Health systems and providers across America are increasingly employing telehealth technologies to better serve medically underserved low-income, minority, and rural populations at the highest risk for health disparities. The Patient-Centered Outcomes Research Institute (PCORI) has invested US $386 million in comparative effectiveness research in telehealth, yet little is known about the key early lessons garnered from this research regarding the best practices in using telehealth to address disparities.This paper describes preliminary lessons from the body of research using study findings and case studies drawn from PCORI seminal patient-centered outcomes research (PCOR) initiatives. The primary purpose was to identify common barriers and facilitators to implementing telehealth technologies in populations at risk for disparities.A systematic scoping review of telehealth studies addressing disparities was performed. It was guided by the Arksey and O’Malley Scoping Review Framework and focused on PCORI’s active portfolio of telehealth studies and key PCOR identified by study investigators. We drew on this broad literature using illustrative examples from early PCOR experience and published literature to assess barriers and facilitators to implementing telehealth in populations at risk for disparities, using the active implementation framework to extract data. Major themes regarding how telehealth interventions can overcome barriers to telehealth adoption and implementation were identified through this review using an iterative Delphi process to achieve consensus among the PCORI investigators participating in the study.PCORI has funded 89 comparative effectiveness studies in telehealth, of which 41 assessed the use of telehealth to improve outcomes for populations at risk for health disparities. These 41 studies employed various overlapping modalities including mobile devices , web-based interventions , real-time videoconferencing , remote patient monitoring , and store-and-forward  interventions . The studies targeted one or more of PCORI’s priority populations, including racial and ethnic minorities , people living in rural areas, and those with low income/low socioeconomic status, low health literacy, or disabilities. Major themes identified across these studies included the importance of patient-centered design, cultural tailoring of telehealth solutions, delivering telehealth through trusted intermediaries, partnering with payers to expand telehealth reimbursement, and ensuring confidential sharing of private information.Early PCOR evidence suggests that the most effective health system- and provider-level telehealth implementation solutions to address disparities employ patient-centered and culturally tailored telehealth solutions whose development is actively guided by the patients themselves to meet the needs of specific communities and populations. Further, this evidence shows that the best practices in telehealth implementation include delivery of telehealth through trusted intermediaries, close partnership with payers to facilitate reimbursement and sustainability, and safeguards to ensure patient-guided confidential sharing of personal health information. People living in medically underserved, low-income, or rural areas; those from racial and ethnic minorities; members of the lesbian, gay, bisexual, and transgender (LGBT) community; and those with limited English proficiency or disabilities often face substantial barriers to accessing needed health care and are at high risk for health disparities ,2. AboutOther populations at particularly high risk for disparities include low-income African Americans in medically underserved urban and rural areas of the South -11, AmerTelehealth modalities have great potential to help overcome geographic, socioeconomic, cultural, and language barriers related to the social determinants of health and enhance access to essential health services for high-risk populations ,25. TeleThe primary purpose of this scoping review was to identify key barriers to telehealth implementation and describe how barriers can be addressed in populations at risk for disparities. PCORI has invested US $386 million in comparative effectiveness research in telehealth, yet little is known about the key early lessons of this research for using telehealth to address disparities. This review describes preliminary lessons from early patient-centered outcomes research (PCOR) experience and literature, using study findings and illustrative case studies drawn from these seminal PCOR initiatives and literature.A systematic scoping review of telehealth studies addressing disparities was performed and was guided by the Arksey and O’Malley Scoping Review Framework , with a The purpose of this scoping review was to describe barriers and facilitators to implementing telehealth or remote interventions in populations at risk of health disparities  within comparative effectiveness research projects funded by PCORI. The specific question addressed was as follows: “How have PCORI-funded investigators overcome barriers to implementing telehealth interventions in populations at risk for disparities?”Between December 2012 and March 2019, PCORI funded 84 comparative effective research projects, in which at least one of the study’s comparison arms used telehealth to improve health outcomes. PCORI defines telehealth as the delivery of health services via remote telecommunication modalities, such as telephonic communication, remote monitoring devices, real-time videoconferencing, and mobile devices. For this scoping review, PCORI staff first reviewed the telehealth portfolio to identify studies aiming to improve health outcomes for populations at risk of or facing disparities. Studies included those that targeted at least one of the following PCORI priority populations at risk for disparities: people living in medically underserved, low-income, or rural areas; those representing racial and ethnic minorities; members of the LGBT community; those with low health literacy/numeracy and limited English proficiency; and those with disabilities . Other POf the 84 PCORI-funded studies in this portfolio, PCORI staff identified 41 studies focused on improving health outcomes for populations at risk of or facing disparities by confirming the target population for each study. Qualifying studies were further reviewed by PCORI staff to determine if any barriers to implementation of the planned telehealth intervention were experienced and whether the investigators were able to mitigate these barriers to successfully implement the study telehealth intervention. A convenience sample of 8 studies was selected based on their representativeness , study phase , and perceived principal investigator availability to provide illustrative examples of successful approaches for working through obstacles in implementing telehealth interventions for diverse populations.We drew on these 8 illustrative examples from early PCOR experience and evidence from the literature to assess barriers and facilitators to implementing telehealth in populations at risk for disparities . Key infFollowing identification of all PCORI studies meeting the criteria for the scoping review n=41), PCORI staff extracted descriptive data for all these studies, including telehealth purpose and modality, population, health condition, budget amount, principal investigator, and institution, using published project descriptions and study papers. PCORI staff and study investigators then extracted additional data from these same sources and interim progress reports (where available) in order to categorize documented barriers and facilitators using a published framework of identified barriers  and facilitators  to telehealth . We then, PCORI sA table including the core descriptive data regarding all PCORI-funded telehealth studies addressing populations at risk for disparities was created. In addition, figures and charts were created to summarize project details about this subset of studies. An assessment of the quality of studies included in this review was not performed. A table was created summarizing the barriers and facilitators identified within each major implementation domain. Finally, major themes regarding how telehealth interventions can overcome barriers to telehealth adoption and implementation were identified through review of both the extracted data and illustrative examples using an iterative Delphi process among the 8 PCORI principal investigators participating in the study to achieve consensus. The Delphi process was facilitated by PCORI staff and a lead principal investigator for the scoping review, and was conducted through teleconference and email communication. PCORI staff and the lead principal investigator initially categorized extracted data and illustrative examples by barrier and facilitator types based on previously published categories . StudiesAs shown in The descriptions, study goals, telehealth interventions studied, settings, number of participants, and targeted study populations for these 41 PCORI-funded studies are detailed in As shown in PCORI-funded studies employed multiple telehealth modalities. The majority of these 41 studies used a mobile device  or were web-based . Fewer studies used real-time videoconferencing , remote patient monitoring , or store-and-forward capability . It is important to note that a similar telehealth modality does not indicate similar studies. For example, studies employing mobile device interventions were diverse and included web-based interventions requiring full internet access, as well as lower-tech interventions that only employed SMS or simple text messaging. All the web-based interventions referenced below required reliable broadband internet access. Real-time videoconferencing was used in 15 studies, and 11 of these studies utilized telehealth to improve access to primary or specialty care. Health conditions involving videoconferencing were cardiovascular diseases (n=2), kidney diseases (n=2), mental/behavioral health (n=4), nutritional and metabolic disorders (n=3), allergies and immune disorders, multiple chronic conditions, neurological disorders, respiratory diseases, and trauma/injury. Remote patient monitoring  was used in 8 studies. Remote monitoring varied from a wearable device  to mobile app monitoring to an in-home device . Health conditions incorporating remote monitoring included cardiovascular diseases (n=3), mental/behavioral health (n=2), nutritional and metabolic disorders, and respiratory diseases. Store-and-forward technology was employed in 5 studies that included the following conditions: functional limitations and disabilities, mental/behavioral health, rare diseases, hearing loss, and skin diseases.Furthermore, the purpose of the 41 PCORI telehealth interventions also varied substantially, with 8 (20%) focused on enabling health monitoring, 20 (49%) focused on improving access to specialty care, 22 (54%) focused on health education, and 27 (66%) focused on promoting chronic condition self-management. The spectrum of chronic conditions studied also varied substantially. The largest proportion of studies focused on behavioral health , and nutritional and metabolic disorders , including obesity and diabetes. In addition, PCORI has funded telehealth projects addressing disparities focused on cardiovascular conditions (n=3), chronic kidney disease , neurological conditions (n=2), reproductive and perinatal health (n=2), rare diseases (n=2), allergies and immune disorders (n=1), cancer (n=1), hearing loss and ear diseases (n=1), functional limitations and disabilities (n=1), infectious diseases (n=1), multiple chronic conditions (n=1), trauma (n=1), and skin diseases (n=1). Although only 1 study focused on patients with multiple chronic conditions, most of the studies included patients with multiple chronic conditions and some additional studies used such presence as an inclusion criterion.Barriers and facilitators of telehealth implementation in PCORI-funded studies using telehealth to address disparities are detailed in Major themes identified across these studies included the importance of patient-centered design, cultural tailoring of telehealth solutions, delivering telehealth through trusted intermediaries, partnering with payers to expand telehealth reimbursement, and ensuring confidential sharing of private information.Illustrative examples of these key themes are reviewed in the sections below, and key system- and policy-level changes needed to address barriers to telehealth adoption and implementation are identified.Patient engagement is a core component of PCORI studies. Some studies go beyond incorporating patient input and feedback on the research and actively engage patients into the study design and overall design of the intervention.The VIGoROUS (Video Game Rehabilitation for Outpatient Stroke) study illustrates how user-friendly consumer-driven design serves as a critical component of sustainable telehealth initiatives ,71. It eSimilarly, the study by Williams et al comparing high-touch and high-tech care in patients with complex care needs utilized a smartphone-based remote care management platform and digital health innovations to support self-directed care management ,97. WhilWhen developing telehealth interventions, it is particularly important to consider the need for culturally competent care, defined as care that respects the diverse characteristics and values of the patients, which can ultimately affect their overall health and health care . This reThe MODEL (Management of Diabetes in Everyday Life) study assessed the effectiveness of tailored motivational text messaging for improving diabetes self-care activities in medically underserved African American adults in the Mid-South, and illustrated the importance of cultural tailoring of telehealth solutions ,45. Thiscondones versus preservativos to refer to condoms). Preliminary findings showed that both providers and study participants reported high satisfaction with the app, as it was easily integrated into the clinic flow, and more importantly, participants showed an increase in knowledge of contraceptive use from baseline to their 6-month follow-up.Similarly, the Health-E You/SaludiTu study demonstrated how cultural tailoring of telehealth interventions can improve their acceptance by populations at risk of disparities ,92. ThisLikewise, early implementation experience from a PCORI-funded study of underserved Asian Americans having chronic hepatitis B suggested that culturally and linguistically competent interventions improve participant retention . This stMany of the studied PCORI telehealth projects successfully employed trusted intermediaries to deliver telehealth solutions and help support people at high risk for health disparities in crossing the digital divide. These projects provided confirmatory evidence to extensive literature suggesting that lay health worker and paraprofessional intermediaries can help build trust among patients and providers in new telehealth modalities. New technologies, including telehealth, that enable the delivery of interventions and diagnostics remotely are emerging, but patients commonly struggle to use these technologies. Patients struggle with both simple technical issues, such as neglecting to ensure that an electronic system is plugged in, and more complex barriers related to social determinants of health  when using advanced technologies such as secure videoconferencing.PCORI telehealth projects consistently demonstrate high patient acceptance of telehealth modalities that connect them to trusted providers, but even with careful attention to patient-centered design, investigators commonly found availability of in-person technological support to be essential. For example, when 1 participant in the aforementioned VIGoROUS video gaming intervention reported that her gaming system would not turn on, a support call revealed that she had not plugged it into an electrical outlet ,71. ThisSimilarly, a study of Native Americans addressing gaps in self-management through home-based CKD care found that continuous care coordination using trained and trusted tribal community health representatives (CHRs) was essential to effective program implementation -88. ThisA study addressing childhood hearing loss in an Alaska Native population similarly found that using trusted lay health workers based in the communities served was essential to telehealth program deployment -103. In Reimbursement for telehealth encounters is increasing. However, lack of adequate reimbursement continues to present a major challenge to telehealth adoption, implementation, and sustainability. Reimbursement barriers to telehealth were acknowledged by nearly all the PCORI-funded study investigators who participated in this study. Despite recent expansions in telehealth reimbursement during the COVID-19 pandemic, variable state and federal reimbursement policies and low levels of reimbursement for services delivered via telehealth continue to challenge telehealth implementation efforts. In response to these challenges, many studies found it essential to partner with payers to expand their support and/or reimbursement for telehealth services.For example, the TEAM UP  study assembled a Payer Advisory Board that explored how military medical sites use telehealth for services such as teleradiology and telebehavioral health . The BoaIn the rural Alaska study addressing childhood hearing loss, the research team developed a streamlined telemedicine consultation process in order to make it financially sustainable in the school setting ,69,104. Similarly, in the Mid-South MODEL study, investigators worked with regional health systems to provide long-term support for a regional registry-based tailored text message system that can assist both large health systems and small independent primary care practices in sending regular motivational messages to interested patients ,45.Early implementation experience from numerous PCORI telehealth studies demonstrates the importance of ensuring confidential sharing of private information in all telehealth interventions considered. The application of telehealth often involves paperless collection and transmission of data at each touch point with participants and with communication between staff at the regional and local levels. This requires careful attention to compliance with local, regional, and federal legal requirements for protection of privacy.For example, the TEAM UP study of alternative childhood obesity treatments for Medicaid-insured individuals employed a secure Health Insurance Portability and Accountability Act (HIPAA)-compliant videoconferencing system to enable remote communication with children and families that protected privacy . WorkingThe VIGoROUS study also had to address challenges related to information security . First, This scoping review of early PCOR evidence suggests that the most effective health system- and provider-level telehealth implementation solutions to address disparities employ patient-centered and culturally tailored telehealth solutions. We found that the development of the most effective telehealth solutions was actively guided by patients themselves in order to meet the needs of specific communities and populations. Early PCOR evidence demonstrates that the best practices in telehealth implementation include delivering telehealth through trusted intermediaries, close partnership with payers to facilitate reimbursement and sustainability, and safeguards to ensure patient-guided confidential sharing of personal health information.The COVID-19 pandemic has given new urgency to these questions regarding telehealth adoption among people at the highest risk for disparities, including those living in medically underserved, low-income, or rural areas; those from racial and ethnic minorities; LGBT persons; and those with limited English proficiency or disabilities. Telehealth modalities have great potential to help overcome these geographic, socioeconomic, cultural, and language barriers and to give populations at risk of disparities enhanced access to essential health services. Although many PCORI-funded studies had to put their research on hold and transfer their institution’s focus on the needs of patients experiencing COVID-19 , the panToward that end, this review places strong emphasis on the importance of deploying patient-centered and culturally tailored telehealth solutions that address the social determinants of health faced by populations at risk for disparities -21. EvenFurthermore, this review revealed that telehealth solutions can take advantage of and expand on existing technological capacities accessible to low-income and other populations at risk for health disparities, and can often be deployed at low cost. The existing PCORI-funded telehealth research suggests that low-cost strategies that employ existing telehealth capacities using standard mobile phones and smartphones may be particularly effective in reaching patients where they are and engaging them in needed care. Although more than 90% of Americans overall carry cell phones and 80% have smartphones , among pThis review also emphasizes the importance of telehealth solutions that work through existing trusted care networks and providers, and that employ trusted lay health worker and paraprofessional intermediaries to introduce and support the use of these technologies by people from the community. Many of the most effective PCORI telehealth initiatives considered in this paper focused their interventions around engaging, training, and deploying trusted intermediaries from the community and/or the population at risk for disparities to be served. This evidence also suggests that personalized human interaction is essential. For example, text messaging interactions were found to be most effective when delivered in real-time, tailored to participant interests and needs, and originated from a trusted known source. The PCORI-funded telehealth projects reviewed suggested that face-to-face videoconferencing and telephone interventions with trusted caregivers are particularly effective.Our review further suggests that substantive reimbursement and regulatory changes are immediately needed to enable low-cost and efficient telehealth solutions. We found that the best practices in telehealth implementation include close partnership with payers to facilitate reimbursement and sustainability. However, ultimately, experience from the existing portfolio of PCORI telehealth projects indicates that permanent changes are needed in national reimbursement policies and regulations to facilitate broader telehealth adoption and implementation. Policies and reimbursement for telehealth were rapidly put into place to allow health care to continue during the severe restrictions early in the COVID-19 public health emergency after March 2020, in order to allow care to continue during the COVID-19 pandemic. However, these changes clearly need to be extended. Needed regulatory changes include expanded reimbursement as well as relaxations of regulations around where the patient or practitioner is located, licensure, and reciprocity across states. For example, since populations at risk for disparities generally have less access to the internet and technology, regulatory solutions need to facilitate and enable the use of existing lower-cost technologies, such as telephone visits using traditional cell phones, rather than requiring video teleconferencing for medical billing. As the pandemic eases, it will be essential to carefully think about how to preserve the progress that was made in expanding telehealth in ways that improve patient care at the federal , state, and individual insurance company levels -118.The existing PCORI telehealth research targeting populations at risk for health disparities is of very high quality and has high applicability to real-world clinical and community settings. Of note, 100% of the PCORI-funded studies in this category employed a randomized controlled trial design and 100% were pragmatic rather than being conducted in highly controlled university settings among highly selected patients. This is a major strength of both the research reviewed in this scoping review and the scoping review itself. Furthermore, the studies included in this scoping review are also notable for their generalizability, given their pragmatic real-world approach. Rather than excluding patients with multiple chronic conditions as is often done in randomized clinical trials, many of the PCORI-funded studies specifically included patients with multiple morbidities given their representativeness of patients most commonly seen in real-world clinical settings.This review is limited by the small numbers of studies that were considered, and its main findings are subject to numerous potential biases. First, since PCORI staff chose studies for an in-depth review based on their perception regarding which study teams overcame notable obstacles to telehealth, selection bias could have occurred. Our expectation is that this bias would most likely lead to the overemphasis of facilitators and solutions as opposed to barriers and challenges. Second, because PCORI staff and investigators comprised the study team, it is possible that there was some bias toward reporting of positive study findings. However, the study team and methods actively sought to address this potential limitation by identifying and categorizing barriers first prior to the discussion of solutions. In addition, some of the telehealth services provided in these studies may be difficult to consistently replicate outside of structured evaluations.The review is also limited because final study results are not available for many of the included studies, and even fewer have detailed analyses of implementation factors and lessons learned available in the peer-reviewed literature. However, the review was facilitated by the detailed project knowledge of PCORI staff and the investigators themselves.Over the past 5 years, PCORI has supported many comparative effectiveness studies of adopting virtual care solutions to manage health outcomes across a wide range of populations at risk for disparities. Through these research initiatives, health care delivery systems are learning to leverage low-cost telehealth solutions to promote better health outcomes by increasing access to care, making care more effective, and continuously engaging patients. PCORI-funded comparative effectiveness research is demonstrating how we can reduce health disparities across the spectrum of disease conditions and populations through low-cost telehealth solutions. This research is identifying key barriers and limitations to implementing telehealth in vulnerable populations and ways to overcome these barriers to help improve health and health care outcomes.In this new era of the COVID-19 pandemic, there has been rapid growth in the use of technology across all industries, but it is proving to be particularly important in the areas of health and health care. Future efforts should focus on fostering collaborations between researchers, payers, and administrators to facilitate widespread adoption of new treatment models that have already demonstrated success through definitive clinical trials. Our review indicates that key system- and policy-level changes are desperately needed to address barriers to telehealth reimbursement. Moreover, further pragmatic research is needed to demonstrate the best approaches for disseminating and scaling these early telehealth findings in broader community practice to promote population health.This scoping review gives strong guidance to health systems seeking to target evidence-based telehealth modalities for health disparities. Results from the studies demonstrate that systems can do so in innovative ways, and in fact, some of the studies identify methods for systems to effectively reach underserved populations in their own communities. These case studies also highlight the critical importance of both supportive infrastructure, and regulatory and reimbursement policies to facilitate telehealth modalities and make them sustainable.However, despite growing evidence that telehealth can deliver care to people at risk for disparities, many major obstacles exist for its implementation. Early PCOR evidence demonstrates that the most effective health system- and provider-level solutions for at-risk populations use patient-centered and culturally tailored telehealth solutions whose development is actively guided by the patients themselves.


The exposome, which is defined as the cumulative effect of environmental exposures and corresponding biological responses, aims to provide a comprehensive measure for evaluating non-genetic causes of disease. Operationalization of the exposome for environmental health and precision medicine has been limited by the lack of a universal approach for characterizing complex exposures, particularly as they vary temporally and geographically. To overcome these challenges, passive sampling devices (PSDs) provide a key measurement strategy for deep exposome phenotyping, which aims to provide comprehensive chemical assessment using untargeted high-resolution mass spectrometry for exposome-wide association studies. To highlight the advantages of silicone PSDs, we review their use in population studies and evaluate the broad range of applications and chemical classes characterized using these samplers. We assess key aspects of incorporating PSDs within observational studies, including the need to preclean samplers prior to use to remove impurities that interfere with compound detection, analytical considerations, and cost. We close with strategies on how to incorporate measures of the external exposome using PSDs, and their advantages for reducing variability in exposure measures and providing a more thorough accounting of the exposome. Continued development and application of silicone PSDs will facilitate greater understanding of how environmental exposures drive disease risk, while providing a feasible strategy for incorporating untargeted, high-resolution characterization of the external exposome in human studies. The past 10 years have seen rapid growth in exposomic studies, providing improved understanding of how environment and other non-genetic factors contribute to disease risk. First defined by Christopher Wild in 2005, the exposome was envisioned as a complement to the genome that aims to define an individuals’ history of exposure and relationship with disease risk, including the influence of lifestyle, diet, and the environment .The cumulative measure of environmental influences and corresponding biological responses throughout the lifespan, including exposures from environment, diet, behavior, and endogenous processes [Recognizing the importance of biological response, the exposome concept has since been revised by Miller and Jones to: rocesses . Within rocesses , 6.By providing a key measure of non-genetic disease risk factors, the exposome has the potential to broaden understanding of how environmental stressors contribute to disease . HoweverThe exposome is unique in ‘omic sciences because it represents an integrated measure across multiple compartments characterizing how non-genetic factors external and internal to the host influence disease risk. As a result, the exposome in its totality requires attention to impacts from internal, external, and psychosocial factors, many of which require separate approaches and study designs to measure. The internal exposome combines measurement of biological activity with internal dose biomarkers that can include levels of the parent chemical, transformation products, and adducts of reactive compounds. The external exposome includes environmental exposures to toxic chemicals, pollution, and radiation, while also incorporating behavioral variables like diet, exercise, and drug use. Non-specific exposures, such as social and psychological stressors, are the third component of the exposome. While these three compartments tend to be measured and considered separately, they are dependent and interrelated. All exogenous biomarkers  originate from exposures that occurred outside the host , and many of these exposures can be both potentiated and varied depending on non-specific factors, such as stress and lack of sleep  .Most efforts to date have focused on the internal exposome due to the availability of biological specimens collected and stored from well-established population studies. These include approaches that aim to identify chemical biomarkers to estimate exposure burden and its relationship to adverse health outcomes, as well as ‘omic approaches that define specific phenotypes of exposure and disease. The most promising approaches for comprehensive measures of the internal exposome include untargeted high-resolution metabolomics, which detects low molecular weight compounds within a biological sample –11. WhilExternal exposome monitoring provides a standalone, but complementary, measure of environmental stressors . Unlike Individual exposome monitoring focuses on characterizing interactions between a person and exposure sources. As a result, multiple strategies are possible, including detection of chemical exposures in food or water and characterization of the indoor and outdoor microenvironments . To measTo better incorporate the exposome into the study of human health, there is a critical need to leverage strategies that enable comprehensive characterization across different exposome compartments. To achieve the power necessary to identify how low-level exposures and associated mixture effects contribute to disease outcomes, it is necessary to use approaches that allow low-cost sampling options and can be deployed in large populations. Current studies show how biospecimens, including blood, urine, and saliva, combined with untargeted assays, provide a solution for internal exposome characterization , 56–59; PSDs are non-invasive, easy to distribute, and can overcome many of the limitations that complicate interpretation of exposure biomarkers in biological samples . While tIdeal PSDs for exposomic studies should have high partitioning coefficients for compounds with a wide range of physiochemical properties, be cheap to manufacture, and be provided in a form that is easy for the participant to use. While multiple strategies have been proposed, the use of commercially available silicone wristbands has shown to provide a versatile, low-cost PSD that enables screening for a broad range of chemical exposures –67. As aOf these studies, wristbands were the most commonly used PSD device, while a limited number used multiple placement strategies to isolate exposure pathways, including brooch samplers for airborne respiratory exposures, and isolated wristbands to minimize dermal contact , 68, 69.Most PSDs 84%) were characterized using targeted approaches, where specific chemical classes were quantified with in-house analytical standards. An additional 18% of the studies included some form of suspect screening. Most exposures included volatile and semi-volatile compounds measured using gas-chromatography (GC), including single- and triple-quadrupole mass spectrometers, with some studies leveraging electron capture detectors for increasing specificity towards halogenated compounds , 71, 72.% were chSince the use of silicone PSDs is a new approach for passive exposure monitoring, 38 of the 44 reviewed studies combined PSDs with validated approaches for assessing exposures, including comparison to biomarker levels and established sampling devices. These include quantification of known exposure biomarkers in blood and urine , 87, 88,Most of the reviewed studies include questionnaires to associate PSD detected chemical classes with behavioral, lifestyle, and demographic patterns that may influence exposure patterns and potential health outcomes. Although few studies focus on biological endpoints, recent applications have attempted to link PSD measurements to health outcomes, including DNA damage biomarkers , thyroidOne of the challenges facing large-scale adoption of silicone-based PSDs for monitoring multiple exposures is uncertainty in partitioning and diffusion rates into the sampler matrix for compounds showing a wide range of physical–chemical properties , 91. ConPSDs are available commercially in a wide range of colors and sizes and can be modified to include text through embossing/debossing. While price varies depending on vendor, amount purchased, color/text options, and size, most are available at low cost (< $0.50 USD) and provide an economical solution for PSDs. However, wristbands purchased commercially often contain a high degree of impurities that can interfere with measurement sensitivity. Before deployment, thorough conditioning and cleaning are required to remove unbound siloxanes and other impurities , 65. DyeWhile solvent washing can remove a significant number of impurities, this approach is expensive, time consuming, and difficult to adapt to large quantities. Recent attempts to lower time and cost of conditioning have been developed, including re-use of solvents and accelerated methods that reduce solvent-washing , 84. HeaKow) may prevent extraction of more polar compounds, while drying steps can result in loss of volatile compounds.Following deployment of silicone PSDs, exposure-related compounds must be removed from the matrix and transferred to a form that is amendable to the chosen analytical method. Since the majority of silicone PSDs have focused on volatile and semi-volatile exposures, most commonly used preparation methods are GC-friendly and include solvent extraction or thermal desorption (TD) Table . The mosTD methods have been used extensively for environmental sampling of volatile compounds, as well as analysis of silicone PSDs , 99. WheAlthough PSDs have received considerable attention for exposure monitoring, their combination with untargeted analysis is limited and most applications have focused on measuring common classes of known exposures. These studies provide important insight into chemical exposures in human populations but do not allow detection of unsuspected or uncharacterized chemical that may be driving health effects , 100. UnThe Fresh Air wristband, which includes a polydimethylsiloxane coated sampler bar located in an enclosed PTFE chamber, has been used extensively with GC-based HRMS methods that include Orbitrap and QTOF-based analyses for sampling exposures . Using uRecently, ultra-high-resolution mass spectrometry methods have been applied to stationary silicone PSDs to evaluate microenvironment exposures, as well as silicone wristbands worn by study participants. In the study by Kalia et al., silicone samplers were placed throughout different rooms within a residential location for a period of 7 days and analyzed using untargeted GC-HRMS . AlthougMany chemicals of emerging concern (CECs) are only detectable using LC-HRMS methods and are known to be volatilized and transported on aerosols; these include per- and polyfluorinated alkyl substances (PFAS). PFAS and many other CECs are ionizable, and partition between the gas and particulate phase depending on interaction with aqueous aerosols and matrices, resulting in unique interactions with PSDs dissimilar from nonpolar SVOCs and persistent organic pollutants . Novel PWhile targeted methods provide excellent sensitivity and can generate new insight into ongoing exposures, costs increase with the number of chemicals analyzed . Thus, dThe number and types of chemicals detected in PSDs can be expanded by combining complementary separation and ionization approaches for HRMS. These include using alternate chromatography strategies, with LC and GC as the most comprehensive platforms for exposome-wide association studies. LC-HRMS platforms are best suited for measurement of polar molecules with ionizable functional groups, or large, non-polar molecules that include lipids, fatty acids, and sterols. However, many exposome chemicals are volatile enough to be introduced into the gas phase when heated and are not detected by LC-HRMS. Thus, GC-HRMS provides the best sensitivity and selectivity for these compounds , 113. MoFollowing analysis of PSDs, chromatograms can be processed using a number of software tools. Key steps include identification of peaks and integration, deconvolution to identify mass spectra, and alignment of peaks across samples. Both commercial and open-source tools are available; however, algorithms optimized for detection of low abundance peaks are best for exposome research –117. DecIdentification of mass spectral signals is one of the key challenges in applying untargeted HRMS. Many detected ions do not match compounds listed in metabolomic or environmental chemical databases and authentic standards are not available. Computational approaches that assign annotation confidence can enhance prediction of chemical identities. These approaches harness multiple lines of evidence to evaluate the quality of annotation and, combined with appropriate databases, improve the number of annotated compounds , 125. MuFor exposome studies designed to evaluate a disease or other adverse outcomes, signals from untargeted profiling must be prioritized to identify which exposures are driving risk . When apMultivariate and data reduction analytic strategies solve two major issues with the traditional exposome-wide association studies by (1) increasing power, since corrections for multiple comparisons are performed on the number of latent features (tens) rather than the number of chemicals (hundreds or thousands), and (2) facilitating determination of networks, since the latent variables are constructed based upon statistical or functional similarity and jointly use information across chemicals. Linear versions of these methods, such as PCA, independent component analysis (ICA), canonical correlation analysis (CCA), linear discriminant analysis (LDA), and partial least squares discriminant analysis (PLS-DA), are popular due to their simplicity of interpretation –143. NonTo realize measurement of the exposome, it is critical to consider exposures across multiple compartments. While most studies use HRMS to characterize the internal exposome using biological samples, personal PSDs provide complementary advantages. First, PSDs are much cheaper to produce and distribute compared with the cost for collecting blood or urine samples. Biologics often require special methods and trained personnel for collection, including clinical visits. These materials must be stored at low temperatures to maintain sample integrity, and different handling and storage procedures can increase variability. PSDs can be provided directly to participants and returned by mail, with limited-to-no contact between study participants and coordinators. This capability is especially important when considering the additional restrictions placed on in-person research due to the COVID-19 pandemic. Long-term, secure storage of biologics can also be costly, as storage in −80 °C is common. In contrast, PSDs can be stored in sealed bags at 4 °C or room temperature, since compounds are stable within the silicone matrix.Although studies to date have only used silicone PSDs with a small number of participants  is one of the primary routes of environmental exposure and must be assessed using other approaches. Biospecimens also allow measurement of alterations across biological levels and long-term maladaptations, an important consideration for evaluating cumulative effects of environmental exposures. Combined with internal chemical and bioeffect monitoring, the use of silicone PSDs provides a strategy for deep exposome phenotyping in human populations Fig. .Fig. 2ToThe goal of deep exposome phenotyping is to provide a systematic framework that operationalizes exposome-wide association studies of human health by combining the key measures necessary to understand the continuum from exposure to disease. Application in longitudinal cohorts can enable in-depth, comprehensive assessment of exposures, and when combined with untargeted HRMS analysis, provides the chemical coverage necessary for characterizing complex mixtures. Integrating external and internal measures of the exposome with multiple “-omic” layers will allow a functional approach to understanding how environment contributes to disease risk laying a foundation for the mechanisms underlying environment-related diseases , 27.Because silicone PSDs are available at low cost, they can be easily incorporated into ongoing longitudinal studies and employed as a tool to estimate temporal changes in exposure patterns through repeated follow-up with new samplers. While the focus to date has been environmental health studies, silicone PSDs also provide a strategy for incorporating the exposome into precision medicine. Environmental factors are widely recognized for their potential to alter treatment efficacy and disease progression . SiliconWhile environment is one of the main drivers of disease risk, the ability to measure the complexity of the exposome is limited by its temporal nature, availability of samples, and the technology to detect complex exposures patterns. While considerable advances have been made in analytical strategies for the internal exposome, comparable methods for the external compartment are not well-developed. Silicone wristbands and other PSDs, which can be combined with untargeted HRMS platforms to characterize the exposome, are a natural way to integrate measures of the external exposome into longitudinal studies. These devices are cheap, non-invasive, and can be easily distributed. Previous studies demonstrate their suitability for many environmental chemical exposures, which is critical for success in exposome applications. By using untargeted approaches, it is possible to detect and identify ongoing exposures that may have not been expected or characterized, supporting pollution control and identification of the primary chemical exposures experienced by humans. Thus, continued development and application of silicone PSDs will facilitate greater understanding of how environmental exposures drive disease risk, while providing a feasible strategy for incorporating untargeted, high-resolution characterization of the external exposome in human studies.


Local drug delivery is an effective strategy for achieving direct and instant therapeutic effects. Current clinical treatments have fallen short and are limited by traditional technologies. Bioadhesive nanoparticles (NPs), however, may be a promising carrier for optimized local drug delivery, offering prolonged drug retention time and steadily maintained therapeutic concentrations. In addition, the possibility of clinical applications of this platform are abundant, as most polymers used for bioadhesion are both biodegradable and biocompatible. This review highlights the major advances in the investigations of polymer-based bioadhesive nanoparticles and their innumerable applications in local drug delivery. Although systemic drug delivery is an advantageous delivery route due to its accelerated onset of action, it is often difficult to maintain the required local drug concentration. Simply administering higher dosages is often not a feasible solution coupled with the restriction of limited extravasation from the bloodstream into the target sites. These factors can lead to higher toxic effect and adverse side effects. Local drug delivery, however, aims to provide an optimal therapeutic effect directly to the disease sites with minimal or no systemic toxicity . An exceThe concept of bioadhesion refers to the interactive forces between the biological or synthetic material and a mucosal surface . SpecifiBioadhesive NPs can not only prolong drug retention time but can also encourage particle uptake and enhance local drug delivery with the combination of their small size and high specific surface area ,10. This“Bioadhesion” is the binding between natural or synthetic biopolymers and mucosa or cell surfaces . When biChitosan, a type of linear polysaccharide, consists of randomly distributed β-linked D-glucosamines and N-acetyl-D-glucosamines. It can be easily extracted and prepared from shrimp and crab shells . It has Gelatin is a generic name for partially acidic or alkaline hydrolysates of collagen from animals . It is gAs is widely known, marine mussels can maintain outstanding clinging properties under extremely harsh marine environments. Recent research has proposed the use of L-3,4-dihydroxyphenylalanine (DOPA) as a bioadhesive functional coating of nanoparticles. DOPA is present in the mussel adhesive protein at high concentrations, with the ortho-dihydroxyphenyl (catechol) functional group. This functional group can form strong bonds with a mass of inorganic or organic surfaces in an aqueous environment ,29,30,31Lectins are naturally occurring glycoproteins or sugar-binding proteins. Lectins can bind directly to the epithelial cells themselves, and therefore they have cytoadhesive properties . This isSargassum. As a polysaccharide consisting of abundant hydroxyl and carboxyl groups, it has natural adhesive properties, as well as excellent biocompatibility and degradability. Due to the innate advantages, the incorporation of alginate has been used widely in the fields of food and medicine. Fernandez et al. used sodium alginate to modify the nanostructured lipid carriers (NLCs) prepared by shea butter and argan oil. These bioadhesive NLCs loaded two antioxidants, and tea oil enhanced the retention of drugs on the skin, effectively releasing them into the wound to facilitate the healing process [Sodium alginate is a by-product of the extraction of iodine and mannitol from brown algae kelp or  process .Apart from natural biopolymers, synthetic biopolymers are also comprehensively applied to local drug delivery systems, owing to their potential of modification, versatility, and low batch-to-batch variation.Polyethylene glycol (PEG) is ubiquitously used in NP coatings due to its low toxicity and its resistance to non-specific binding of biomolecules. PEG coatings could considerably prolong the circulation of blood of NPs. However, most intravenously injected PEG-coated NPs are still observed to accumulate in the liver ,38. In c4 treatment. The aldehyde groups will form Schiff base bonds with amino groups, leading to bioadhesion on protein-rich surfaces  by the oxidation of surface vicinal diols into aldehydes with NaIOsurfaces . These B50 in HCT116 cells, being advantageous in the development of a formulation for colorectal cancer treatment [Poly (acrylic acid) (PAA) is generally regarded as safe by the U.S. Food and Drug Administration (FDA) when administrated orally, showing low toxicity and irritation . Carboporeatment . Recentlreatment .Helicobacter pylori (H. pylori) in a C57BL/6 mouse model was significantly decreased following oral administration of ethyl cellulose (EC) NPs encapsulating clarithromycin. The superior treatment effect of encapsulated clarithomycin is due to the improved adherence of EC NPs to the stomach mucosa of treated mice, which may suggest a potential use of these NPs for the oral delivery treatment of H. pylori infections [As the major constituent existing in plant cell walls, cellulose has been widely investigated for medical applications due to its biocompatibility. However, its main disadvantage is its poor processability, owing to its weak solubility in common organic solvents. Cellulose derivatives are feasible alternatives compared to pure cellulose . Cellulofections .There are a series of test models established for the characterization of bioadhesive properties. In in vitro models, Deng et al. utilized poly(L-lysine)-coated slides to stimulate the protein-rich tumor microenvironment and tested the performance of bioadhesive nanoparticles. They immobilized the surface of slides with unloaded NPs, EB-loaded NPs (EB/NNPs and EB/BNPs), free EB, or PBS and evaluated the in vitro efficacy in order to suppress the growth of USC cells. They observed that only slide regions pretreated with EB/BNPs significantly suppressed the growth of tumor cells . In addiOne of the primary purposes of designing and applying bioadhesive materials is to achieve slow and sustainable drug release at the target site by enhancing cargo retention . It is kThis review summarizes the recent advances of bioadhesive NPs for local drug delivery, mainly introducing a variety of polymers exerting bioadhesive properties as well as their related local applications. In conclusion, the bioadhesive NPs possess dual advantages of being nanocarriers and bioadhesives, making this platform more suitable for local therapies. Meanwhile, novel and multifunctional delivery systems based on bioadhesion are rapidly being developed in combination with nanotechnologies. For instance, tissue adhesives show excellent antibacterial and hemostatic properties when combined with the metal NPs, among which silver and gold are the most studied. Nano-compounds encapsulating growth factors or genes, or those combined with stem cells, have attracted great attention and interest from researchers, potentially providing novel therapeutic strategies in the near future . In addi


PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.The ancient paralogs premelanosome protein ( GPNMB (Glycoprotein nonmetastatic melanoma protein B)  and PMEL (premelanosome protein)  have generated increased interest from disparate biomedical fields. GPNMB and PMEL are related genes that encode for proteins with a common functional domain architecture: a signal peptide, core amyloid fragment, Kringle-like domain and a single transmembrane domain XXXL[LI] sequence D 130]. . 130]. AThe strong conservation between the PKAT family members may be considered surprising, if one interprets the phenotypic effects as relatively minor in these loss of function mutations across a variety of species (The repeat domain (RPT) is a low complexity region unique to PMEL, and in mammals, it contains a variable number of proline/serine/threonine-rich imperfect repeats . The RPTTo investigate the evolutionary relationship between orthologous PMEL RPT domains, we used RADAR predictions ,133 and The primary sequences of individual repeats are also drastically different between species. The human RPT contains two motifs that we noted are highly conserved amongst the repeats: GTT and PTXE; in contrast, the chicken RPT domain has a much more divergent TXXXTXX[DE] motif, despite also being a similar 12-mer-repeating unit. These striking differences in sequences are especially remarkable considering that the chicken RPT domain is sufficient to rescue the fibrilogenesis defects of human PMEL lacking a RPT domain, while the zebrafish RPT domain can provide a partial rescue . These dd-galactopyranoside causes a collapsed PMEL amyloid  ladder” , blunt-snouted clingfish (Gouania willdenowi) and Atlantic herring (Clupea harengus) each possess many repeat units that are almost perfectly identical in amino acid sequence within the species. However, comparing the near-perfect regions between species reveals that these closely related species underwent considerable sequence divergences (An interesting observation made during the analysis of the RPT domain sequences was that some species had near-perfect repeating protein sequences. For example, the RPT domain of zebrafish (ergences A. We theergences B. For thOne might speculate that these near-perfect repeat units confer an evolutionary advantage to the species examined or that imperfect repeats confer an evolutionary disadvantage. However, a far greater number of sequences from individual animals than are currently available would be required to test this hypothesis. Despite this divergence, these three species did have regularly spaced D/E ladders within all of the repeats, separated by speciation events. The PKAT family of genes are linked by a common ancestor and share a common domain architecture. However, the functional roles of PMEL and GPNMB have few commonalities, whereas practically nothing is known about the most ancient member, TMEM130. In this manuscript, we investigated the commonalities in the mutant phenotypes, evolutionary origin and functional domains between GPNMB and PMEL and analyzed the major difference: the RPT domain. Furthermore, we established that TMEM130 is well-suited to serve as an outgroup for future analyses comparing PMEL and GPNMB functions. We hope that this analysis assists with improving the understanding of the PKAT family and encourages further investigations into their evolution and diversification to perform such disparate functions.


Brain states such as arousal and sleep play critical roles in memory encoding, storage, and recall. Recent studies have highlighted the role of engram neurons–populations of neurons activated during learning–in subsequent memory consolidation and recall. These engram populations are generally assumed to be glutamatergic, and the vast majority of data regarding the function of engram neurons have focused on glutamatergic pyramidal or granule cell populations in either the hippocampus, amygdala, or neocortex. Recent data suggest that sleep and wake states differentially regulate the activity and temporal dynamics of engram neurons. Two potential mechanisms for this regulation are either via direct regulation of glutamatergic engram neuron excitability and firing, or via state-dependent effects on interneuron populations–which in turn modulate the activity of glutamatergic engram neurons. Here, we will discuss recent findings related to the roles of interneurons in state-regulated memory processes and synaptic plasticity, and the potential therapeutic implications of understanding these mechanisms. This phenomenon is easily observed using immediate-early gene (IEG) expression , as a readout measure. Principal neurons in both neocortex and hippocampus also undergo ultrastructural and intracellular molecular changes in the hours to days following learning  promoter  was more active when mice were discriminating between the CFM context and a similar context during recall. They also found that chemogenetic suppression of activity in the Npas-expressing population (but not the Cfos-expressing population), disrupted context discrimination. Conversely, inhibition of the Cfos-expressing engram population (which received less inhibition) increased contextual fear discrimination, and activation of this population increased contextual fear generalization , one would be forgiven for concluding that memory encoding, consolidation, and recall are the domain of glutamatergic circuits. However, GABAergic interneurons–the main source of inhibition in the hippocampus and neocortex–also play a critical role in mnemonic processing. Indeed, one of the IEGs described above, ectivity . A more not both . Intrigulization . These fGABAergic interneurons represent about 15–20% of total neurons in hippocampus and neocortex, and are highly heterogeneous. Various interneuron types have been classified based on anatomical location, structural morphology and biochemical properties . Within As mentioned above, a large proportion of GABAergic interneurons in both the neocortex and hippocampus are either PV+ or SST+. These two interneuron populations both provide strong inhibition to neighboring excitatory  neurons’ cell bodies/axon initial segments and dendrites, respectively . As discN-methyl-D-aspartate (NMDA) receptors in PV+ interneurons disturbs working, short-term, and long-term spatial memory  leads to impaired consolidation of multiple hippocampus-dependent forms of memory . A recenThis interpretation of our data is consistent with recent neuroanatomical findings from mouse neocortex in the context of consolidation of a recently learned motor task . The autArc’s messenger ribonucleic acid (mRNA) and protein expression is reduced in DG after a period of SD , we identified known clusters of co-regulated transcripts with abundance that varied in the hippocampus as a function of prior sleep and wake amounts. We found that two clusters of known co-regulated mRNAs are upregulated together on phosphorylated ribosomes, in proportion to prior wake time. We next compared these wake-driven, clustered transcripts with previously described cell type-specific transcript profiles, using cell type-specific enrichment analysis (CSEA). We found that mRNA markers of SST+ and NPY+ interneurons (but not VIP+ or PV+ interneurons) are selectively increased after SD  after sleep or SD. mRNAs which were associated with phosphorylated S6-containing ribosomes were sequenced from hippocampi of mice allowed after SD , as are after SD . Conversad lib sleep  during REM sleep . While bReactivation of neurons active during prior experience (in the hippocampus and other structures such as the neocortex) has been proposed as an essential component of systems-level memory consolidation . Such reThe function of reactivation/replay in memory storage (and as a mediator of sleep-dependent memory storage) has been a matter of debate . A majorcfos promoter drives expression of an estrogen receptor-fused CRE recombinase. Using this genetic tool, CRE-dependent expression of fluorescent proteins or opsins was targeted in sensory engram neurons in V1, by presenting the visual stimulus in close temporal proximity to administration of tamoxifen. After genetic targeting, these same neurons were activated in V1 in the context of fear conditioning, using the same visual stimulus as a repeated cue for foot shock. By expressing a fluorescent protein in these “visual engram” neurons and characterizing IEG expression in V1 following conditioning, we found that engram neurons are selectively reactivated during sleep-dependent consolidation of visually-cued fear memory. To test the functional role of this reactivation, we expressed the inhibitory opsin archaerhodopsin in the V1 engram neuron population. Inhibition of engram neurons in V1 during bouts of sleep in the first few hours following conditioning was sufficient to disrupt visually-cued fear memory consolidation. Together these data suggest that neurons encoding new associative memories are selectively reactivated during subsequent sleep, and that this sleep reactivation plays a necessary role in memory consolidation.While the role of hippocampal and neocortical experience-encoding neurons in memory recall or perception has been well described , very liOne plausible explanation for the preferential occurrence of reactivation and sequential replay during sleep states is that sleep oscillations appear to play a critical role in promoting their occurrence. Sequential replay events  have been reported to occur in the context of sharp wave-ripple events  and theta oscillations . In neocortical networks , sequential replay has also been observed , althougHow do replay and reactivation contribute to long-term memory storage? Recent work from our lab suggests that firing of learning-activated ensembles is sufficient to drive reliable downstream spiking in post-synaptic neurons . This poWhile the relationship between spike timing-dependent plasticity in neural circuits and memory consolidation is still a matter of speculation , availabInterneurons in both the hippocampus and in thalamocortical circuits play an essential role in regulating sleep oscillations. Within the hippocampus, PV+ interneurons in CA1 are essential for the generation of both theta oscillations  and rippParvalbumin interneurons in the neocortex also play a role in coordinating sleep oscillations. For example, chemogenetic activation of motor cortex PV+ interneurons suppresses NREM slow wave activity (and other oscillations), but, intriguingly, simultaneously augments REM oscillations . On the Beyond this, PV+ interneurons in other brain regions are essential contributors to sleep oscillations present in both the hippocampus and neocortex. For example, generation of spindles in NREM is critically dependent on PV+ interneurons in the thalamic reticular nucleus , which ain vivo recordings have demonstrated that some subclasses of SST+ interneurons show generally suppressed firing during sleep relative to wake, and are further suppressed during the occurrence of sharp wave-ripples  . Criticaafter SD . This nNafter SD . A seconafter SD , these nafter SD .As mentioned above, interneurons appear to be important not only for coordinating oscillations within neocortical columns or hippocampal subregions, but across the extent of both structures, and even between neocortex and hippocampus. Because the spatial coordination of certain oscillations–such as hippocampal-cortical ripples or neocortical slow waves–is so extensive, it is tempting to speculate about how such synchrony is possible. While coordination via cortico-thalamo-cortical loops appears to play a critical role , direct While the role of interneurons in regulating sleep-associated network oscillations may be critical for memory storage, recent work has highlighted other, additional potential roles for interneurons in the process of consolidation. There is considerable evidence that sleep (and sleep loss) modify excitatory/inhibitory balance in neural circuits. Both electrophysiological  and moreOur recent work in the hippocampus likewise demonstrates that that excitatory/inhibitory balance is dramatically affected by brain state. Over the course of a few hours of SD, SST+ interneurons in the hippocampus become selectively activated, suppressing activity in surrounding DG granule cells and CA1 and CA3 pyramidal neurons . As mentex vivo and in vivo neocortex, SST+ interneuron activation during rhythmic upstates plays a significant role in constraining pyramidal neuron firing  partially understood, and a better understanding of these mechanisms would profoundly impact the targeting of therapeutic strategies. Critically, impaired functioning of GABAergic interneurons has been implicated in several neurodegenerative and neuropsychiatric disorders .Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and one of the most common causes of dementia. Although the underlying mechanisms of AD are still not completely understood, disrupted interneuron function has been associated with AD . For exaNpas4 expression to the prefrontal cortex of mice with schizophrenia-associated 16p11.2 microduplications led to a rescue of neuronal excitatory/inhibitory balance, and behavioral phenotypes, in this mouse model  is another multifaceted condition, typically characterized by low mood, anhedonia, and cognitive deficits including attention and memory problems . Sleep dAltogether, the available data suggest that interneurons likely play an important role in the development or progression of many psychiatric disorders, and that these may be mediated in part through effects on sleep oscillations and sleep-dependent circuit plasticity.Available data suggest that interneurons play vital roles in gating and timing the activation of engram neurons in the context of memory encoding. Our recent data suggest that they likely play similar roles during the process of memory consolidation–either permitting or suppressing reactivation of these memory-encoding neurons during subsequent sleep. Beyond this, PV+ and SST+ interneurons regulate sleep oscillations that play a vital role in sleep-dependent memory consolidation. Recent data from our lab and others indicated that hippocampal memory consolidation is highly sensitive to post-learning manipulations of interneuron activity. Future studies will be needed to clarify what aspects of NREM and REM sleep regulate the seemingly distinct roles PV+ and SST+ in consolidation, as well as how interneuron populations contribute to communication across various brain structures during sleep. Because interneurons are so functionally and structurally diverse, a better understanding of their subtypes and interactions across brain states is also needed. Finally, it will be important to understand how biological factors such as age, sex, and stress–which can alter both sleep and cognition–contribute to interneuron-mediated regulation of memory processing. An understanding of these mechanisms will have broader implications for our ability to diagnose and treat neuropsychiatric disorders which are associated with both sleep and cognitive disruption, as well as underlying pathological changes to interneurons.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Although it is generally accepted that physical activity reduces the risk for chronic non-communicable disease and mortality, accumulating evidence suggests that occupational physical activity (OPA) may not confer the same health benefits as leisure time physical activity (LTPA). It is also unclear if workers in high OPA jobs benefit from LTPA the same way as those in sedentary jobs. Our objective was to determine whether LTPA and leisure time sedentary behaviour (LTSB) confer the same health effects across occupations with different levels of OPA.Searches were run in Medline, Embase, PsycINFO, ProQuest Public Health and Scopus from inception to June 9, 2020. Prospective or experimental studies which examined the effects of LTPA or LTSB on all-cause and cardiovascular mortality and cardiovascular disease, musculoskeletal pain, diabetes, metabolic syndrome, arrhythmias and depression among adult workers grouped by OPA  were eligible. Results were synthesized using narrative syntheses and harvest plots, and certainty of evidence assessed with GRADE.The review includes 38 papers. Across all outcomes, except cardiovascular mortality, metabolic syndrome and atrial fibrillation, greater LTPA was consistently protective among low OPA, but conferred less protection among moderate and high OPA. For cardiovascular mortality and metabolic syndrome, higher levels of LTPA were generally associated with similar risk reductions among all OPA groups. Few studies examined effects in standers and none examined effects of LTSB across OPA groups.Evidence suggests that LTPA is beneficial for all workers, but with larger risk reductions among those with low compared to high OPA jobs. This suggests that, in our attempts to improve the health of workers through LTPA, tailored interventions for different occupational groups may be required. More high-quality studies are needed to establish recommended levels of LTPA/LTSB for different OPA groups.CRD42020191708.PROSPERO #The online version contains supplementary material available at 10.1186/s12966-021-01166-z. Physical activity and sedentary behaviour play key roles in the prevention of non-communicable chronic conditions and mortality –3. AlthoThe postulated health sequelae of OPA are attributed to postures and movement patterns undertaken during occupational tasks  , that arThe primary objective of this systematic review was to determine whether LTPA confers the same health benefits among workers with different levels of OPA. The secondary objective was to examine the health effects of leisure time sedentary behaviour (LTSB) across OPA levels.https://osf.io/5angw/), and adheres to the PRISMA statement [This review was prospectively registered with PROSPERO CRD42020191708) and documented on Open Science Framework (1708 and Adult workers (mean age: 18–65 years) with adequate information on OPA, who were generally healthy and did not report the outcomes of interest at baseline , except if baseline values were controlled for in the analyses.The primary exposure was LTPA. Physical activity is defined as “any bodily movement produced by skeletal muscles that results in energy expenditure”  > 1.5 meTo meet the second objective studies were required to provide a quantification of LTSB. Sedentary behaviour includes non-sleeping activities undertaken at ≤ 1.5 metabolic equivalents while sitting, lying or reclining . Also, aAll-cause and cardiovascular mortality, diabetes, metabolic syndrome, arrhythmias, cardiovascular disease, musculoskeletal pain, and depression.Intervention/experimental, retrospective or prospective cohort studies, and case-control studies. Cross-sectional studies were excluded as they were unable to assess some level of causation.Publications in English, French, Danish, Norwegian, or Dutch were eligible based on authors’ language capacity. Published studies were eligible provided they included sufficient information. All literature, regardless of date of publication, was considered.The search strategy was created by a research librarian (KM) in collaboration with other members from the authorship team. The search was first created in Medline using a combination of index terms/unique subject headings and keywords related to occupations, physical activity, sedentary behaviour, study designs, and adult populations. After trial searches, search strategies were modified to ensure that pre-identified key papers were captured. Once the strategy was finalized   and modiy graphs . Althouglow OPA/sitters: tasks largely conducted while sitting with little OPA; (2) standers: tasks largely conducted while stationary standing; (3) moderate OPA/intermittent movers: tasks largely involving frequent postural changes (e.g. sitting to standing) with low-intensity OPA and without heavy laborious tasks; and, (4) high OPA/heavy labourers: tasks involving moderate-to-high intensity OPA and may include carrying/lifting/pushing/pulling heavy loads, and extensive walking. From henceforth, we refer to the four groups as low OPA, standers, moderate OPA, and high OPA.Classification of OPA was based on the study’s description of physical job demands, job title and/or self-reported or device-based OPA. OPA for each study was assigned by one independent reviewer and, once completed; a second reviewer verified for accuracy. Workers were grouped, where possible, based on their most frequent occupational tasks and OPA into the following four groups: (1) p < 0.05) where possible [Standardized data extraction forms were developed and completed using Google Forms. One reviewer independently extracted data and a second independent reviewer verified the accuracy of the extractions. Effect estimates  for the association between LTPA or LTSB and each outcome were extracted and included results from fully adjusted models. A narrative synthesis summarizes and describes trends in risk associated with lower compared to higher quantities of LTPA across all outcomes within each of the OPA groups. Although a meta-analysis was initially planned in the protocol, it was precluded due to much heterogeneity between studies resulting from differences in populations, measures of LTPA and OPA, and reference and comparison categories used in analyses. Harvest plots display the data visually in terms of risk (higher/lower/no risk) associated with LTPA among the OPA groups, and are based on statistical significance  approach adapted for narrative syntheses , 29. TheThe search of electronic databases identified 7123 potentially relevant papers  from the past 10 years. Fifteen countries were represented, with most papers reporting on data from Finland (26%) and Denmark (26%); two studies , 64 captRisk of bias is summarized in Supplemental Fig. Among cohort and case-control studies, 20% had a high risk of selection bias, largely due to convenience samples limiting generalizability of findings; for another 20% the risk of selection bias was unclear. Approximately 30% had a high risk of performance bias due to the use of self-reported LTPA measures that were non-validated, an additional 25% were unclear. The majority (80%) had a low risk of detection bias as most outcomes were assessed objectively using medical records and registries. Most had a low risk of attrition bias with < 20% loss to follow-up. For half of the papers, it was unclear if selective reporting bias was present due to a lack of clarity on whether all available data were reported. More than half of the papers had a high risk of ‘other’ biases largely attributed to a lack of adjustment for the potential for reverse causality.Eleven papers (9 studies) of variable quality, examined the interaction between OPA and LTPA and all-cause mortality using prospective cohorts Fig. . Among lOne study examined the interactive effects of LTSB and OPA on all-cause mortality. This study only included low OPA; therefore, comparisons across OPA groups could not be made.Ten papers  of moderate-to-high quality examined the interaction between OPA and LTPA and the risk of cardiovascular mortality Fig. . RegardlEleven papers , examined the interaction between OPA and LTPA and cardiovascular disease Fig. . All stuEleven papers  examined the interaction between OPA and LTPA and the risk of musculoskeletal pain Fig. . Six papOne high quality cohort study assessed the association between LTPA and OPA for diabetes risk . A signiOne moderate quality cohort study assessed the interaction between LTPA and OPA and the risk of metabolic syndrome . No statOne low quality cohort study examined the risk of depression . Among lOne high quality cohort study examined the risk of arrhythmias; specifically atrial fibrillation . There wThis systematic review offers insight into whether LTPA confers the same health benefits for workers with different OPA levels. Across various health outcomes , greater LTPA was consistently protective among low OPA, but conferred less protection among moderate and high OPA. However, no clear statements can be drawn due to the relatively few studies and low-to-very low certainty of evidence. The most consistent evidence was found for cardiovascular mortality and metabolic syndrome, showing a protective effect of LTPA across the OPA groups. No evidence was available regarding health risks from LTSB across OPA groups. These findings support the need for more studies investigating the effects of LTPA and LTSB on health risk among workers with different OPA levels. Moreover, while there is little doubt that LTPA is beneficial for workers in sedentary jobs, we still lack high quality evidence to give recommendations of LTPA among workers in jobs involving moderate and high OPA levels or standing.Previous systematic review findings complement those observed in the present review, suggesting that LTPA confers a stronger independent protective effect compared to OPA for type 2 diabetes , cardiovThere appeared to be differences between men and women on the effects of LTPA on cardiovascular disease, especially among high OPA. Differences may be attributed to a mix of sex and gender-specific considerations such as: differences in the tasks or occupational exposures of men and women in the same jobs/workplaces; effects and exposure to toxic workplace substances; hormonal and other factors implicated in the development of cardiovascular disease; employment status and hours worked; interactions between tasks undertaken at work and home; or, high OPA representing different types of occupations with different types of demands for women . It is aPhysical inactivity is among the most recognized modifiable risk factors for the maintenance of good health and longevity . RecentlWe found very few studies examined the health effects of LTPA on workers with prolonged occupational standing. Prolonged occupational standing has been associated with muscle and psychological fatigue , 80, lowOur findings support that LTPA is essential to the overall health of workers. One of the ways in which physical activity promotes better health is through its ability to improve cardiorespiratory fitness . While LThe strengths of this review include a very rigorous approach, an a priori protocol, a comprehensive and peer-reviewed search strategy, the use of harvest plots that considered both statistical and clinical significance, and the use of GRADE to assess the certainty of evidence. The review was, however, limited to studies where occupations could be categorized into one of the predefined OPA groups. Secondly, the measures of OPA and LTPA were all self-reported and varied widely. This led to a large amount of heterogeneity in the OPA and LTPA groups and likely misclassified physical activity intensity. Thirdly, while we did include data from fully adjusted models, it is possible that residual confounding remains, either because of misclassification within considered confounders, or from other factors that were not considered. Although our inclusion criteria required a longitudinal or experimental design to assess some degree of causality, the assessment of OPA and LTPA at one point in time may not accurately capture lifetime exposure. We attempted to reduce this bias by including a requirement for the analysis to adjust for reverse causality by removing at least the first 2 years of follow-up data. Unfortunately, most prospective studies did not undertake this approach. Lastly, most of the literature has come from Scandinavian and Western European countries. A recent study explored domain-specific physical activity and found a wide variation in levels of LTPA and OPA across countries ; it is pLow certainty evidence from this review suggests that LTPA is overall beneficial for all workers, though the magnitude of benefit may depend on OPA levels. Across most health outcomes , greater LTPA was consistently protective among low OPA, but conferred less consistent protection among moderate and high OPA. For cardiovascular mortality and metabolic syndrome, we generally found more consistent evidence for a protective effect of LTPA across all OPA groups. These findings suggest that LTPA interventions may need to be tailored based on the OPA level of workers. We still lack high quality evidence to provide specific LTPA recommendations for workers with different types of occupational physical demands .Additional file 1.


Mast cell disease is an epigenetically and genetically determined disease entity with very diverse clinical manifestations in potentially every system and tissue due to inap pro priate release of variable subsets of mast cell mediators together with accumulation of either morphologically normal or altered mast cells. Easy bruising, excessive bleeding, and aberrancies of erythropoiesis can frequently be observed in patients with mast cell disease. A thorough history, including a family history, will guide the appropriate work-up, and laboratory evaluations may provide clues to diagnosis. In recent years, our understanding of the involvement of coagulation and anticoagulant pathways, the fibrinolytic system, and erythropoiesis in the pathophysiology of mast cell disease has increased considerably. This review summarizes current knowledge of the impact of the disturbed hemostatic and erythropoietic balance in patients with mast cell disease and describes options of treatment. Mast cells (MCs) are hematopoietic tissue immune cells that act both as effector and regulatory cells  in adapPrimary MC disease comprises a group of historically defined different disease entities  and cellular components  as well as clotting and fibrinolytic factors  system by thrombomodulin-bound thrombin . The PC/MC tryptase could have opposite effects on hemostasis as both an anticoagulant and a procoagulant (see below). The antithrombotic function is mainly due to the ability of tryptase to proteolytically degrade fibrinogen, predominantly by cleavage of the C-terminal α-chains before thrombin can convert fibrinogen to fibrin and subsequently impair fibrin polymerization , should be carried out for diagnostic reasons  and befoSince MCs represent the main source of endogenous heparin , it is iAt first, baseline heparin (anti-Xa) levels should be sampled. For proving MC-mediated release of heparin, as a provocation test venous occlusion of the upper arm for 10 minutes  can be performed, using a blood pressure cuff inflated 10 mm Hg above diastolic pressure. This standardized mild non-pharmacological test might be able to stimulate MC activation and degranulation by hypoxia and increased compartment pressure ,27,48,49Hyperfibrinolysis in MCAS patients with bleeding diatheses can be identified by increased levels of tPA and of plasmin–antiplasmin complexes (PAP)  using th1 blocker or cromolyn for epistaxis, or a vaginal douche or suppository of such drugs for menorrhagia [In the absence of placebo-controlled trials, hemostatic treatment of mild bleeding in MC disease patients is based only on clinical evidence. Before and after surgeries, MC activity should be reduced as much as possible by medications and other maneuvers  ideally determined at an earlier, non-emergent point to be ideal for this task. Of note, though systemic medications may be sufficient, sometimes topical medications may be needed, or at least more helpful, for controlling bleeding in topically accessible sites . Routinorrhagia , so alteAntifibrinolytic drugs such as TXA or epsilon aminocaproic acid (EACA) often are effective due to several mechanisms: (I) One of the major bleeding signs in patients with MC disease is mucocutaneous bleeding from local hyperfibrinolysis in well-vascularized tissues, e.g., endometrium, bladder, gums, or regions of the ear, nose and throat.(II) Antifibrinolytics target endogenous fibrinolysis, which is involved in increased pathologically irritable MC activation. The synthetic lysine-analogue TXA and also EACA competitively block the binding of plasminogen to fibrin via its lysine-binding sites and, therefore, inhibit the tPA-mediated activation of plasminogen to plasmin . The bin(III) Given the link between MC-mediated inflammatory response and increased fibrinolysis, TXA feasibly attenuates the inflammatory response. This possible anti-inflammatory effect of TXA has been demonstrated in randomized controlled trials in cardiac surgery, measuring biomarkers  and cardiac markers , respectively ,54.(IV) Finally, TXA may inhibit the complement system, as has been observed in patients with hereditary angioneurotic edema , e.g., bDepending on the clinical circumstances, TXA can be administrated topically, orally, or intravenously. The risk of arterial or venous thromboembolism using TXA remains unclear but appears to be low with the dosages usually applied. TXA should be administrated with caution in patients with renal impairment to treat bleeding of the urinary tract because the ureter does not provide tPA release intraluminally. Thus, in the case of urinary tract bleeding, the fibrinolysis of clots might be irreversibly blocked by TXA leading to ureter and/or urethral obstruction.In MC disease patients with vitamin K deficiency, vitamin K could be applied orally in high doses (5–10 mg p.o.) or intravenously in case of disturbed absorption. Thus, vitamin K substitution should be considered in  patients with severe bleeding symptoms and/or patients undergoing surgery with a high bleeding risk.In case of very high plasma heparin levels and clinically overt bleeding, the application of protamine chloride to neutralize heparin should be considered. Protamine chloride is derived from salmon melt and is used to reverse anticoagulation with unfractionated heparin (UFH) and—less effectively—low-molecular-weight-heparin (LMWH). However, adverse reactions to protamine, such as hypotension, might be related to the release of inflammatory mediators, including histamine, by MCs. Therefore, protamine use should be limited to MC disease patients with life-threatening bleeding in presence of measurable endogenous heparin. In these situations, heparin could be neutralized by protamine titration to the patient’s heparin levels. In rare cases of adverse protamine effects, administration of recombinant activated factor VII (rFVIIa) might be used as an alternative in case of life-threatening bleeding. Apart from the possibility of bleeding, in patients with MC disease, LMWH, UFH or fondaparinux as thromboprophylaxis should not be avoided when thromboprophylaxis is required because of internal or surgical treatment.In parallel with inducing a bleeding diathesis, MCs are thought to contribute to venous thrombo embolism and atherosclerosis ,58 throu1 histamine receptor antagonist rupatadine may theoretically be the best choice of such antagonists for MC disease patients with thrombotic tendencies because it also inhibits PAF.For reducing the risk of thrombosis and bleeding in patients with primary MC disease in their everyday lives, MC activation should be reduced as much as possible by the profile of medication found best in the individual patient. Given the extreme combinatorial epigenetic and genetic complexity of primary MC disease, resulting in extreme interindividual heterogeneity in aberrant constitutive and reactive mediator release and thus extreme heterogeneity in clinical presentation, it is unsurprising that the optimal treatment profile is unique to the individual patient, and it also is unsurprising that a very large array of treatments has been found helpful in various MC disease patients . Of noteAbnormalities in quantity or function of any of the molecules (many of which can be found in the large repertoire of mast cell mediators) directly or indirectly affecting any of the many steps in the erythropoietic (and red cell degradation/recycling) processes have potential for driving not only abnormal quantities (high or low) of circulating erythrocytes but also abnormal erythrocytic qualities. .As one could accurately say about the development of any cell population in eukaryotic organisms, normal erythropoiesis is the result of an exquisitely choreographed ballet of a great many distinct molecules, in the case of erythropoiesis resulting in production of not only normal structured individual erythrocytes  but also normal numbers of such erythrocytes , which then are released from the marrow into circulation at rates appropriate to the prevailing physiologic circumstances in the body—and later (on average a bit more than three months after emergence from the marrow) are normally decomposed (with subsequent recycling of component molecules).Given the huge menagerie of mediators produced and released by the MC, and with most MC mediators naturally driving a wide range of direct and indirect, local and remote effects, the range of symptoms which potentially can be produced by chronic aberrant MC activation is vast. Yet, three general themes become apparent on study of the matter: inflammation, allergic-type phenomena, and dystrophisms —and though various MC disease patients may have highly varying extents of allergic and dystrophic issues, chronic multisystem inflammation clearly is the “universal constant” of MC disease. Depending on degree and chronicity, inflammation usually drives relative or even absolute anemia (the latter seen in about two-thirds of MCAS patients ). HoweveAlthough palpable splenomegaly is not uncommon in systemic mastocytosis, it is uncommon to find splenomegaly detectable by either palpation or imaging in MCAS patients . As such, sequestration is rarely a mechanism of anemia in MCAS. Hemolysis, too, appears to be uncommonly driven by MC disease, though of course MC disease does not prevent other hemolytic ailments from emerging independently of a patient’s MC disease. MC disease seems to have some propensity to spur the humoral immune system to errantly/inappropriately produce a wide variety of antibodies , and as such antibodies usually are randomly targeted, most probably are not detectable by assays for specific antibodies, and many of the detected antibodies clearly  are not “on-target” antibodies reflecting the true presence of infectious or autoimmune diseases—but occasionally such an errantly produced antibody has sufficient specificity to drive a clinically apparent autoimmunity, such as an autoimmune hemolytic anemia, an anti-phospholipid antibody syndrome (driving thrombosis or bleeding), or autoimmune rheumatologic, endocrinologic, or neurologic diseases. Standard treatments for such diseases are needed, but concomitant control of the MC activation may yield even better outcomes (as is sometimes seen in the setting of SM with associated hematologic neoplasms (SM-AHN) and in MCAS-associated cancers, too ).1 ± H2 histamine receptor antagonists) not uncommonly is needed prior to parenteral iron treatments. In addition, intravenously applied iron is stored in the reticuloendothelial system for long periods of time. Ergo, if iron is activating a MC disease patient’s dysfunctional MCs, this activation will persist for a long period. MC disease patients often react to medication product excipients, so a patient’s failure to tolerate one iron oral or parenteral iron supplement does not ensure intolerance of other supplements mixed with different excipients. Organic oral iron polypeptide formulations sometimes are better tolerated than inorganic iron salts . Occasionally, appropriately matched transfusion of packed washed leukocyte-poor red cells is the only way to tolerably address iron deficiency . In this context, however, although transfusion therapies usually are well tolerated in MC disease patients, one must also consider that transfusion therapies may elicit immunological effects which are able to activate MCs, thereby aggravating MC disease.Although inflammation-driven anemia  is expected and common in MC disease, other (MC activation-driven and non-MC activation-driven) causes of anemia can also be present and should be suspected and further evaluated when red cell indices seem inappropriate for an inflammation-driven anemia vis-à-vis the extent of clinically apparent inflammation. With regard to microcytic anemias (seen in 24% of MCAS patients ), iron dMoreover, therapy-refractory anemia may be induced by MC disease-induced kidney disorders  that mediators inappropriately released specifically from proximal luminal gastrointestinal tract MCs were (directly or indirectly) intensively inhibiting erythropoiesis. In another unreported example highlighting the diversity of MC mediators and their effects, author LBA identified an MCAS patient also suffering both sickle cell anemia and dialysis-dependent end-stage kidney disease resulting in unusually severe anemia occasioning transfusion dependence despite high-intensity erythropoietin treatment—and in whom treatment with montelukast for chronic mild asthma quickly resolved the excess anemia and the transfusion dependence. Additionally, in yet another example [Sometimes, specific proximate causes of anemia or polycythemia can be identified, but then the typical roots of those proximate causes cannot be found , in which case the possibility that MC activation may be the underlying issue needs to be considered. For example, idiopathic pure red cell aplasia (PRCA) has been reported in association with MCAD  is common in MCAS (about 29% of such patients ), and thSince MCAS is common, most aberrant erythropoiesis in primary MC disease  is due to aberrant MC activation. However, a tiny fraction of the total MC disease population suffers substantial MC neoplasia, and though aberrant MC activation in some fashion, to some extent, is “part and parcel” of every neoplastic MC disorder, it must be remembered, too, that high marrow tumor burdens observed in some types of MC disease  of course can further contribute to anemia from simply physically compromising the amount of space in the marrow available for erythropoiesis. In such cases, therapies which are able to reduce MC numbers in the bone marrow  are indicated.Disturbances of hemostasis and erythropoiesis often occur in primary MC disease and may or may not be consequential to the MC disease. Specific cause(s) of any given such disturbance should be sought and specifically treated, which may require MC-specific or non-MC-specific interventions. Prophylactic interventions may be warranted in certain  circumstances.