be used for the design of patient-specific assays with reference to previous knowledge of the tumor type.CtDNA analysis relies on the detection of somatic mutations that are not present in normal DNA. Therefore, prior knowledge of the genetic landscape of each patient’s underlying tumor is used for the detection through targeted assays, namely PCR methods . Third gNGS is a valuable tool for identifying more comprehensive mutations across wider genomic regions . CurrentKnowledge of the original tumor is essential for serial disease monitoring and detection of resistance mutations with the potential for detection of subclonal disease. For the detection of low frequency clinically relevant somatic alterations (0.004%), Newman et al. used cancer-specific profiling by deep sequencing (CAPP-Seq) as an economical and ultrasensitive method with broad patient coverage . HoweverThe ability to sequence individual patient tumors is attractive in the field of personalized medicine as it is an unbiased approach for the detection of targets that were not previously associated with the individual’s tumor type. These methods typically involve sequencing of the patient’s tumor, from which the data are then used to design targeted assays, pending validated tumor-specific data. . With blIn childhood cancers, where the ctDNA burden is expected to be different across underlying cancer types and where tumor burden is below 0.01% of total cfDNA, the available ctDNA is likely to contain less than one cancer genome . TherefoAs solid tumor sampling and low levels of tumor burden are ongoing challenges for the assessment of disease in children with cancer, it is becoming more apparent that ultrasensitive and low-cost techniques for ctDNA detection can improve these limitations. Biosensing technology presents a unique alternative approach to conventional techniques of ctDNA detection, in part due to the lack of DNA amplification, that is, traditionally required for PCR methods. Biosensors are tools that produce a biological signal when detecting target analytes such as ctDNA, which is then converted into a quantifiable signal response . ElectroEGFR detection in NSCLC – the QIAGEN therascreen EGFR RGQ PCR kit and the cobas EGFR Mutation Test. These tests have been approved as companion diagnostics for solid tumor patients and have shown sensitivities of 0.81 and 3%, respectively, calculated as true positives and false negatives defined in accordance to the tissue-based test  which screens for fetal aneuploidy in pregnant individuals . To datesed test . The QIAsed test . So far,More recently, an NGS-based FoundationOne Liquid CDx test was approved as a companion diagnostic technology for mutations in NSCLC and prostate cancer to indicate eligibility for treatment . GuardanEGFR and KRAS, both of which now have regulatory approval as companion diagnostics in Europe and the United States  . NakamurTo date, ctDNA analyses in children with ALL have identified assays that are not sufficiently sensitive to inform prognosis . HoweverA retrospective study by Jimenez et al. investigated plasma samples collected at the time of diagnosis of different types of kidney tumors in 18 patients. The results demonstrated sensitive detection of plasma tumor-specific copy number and/or single-nucleotide changes . As renaEWSR1 gene, most typically EWSR1-FLI1 and EWSR1-ERG rearrangements, are the diagnostic hallmark for Ewing sarcoma. The specificity of key fusion genes such as EWSR1 allows for robust detection in ctDNA assays, however whether detection in blood can inform clinical decisions remains to be demonstrated. Recurring or increasing levels of the EWSR1 fusion gene copy numbers were previously shown to be associated with relapse in 234 blood samples from 20 patients  and standard operating procedures  for ctDNA analysis. However, there are agreed conditions that have been shown to affect sample quality and stability of ctDNA. The impact of various cell-stabilizing blood collection tubes on cfDNA extraction was investigated by Alidousty et al., who demonstrated that cfDNA collection tubes containing preservatives stabilized total cfDNA and intact cells for up to 1–2 weeks at room temperature, which is more feasible for shipping, storage and batched processing protocols . This waFurthermore, lack of standardization for the type of sample  collected for ctDNA analysis is an issue often overlooked in many studies. However, studies performed by Thierry and Bronkhorst have since demonstrated that levels of ctDNA are much higher in plasma . Lee et The sensitivity of ctDNA detection techniques should always be considered for clinical validity in an MRD or early detection setting. For acute leukemias, MRD for treatment response assessment is most beneficial at early timepoints, when adequate numbers of tumor cells are present . SensitiFor current MRD measurement, routine analysis requires a threshold to determine the MRD status of the patient to determine the sensitivity and specificity of the MRD assay, which may be adjusted for every patient depending on the clinical question. For every cancer, excluding ALL, AML and chronic myeloid leukemia (CML), there is no gold standard for such a threshold. In the past, sensitivities were established by a serial dilution of cells in a sample from a healthy individual  to deterGuidelines for the most suitable unit of measurement for ctDNA have not been established and this is partly due to the purpose and type of ctDNA detection method. CtDNA concentration determined by ddPCR is expressed as ng/mL or haploid genome equivalents (copies)/mL. With NGS, Abbosh et al. measured genomic alterations in ctDNA as a fraction of total cfDNA   whereas In general, while targeted approaches were shown to have better analytical sensitivity than untargeted, both methods have been used extensively in clinical studies of pediatric cancer in adult cancers. They are required for: 1) investigating ctDNA biomarkers and their biological and clinical significance; and 2) designing personalized assays for ultrasensitive ctDNA detection. It is evident that standardization of units for ctDNA measurement in cancer, irrespective of adult or childhood, is challenging. Ultimately, the choice of ctDNA of biomarker and units of ctDNA analysis should always answer the clinical question at hand.Recent liquid biopsy studies in childhood cancers have provided novel insights into the biology and dynamics of ctDNA. While the sensitivity and analysis of ctDNA measurement are improving, the clinical application of ctDNA is yet to inform prognosis and evaluate treatment response in pediatric patients. CtDNA applications for pediatric cancers are not as advanced as in adult cancers and the studies summarized above are retrospective studies in small cohorts. Despite the challenges of ctDNA analysis, these studies have shown the potential for ctDNA tests in pediatric tumors with high turnover of immature cancer cells. Furthermore, there were previously limited clinically relevant experimental models to mimic childhood cancer, relapse and treatment resistance, however this has significantly improved with patient-derived models now used in large translational programs in pediatric cancer . There iThe dynamics of ctDNA in the context of MRD or clinically-defined disease stages are poorly understood. For clinical implementation, robust guidelines for standardised sample collection and processing, as well as correlation to clinically significant outcomes in large clinical trials are needed. Comparisons between ctDNA analysis and current standard of care response assessments such as conventional MRD monitoring in ALL, are also necessary to understand the value that ctDNA provides. To our knowledge, there are few studies that investigate ctDNA for cancer screening or early detection, and the available data show ctDNA assays with low sensitivities for early cancer diagnosis. With cfDNA prenatal testing for certain fetal chromosomal abnormalities now standardized for pregnant individuals, identification of high-risk mutations for cancers for cancer predisposition syndromes is a potential application of ctDNA however, this will need to be validated in prospective studies. Different approaches to ctDNA detection have recently been developed, with promising technology in the field of nanomedicine for ultrasensitive ctDNA detection without the need for DNA amplification. For sensitive ctDNA assays to succeed as disease monitoring tools, they must demonstrate strong value in improving patient care as is the case for regulatory approval of novel drugs. CtDNA in combination with other liquid biopsy constituents including CTCs and exosomal miRNA may better reflect the cancer in one blood sample as accurately as possible. Information from all these samples may be worthwhile in providing a more detailed comparison of the genomic alterations present in ctDNA. The overarching challenge is to develop a liquid biopsy test, that is, rapid, patient-specific, low cost, clinically translatable and applicable to a wide range of pediatric cancer patients.


There are substantial differences in the progression of aging between males and females including in progression and prevalence of disease and longevity. Not all can be explained solely by sex-specific endocrine regulation and growing evidence suggests there are basic biological and genetic differences in sex that drive disparity in physiological function. In this study, we describe metabolic differences at the cellular level that both define some of these biological differences as well as provide a potential mechanism for delineating relevant molecular mechanisms of aging. Using HET3 mice, a genetically heterogeneous model with a consistent female advantage in longevity, we show that primary fibroblast lines retain functional metabolic characteristics, including mitochondrial response and stress resilience, which are representative of the sex of the donor animal. These differences persist even after several rounds of passage using standard culturing techniques suggesting these differences are not driven by direct impact of circulating sex hormones. Moreover, we find that differences in these cellular responses have some predictive power to determine both sex- and individual-specific responses to physiological challenge including obesity and longevity. In addition, we are able to use this model to delineate how donor sex affects cellular responses within defined pillars of aging including proteostasis, metabolic function, and adaption to stress. Overall, our model then provides valuable in defining the cellular responses that contribute to the mammalian aging process.


Epilepsy can be interpreted as altered brain rhythms from overexcitation or insufficient inhibition. Chemogenetic tools have revolutionized neuroscience research because they allow “on demand” excitation or inhibition of neurons with high cellular specificity. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are the most frequently used chemogenetic techniques in epilepsy research. These engineered muscarinic receptors allow researchers to excite or inhibit targeted neurons with exogenous ligands. As a result, DREADDs have been applied to investigate the underlying cellular and network mechanisms of epilepsy. Here, we review the existing literature that has applied DREADDs to understand the pathophysiology of epilepsy. The aim of this review is to provide a general introduction to DREADDs with a focus on summarizing the current main findings in experimental epilepsy research using these techniques. Furthermore, we explore how DREADDs may be applied therapeutically as highly innovative treatments for epilepsy. Epilepsy is a disorder that is generally perceived as an imbalance between excitation and inhibition in the brain. According to the International League Against Epilepsy, this disorder affects approximately 65 million people worldwide  that is solely activated by the insect peptide allatostatin. Delivery of allatostatin to ex vivo ferret brain slices induced hyperpolarization of mammalian cortical neurons expressing allatostatin receptors in a reversible manner without endogenous receptor cross-reaction . GPCRs are the largest group of cell surface receptors in the central nervous system  potential side effects of CNO in non-DREADD-expressing animals; and (ii) the injection of a vehicle, such as saline, within DREADD-expressing subjects  intracerebral injection of adeno-associated virus (AAV) vectors encoding the engineered receptors to a genomic sequence, with the genomic sequence linked to cell-type specific transcription factors, which results in receptor expression in a targeted subset of cells; and (ii) using transgenic mice that express the engineered receptors in cell populations genetically defined by the Cre-driver mouse line  AND (“epilepsy” OR “seizure” OR “spasm” OR “epileptogenesis”). Publications that were classified as reviews were excluded. References were screened for additional relevant articles. Title/abstract and full text screenings were performed. Articles used for full text analysis were those using chemogenetics to investigate the pathophysiology of epilepsy.in vitro were excluded from the table but are included in the text. Three (3) publications on chemogenetics with potential for additional development were included in the “Discussion” Section.Only primary or original research was considered in scope of this review. Our initial search returned 34 publications from PubMed, 385 from Scopus, 124 from ScienceDirect, and 30 from Web of Science. After removal of duplicates, 470 articles were included in our review. Publications were included if DREADDs were used to evaluate mechanisms of epileptogenesis or the epileptic phenotype. Additionally, publications were included if DREADDs were used to analyze biochemical and molecular mechanisms involved in epilepsy, even if results did not directly demonstrate seizure induction or seizure control. Publications were excluded if they evaluated seizures or spasms unrelated to epilepsy  or focused purely on chemogenetics development . After reading the abstract and applying inclusion and exclusion criteria, we identified 63 articles. After reading the full text and applying the same criteria, we included 25 articles in our analysis. Two (2) additional articles were identified by reference mining  resulting in a total of 27 articles. See We divided the articles included in this review into two main categories, i.e., those investigating hippocampal and extrahippocampal networks. Within the section on hippocampal networks, examples of seizure potentiation and induction by DREADDs are reviewed, followed by studies demonstrating seizure reduction using hM3Dq and then studies describing seizure reduction using hM4Di. A similar structure is used for the section on extrahippocampal targets, with examples of seizure induction described, followed by seizures reduced by hM3Dq and then by hM4Di. Finally, we discuss the experiments that use DREADDs to evaluate biochemical changes and then comorbidities associated with epilepsy.via the subiculum. Two other loops that bypass the dentate gyrus are an intermediate-length loop from EC to CA3 to CA1 to subiculum/EC; and a short loop that projects directly from EC to CA1 , which is characterized by a seizure onset zone located in the temporal lobe  interneurons (INs) in the DG -, somatostatin (SOM)-, cholecystokinin-, and vasointestinal peptide-expressing INs  subcutaneous clozapine injection resulted in decreased acute seizure frequency in rats with epilepsy induced by systemic KA. Additionally, clozapine or olanzapine were infused continuously for 7 days using osmotic minipumps, which resulted in significant seizure suppression during the first 4–5 days of treatment. However, seizure frequency increased to pre-treatment levels in the last 2 days of treatment with seizure duration exceeding baseline 3–4 days into treatment. Furthermore, after the removal of the minipumps, the animals showed a rebound effect both in seizure frequency and duration, reaching levels above the baseline observed before the onset of the treatment with CNO  for DREADDs activation, these results show the potential of generating acute generalized seizures in an “on demand” way with high cellular selectivity. By targeting hM3Dq or hM4Di to specific regions and subsets of cells, DREADDs reduce off-target effects of chemoconvulsants since CNO is inert to receptors other than the expressed DREADD. Furthermore, the effect of DREADDs may be titrated both at the level of receptor expression and dose of ligand administered, which may allow for tighter control over the severity and duration of seizures produced in animals.In addition to activating cells within the hippocampus, hM3Dq-mediated excitation has allowed for evaluation of extrahippocampal neuronal circuits on seizures originating from within the hippocampus. The studies presented in this section are summarized in The parafascicular nucleus of the thalamus is known to be involved in the generation of physiological oscillatory rhythms and in the control of epileptic seizures due to its projections from and to areas implicated in seizure generation such as the cortex, thalamus, hippocampus, and especially the striatum  completely blocked seizure activity in a subset of animals , which results in much slower onset of their effect  to have higher sensitivity to glutamate. Pilocarpine-induced epileptic mice that had these enhanced glutamate-gated chloride channels delivered into cortical pyramidal neurons showed attenuated acute seizures and a progressive decrease in the number and frequency of seizures in the chronic period of epilepsy (Lieb et al., Fifth, ligands for DREADDs activation will also need to cross the BBB. Recent publications provided evidence that CNO is reverse metabolized peripherally into clozapine and N-desmethylclozapine. Instead of CNO, these metabolites cross the BBB and possess higher binding affinity to muscarinic DREADDs than CNO (Nawaratne et al., Finally, another important consideration when applying DREADDs to treating patients with epilepsy is the longevity of the treatment. The substantial effect and relatively long duration of 8 h after administration of ligand currently make DREADDs an ideal rescue treatment upon administration of the ligand. Long-term dosing of the ligand as a chronic treatment requires further development and consistency across researchers in designing experiments. It is unknown whether the tolerance effect presented by Goossens et al.  above coPrior to clinical use, DREADDs must be demonstrated safe and efficacious in non-human primates. As a first step, researchers investigated whether the use of rodent-optimized viruses can transduce and drive the required levels of DREADD expression in these primates. To evaluate this, Galvan et al.  performeIn summary, DREADDs have proven to be powerful tools for improving our understanding of the pathophysiology of epilepsy. DREADDs have the potential to become new treatment modalities for patients suffering from this disease. Further investigations are needed in order to apply the laboratory findings to improve treatment outcomes for these patients.All authors contributed equally to the planning, research, writing, review, and development of this work. 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.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.