Supplemental Digital Content is available in the text. The promotion of physical activity is inextricably dependent on the environment. This systematic review summarizes studies that used geographic information systems to account for the role of geographic features in the design, implementation, or evaluation of interventions that promoted physical activity.Pubmed, ProQuest/PsycInfo, and Cochrane Library were searched for physical activity interventions that employed geographic information systems. The search was conducted with an algorithm that included 10 geographic, 28 intervention, and 9 physical activity search terms. Data were systematically reviewed using a standardized form based on the PICOS framework . Quality of the studies included was independently rated on 14 criteria (Registration number CRD42016046011).Search yield 12,518 published articles, of which, 19 studies satisfied our inclusion criteria. Proximity to recreational areas and neighborhood walkability were the most common geographic factors measured in studies of physical activity interventions. Interventions focused on supporting physical activity by providing recreational spaces and adequate infrastructure to participants. Fifteen intervention studies assessed socioeconomic environmental factors along with physical environmental factors. Support groups were introduced by 4 of the interventions to offset social environmental barriers in the geographical environment. Given the lack of consistency in measurements of physical activity, and long-term assessments, it was not possible to determine if findings are attributable to the geographic environment or a novelty effect.More research is needed to better understand the physical and social factors within the geographic environment that work as barriers or facilitators of physical activity changes. The identification of geographic variations in health disparities suggests that physical activity cannot be solely attributed to individual factors. Efforts to prevent chronic diseases and reduce health disparities need to consider geographic barriers and opportunities for healthy lifestyles.,7Increasing physical activity, particularly among the most inactive and vulnerable populations is a public health priority listed in the “Global Noncommunicable Diseases Action Plan 2013–2020” and “Healthy People 2020.” Geographic physical and social characteristics of disadvantaged neighborhoods including lacking sidewalks and green areas, traffic safety, limited availability of healthy foods, land use, urban sprawl, and housing insecurity have been linked to increased rates of noncommunicable diseases and health disparities.–6,9–12 These geographic attributes have been identified by several cross-sectional reviews as key barriers for physical activity.,12,13 Yet, the benefits of using geographic information to guide physical activity interventions and empirical connections between changes to geographical environments and physical health outcomes are scarce.Physical activity is inextricably dependent upon features in the geographic environment. In this earlier review, Sallis et al concluded methodological difficulties hindered the implementation of environmental approaches in physical activity interventions. Since then, technological advances have made geographic software programs more accessible for health practitioners. These improvements have enable researchers to analyze the influence of geographic factors on physical activity implementing diverse tools to layer geographic physical and even social information.An earlier systematic review that examined the effects of environmental and policy interventions aimed at increasing physical activity encouraged researchers to use objective measurements of the environment to account for geographic effects. Unfortunately, beyond epidemiological and cross-sectional studies, the application of geographic information through GIS or similar methodological approaches has been limited.From its inception, Geographic Information Systems (GISs) were designed to traverse a variety of disciplines. The purpose of GIS is to layer diverse types of data over a geographical map to illustrate relationships with data and locations. GIS ability to address the multifaceted and complex relationship between temporospatial variables lends itself well to the study of health. In health studies, GIS has been essential to track the location and movements of diseases and has been a major tool in the control and study of communicable disease outbreaks.In this review, we aim to systematically examine the implementation of geographic programs or GIS to include geographic information in the design, implementation, or evaluation of interventions that promoted physical activity. For the purpose of this review, geographic information will refer to any objective or subjective assessment of environmental factors with a spatial location, which can support, moderate, mediate, or inhibit physical activity. Whenever we are referring to objective measurements, we are including all measurements of physical activity or geographic information collected using a given instrument , to systematically calculate the repetition of a given unit . In contrast, when we refer to subjective report tools, subjective instruments, or subjective measurements, we include assessments that relied on individual’s perceptions and recall abilities. Instead of being directly measured, subjective assessments are collected from individual-reporting using questionnaires which may or may not have undergone validation. Geographic factors examined as geographic information also include any changes in the built environment and any environmental factors for which location is assessed and considered to influence participation in physical activity.The aim is to summarize the different approaches employed to operationalize geographic information in the promotion of physical activity. Findings from this review introduce empirically tested approaches that we hope will serve as a resource that informs clinicians, researchers, and stakeholders about options to benefit from geographic information.The protocol for this systematic review is registered in PROSPERO International Prospective Register of Systematic Reviews (Registration number CRD42016046011). Upon completion of the piloting of the study selection process, the protocol was amended to include participants of all ages, and variuos programs, including GIS, which have been used to examine geographic information.http://links.lww.com/PP9/A4Supplemental Digital Content 1, ). Whenever possible, we limited our search to: (1) studies published after January 2000 , which triggered the proliferation of diverse tools that enable researchers to analyze the influence of geographic factors on physical activity) and (2) human species only. The third database was selected to include bibliographies from similar systematic reviews. Bibliographies from similar systematic reviews were hand searched to identify additional studies that satisfied inclusion criteria. We only included publications available in (3) English and (4) published in peer-reviewed journals. Editorials, policy briefs, letters, and commentaries were excluded.This literature review examined scientific articles describing or evaluating physical interventions published before April 2017. The review began with a systematic search in 3 databases: Pubmed, ProQuest/PsycInfo, and The Cochrane Library . The first 2 databases were selected because of their wide breath of health information, and because our aim was to introduce this review to health professionals who may not necessarily be geography or urban planning experts, but are interested in uses of geographic information to promote physical activity. The search was conducted with an algorithm that included 47 search free text and Medical Subject Headings. Our search included articles that resulted from any combination of 3 terms, one term from each search category: (1) “Geographic Information” (10 terms); (2) “Health/Wellness interventions” (28 terms); and (3) “Physical Activity” (9 terms) ; and use any geographic software or similar methodology to consider geographical information in the design, implementation, or analyses of the physical activity intervention. For eligibility purposes, geographic information referred to the assessment of the spatial location of any objective or subjective factors considered to support, moderate, mediate, or inhibit physical activity.One researcher completed an initial screen of article titles. The title review yielded 1614 references that fit the inclusion criteria or were otherwise unable to be deemed irrelevant based on the title alone. Examples of studies eliminated in this stage included cross-sectional studies, animal studies, or studies not available in English.http://links.lww.com/PP9/A5Supplemental Digital Content 2, ).In the next step, 2 researchers screened 1614 references independently based on abstracts and methodology sections. Studies were reviewed based on methodology, specifically to ensure that geographic information was included. Systematic reviews were examined to determine if there were any additional studies that satisfied inclusion criteria. From these steps, researchers identified 34 studies that satisfied the inclusion criteria. Upon completion of the title, abstract, and methodology review, an agreement rate of 85% (28/33) was achieved among 2 reviewers. A third reviewer was included to complete full-text reviews of the 33 studies identified, and limit biases introduced by reviewers. Discrepancies were resolved by consensus among all reviewers. The final list included 19 studies , study design, comparison group, outcomes, geographical factors reviewed and tools used to approach geographical environments, outcomes attributable to geographic information, length of intervention, and duration of follow-up. If any information needed to complete the standardized form was not available in the selected manuscripts, additional publications were reviewed, and if information was not found, or other publications were not available, corresponding authors were contacted.A standardized form was created based on the PICOS framework  to systematically extract data.We used 4 different criteria to examine the different approaches to geographic information in physical activity interventions. First, we examined whether studies: (1) measured or (2) modified factors in the geographic environment, and then whether the interventions’ interactions with the environments addressed (3) primarily physical factors, or (4) if the location of social factors was also considered.,17To assess the quality of the studies included in this review, interventions were independently rated on 14 criteria by 2 researchers. The rating scale developed was adapted from the Cochrane review tool, and from a scale designed to assess study quality of public health intervention studies.Criteria scored included: description of study aims, details of target population, report of attrition rates, description of the context where the intervention took place, description of field work, duration of the intervention, follow-up assessments and length of follow-up assessment, use of objective data to assess physical activity, use of subjective instruments to assess physical activity, use of validated assessment of physical activity, detailed description of data analyses, power analyses considerations, tailoring intervention to participants settings or culture, and acknowledgment of limitations. An additional point was added to the quality score of each study for additional follow-up assessments and for detailed descriptions of the intervention field work. Possible scores ranged from 0 to 17, with higher scores representing higher quality of evidence reported.To reduce risks of bias in the review and quality assessment, 2 reviewers worked independently, and agreement was assessed by a third reviewer. Discrepancies were discussed in consensus with the third reviewer until agreement was reached among all reviewers.,29,32 these studies were included because their methods to measure physical geographic features were described in detail, and presume the use of a geographic program or introduce a methodology that can be replicated using geographic information systems.A total of 19 studies meet inclusion criteria for this systematic review  examined subjective physical and social geographic barriers and facilitators of physical activity.,32,36,37 Three interventions increased physical activity by modifying the infrastructure through construction projects .,30,34 Other physical activity interventions implemented physical infrastructure modifications through policy changes, which impacted the geographic environments by providing temporary access to recreational spaces. Examples of these policy approaches included “Play Streets,” and government-sponsored swimming sessions.,28,32,36Interventions that focused primarily on physical geographic factors aimed to provide spaces to encourage physical activity.,32,33,36 Of the 13 interventions that considered the influences of social factors, 9 interventions (42%) implemented strategies to limit the negative impact of social barriers on physical activity .–27,29,32 Six of these studies that modified social factors, organized trainer-led activities to enhance social support among the participants.,27,29,32 Most of the interventions that enhanced social support yielded positive improvements in physical activity,,30,32,38 but not all.Thirteen interventions included in this review (68%) used a geographical approach to investigate social factors that may influence physical activity .,29,32,36 and 2 interventions complemented their assessments by analyzing physical geographic factors with the location of neighborhood crime.,22We identified 8 interventions that assessed both, social and physical geographic factors Table . Proximi,38 introduced police-patrolled walks as an innovative approach to effectively offset the objective and subjective safety concerns previously identified as an environmental barrier to physical activity in the target community.,38 Although police-patrolled walking interventions introduce an innovative approach to address concerns of neighborhood safety, participation in walking interventions was only marginally affected by crime-related safety approaches.,38All interventions that modified social environmental factors to support physical activity did so by organizing support groups within worksites or the community settings. In addition to fostering a support group, Wilson et al,31,32,36 Five interventions promoted walking (26%),,27,29,33 3 interventions promoted walking and cycling (16%),,34,35 and 1 intervention promoted swimming (5%).Different modes of physical activity were promoted Table . Ten int,25–27,31 Seven interventions (37%) used self-reported, subjective assessments,,30,34–36 or proxy measurements, like attendance data.,28,29 A combination of objective and subjective measurements of physical activity was used in 3 studies.,32,33 Of the 9 studies that collected objective measurements of physical activity, 6 (25%) used accelerometers,–27,31,32 1 intervention (8%) used pedometers, 1 (8%) used Fitnessgram , and 1 used heart rate monitors. Subjective measurements of physical activity used included researcher observations with a validated tool, and different self-reported validated instruments that assessed frequency, intensity, and duration of physical activity. Studies that combined objective and subjective approaches relied on pedometers with self-reported 7-day recall, data collected using heart rate monitors and walking logs, and 3-day accelerometers data complemented by answers from self-reported questionnaire of children’s wellbeing.All studies measured physical activity, 6 (32%) used objective measurements. on multisector policy intervention, all interventions included had been administered and completed at the time of our review. Yet, 4 of the intervention studies reviewed had not yet published all available results,,25,27,37 and 2 had published only results of data collected during the pilot assessments.,32With the exception of one study,31,32,37 Eleven interventions (58%) targeted adults 18 years of age and older,–36 2 of which targeted older adults (8%).,38 Out of the 19 interventions, 9 (47%) included more than 1000 participants,–28,30,31 and 10 (53%) interventions included between 79 and 939 participants.,29,32–36There was great diversity among the populations targeted by the interventions examined Table . Eight i,26,29,36 Four of these interventions that targeted racial or ethnic minorities were conducted in the United States, 2 of them exclusively focused on African Americans,,22 and 2 on participants living in ethnically diverse disadvantaged neighborhoods.,23 A fifth intervention conducted in an ethnically diverse community took place in Cali, Colombia. Although the authors did not specify whether the neighborhood setting was economically disadvantaged, the program reported the inclusion of an ethnically diverse, socially disadvantaged community. The sixth intervention targeting a specific ethnic or racial group was conducted in Denmark. This study focused exclusively on schools where the majority of students were native Danish.Seven interventions (37%) focused on promoting physical activity among specific racial or ethnic groups. However, the analysis of this walking program demonstrated this physical activity intervention had limited participation from non-White minorities, and significantly less walks were offered in ethnically diverse and disadvantage areas.A national walking program implemented in the United Kingdom included a diversity of ethnic and racial groups.[ Eight of the interventions relied on a pre–post design,,32,34,35 and 3 used a post-hoc comparison between groups to examine intervention effects.,29,36 All studies (79%) collected quantitative data, 4 of these collected both quantitative and qualitative information (21%).,25,27,33 Seventeen interventions (89%) focused exclusively on increasing physical activity among participants,,27–36,39 with only 2 multicomponent interventions (11%) that aimed to increase physical activity and improve nutritional habits.,21A quasi-experimental controlled research design was utilized by 8 (42%) interventions.–33,36,39 Two interventions (11%) focused primarily on weight-related measurements,,21 2 (11%) on campaign awareness,,35 and one ongoing study had not published information regarding their primary outcome by the time our review was completed.There was great heterogeneity among measurements of physical activity, characterizations of geographical environments, and primary outcomes reported Table . Physica,27,31,33 Follow-up and long-term assessments were planned or conducted in only 47% (9) of the intervention studies reviewed.[Overall, study quality was high. Quality scores ranged from 8 to 17, with 17 being the highest possible score, and a mean score of 12.6 ± 2.4 .,30,33–36 Among the 8 physical activity interventions that targeted children primarily, 3 were school based (16%),,26,31 3 were community based (16%),,28,32 and 2 included multiple settings in their communities with schools as focal points supported by primary healthcare clinics and by multisector approaches.The interventions were delivered through a variety of settings such as: workplace, schools, and neighborhood/community, with a large majority of them using a community or neighborhood based approach. Of the 11 interventions that targeted adults (58%), 2 were implemented in worksites (11%), and Promoting Activity and Changes in Eating (PACE) physical activity interventions shared a multicentered cluster randomized-control trial design. In the “Walk-to-Work” intervention geographic information was collected to separate walks to work from other walks and evaluate the effects of the intervention that promoted walking to work. The PACE intervention implemented a geographic inventory of physical activity opportunities, and food services available in the 34 worksite neighborhoods where the intervention was administered. In addition to promoting increasing walking rates among employees, the PACE intervention also promoted increasing other modes of physical activity , and improving dietary habits. The geographic information from the neighborhood inventories was used to examine the role of availability of physical activity opportunities, and food services, in relation to behavior changes . Disparities in worksite neighborhoods explained intervention effects. Worksites in more affluent neighborhoods supported improvements in physical activity and healthy eating, whereas underserved areas were associated with lower frequency of walking and lower fruit and vegetable intake.Two of the 19 interventions targeted adult populations in different worksites. The “Walk-to-Work,” Two studies examined the impact of “Play Streets” on physical activity changes among children,32 and a third “Play Streets” study examined physical activity effects among children and adults.Community settings were considered in 63% of the physical activity interventions that measured or modified physical geographic factors. Community approaches that targeted children were all policy-driven changes with a wide range of complexity. One community approach examined the geographic and social differences in the effects of a policy initiative that provided swimming sessions to children under the age of 15.,32,36 Three of the studies included in this review reported positive effects on the physical activity of children and adults who participated in “Play Streets” programs in the streets of Colombia, Belgium, and the United States. In addition to providing car-free play zones, these approaches introduced recreational equipment and activities to support the promotion of physical activity. Examples of equipment included a box with play equipment, a climbing wall, and bicycle ramps, and recreation zones, with “aerorumba” (aerobic sessions with Latin dance rhythms). Studies assessing the effects of “Play Streets” found that these programs yielded positive physical activity changes among children and adult participants,,32,36 and improved social participation in the community.,32“Play Streets,” or “ciclovias,” introduced a novel policy approach to address geographic structural or financial barriers that may limit the provision of recreational spaces in park-deprived neighborhoods. In these physical activity interventions, streets in neighborhoods with limited recreational spaces were temporarily assigned to be car-free play areas for children and adults.,35; increments in physical activity attributable to changes in the built-in-environment after infrastructure modifications,,30,34 and outcomes resulting from walking programs.,22,29,33Other community-based interventions included in this review examined the geographic differences in cognitive effects of physical activity campaigns,35 Merom et al examined the influence of geographic factors on the effects of a physical activity campaign, and the physical activity outcomes associated with infrastructure improvements. Although proximity was found to positively influence cognitive effects and yield increases in cycling, remoteness not only was associated to lower awareness of the physical activity campaign but also to decreases in cycling.Access and proximity to walking infrastructure were found to positively influence cognitive effects of physical activity campaigns.,30,34 In all 3 studies, increases in the amount of time spent walking and cycling were associated with the use of the new infrastructure, which benefited mostly residents living in proximity to the enhanced routes.,30,34Three studies examined physical activity changes attributable to changes in the built-in-environment after infrastructure improvements.–22,33 and a fourth study examined the social differences in the provision of walks hosted by a national non-for-profit walking program. These studies found that although social life and income are important predictors of participation in organized walking groups, greater need is not naturally associated with greater provision of services. As noted by Hanson and Jones, unless measurements are set in place to promote equity, physical activity interventions can lead to greater inequity by benefiting more active, and wealthier individuals.Among the interventions included in this review, walking was the only physical activity promoted by organized facilitators. Three quasi-experimental research interventions based on community designs introduced facilitator-led group walks among underserved communities, Two studies used physical and social geographic information primarily to improve their sampling strategy by considering geographic physical and social factors from schools as control or comparison sites during the match-pair design of intervention evaluation.,26 One study used the geographic information to assess if changes in the infrastructure of the environment had triggered changes in the places where children are physically active. Last, one intervention did not provide specific details about the uses of geographic data beyond their plans to rely on policy changes to modify social and physical environments.There was great heterogeneity among 5 school-based interventions included in this review, yet all school-based interventions assessed existing physical geographic factors of the environment before the implementation of their interventions. One study used the geographic information collected during the formative stage to inform lessons on places that are most conducive to physical activity among targeted children.–26 Among the 3 interventions that proposed physical environmental improvements, only the intervention by Toftager et al had published outcomes from the intervention. In this assessment, although all participating schools upgraded their playground areas and implemented sport programs, no significant changes in physical activity behaviors were found among children ages 11–13 years of age.All of the school-based interventions proposed changes to the social  environment to increase physical activity among children, with 3 school-based programs also proposing to implement improvements in the physical environment, particularly in the infrastructure of the playground areas.,31 teacher-led physical activities,,26 and 2 physical activity interventions that focused on policy changes, such as recess length regulations, and after school policies.,25The social environmental changes introduced to promote physical activity in schools included peer-mentoring interventions, The other peer-mentoring model matched children ages 11 and 12 years old with older peers (ages 13–14 years old). In this intervention, older peers were instructed on how to encourage and monitor physical activity of their younger peers. The peer-mentoring approach was implemented by itself in one subsample. A second subsample examined a peer model complemented with geography lessons. In the geography lessons, children learned about places identified during formative research to be conducive to physical activity. Modest physical activity improvements were found among participants of the peer-model intervention that was complemented by geographic lessons. None of the peer-mentoring programs yielded significant physical activity changes.,31One peer-mentoring approach, complemented peer mentoring with improvements in infrastructure and other organizational changes that supported physical activity in schools.,33,35,36 measuring the distance to resources (proximity),,32,36,38 or by providing spaces that support physical activity in areas with limited opportunities for physical activity.,32,34,36 Across the studies reviewed, it was consistently observed that infrastructure improvements alone do not lead to sustainable changes in physical activity if perceptions of the neighborhood are not modified, or awareness is low.The usefulness of geographic information in the promotion of physical activity is becoming increasingly clear. All intervention studies examined acknowledged the importance of geographic factors to physical activity by either measuring resources available ,,31,32,36 walking programs were most often organized by interventions targeting adults.,29,33,38 Free play and walking programs inherently combined physical and social geographic factors.The development of active-friendly neighborhoods needs to consider the resources and barriers in their surroundings, and the different ways in which geographic factors impact the perceptions and behaviors of communities. An environment conducive to physical activity needs to be one where not only physical infrastructure is available but also where the social context provides a supportive setting. Although “free play” was the activity most often promoted to increase children’s physical activity, observed that the study of geographic factors associated to physical activity is well advanced. However, most studies of geographic factors associated with physical activity are cross-sectional, and the implementation of physical activity interventions that verify the causal role of geographic factors remains scarce. In this systematic review, we documented a wide variety of approaches that researchers implemented in different stages of physical activity interventions to account for the effects of a variety of objective and subjective geographic factors. Although distance, traffic speed, density, diverse housing types, and mixed land use have been documented to influence walking and cycling in previous systematic reviews of cross-sectional studies,,40,41 this systematic review adds to the literature by summarizing the available causal evidence of physical activity changes achieved by addressing a wide variety of physical and social geographic factors previously identified in the literature. Although the causal evidence supporting the creation and modification of the geographic environment to resolve the lack of recreational spaces was limited, positive effects were consistent.In an umbrella review of systematic reviews, Bauman et al,32,36 Although there was no overlap in the evaluation designs implemented by the “Play Streets” assessments, it can be noted that these interventions were associated with positive physical activity outcomes in children,32 and adults. Moreover, these approaches proved to be successful despite documented differences across countries in the geographic factors associated with physical activity. Besides increasing physical activity, these temporary programs offered opportunities for community engagement and social interaction with potential benefits to social and mental health. A key recommendation for researchers is to further document the physical and social health benefits and challenges of the “Play Streets” interventions using an international framework that takes into account cultural, economic, and geographic differences across countries. The work by the International Physical Activity and the Environment Network study can guide this analytical framework.Within the different approaches undertaken to create recreational spaces, the temporary provision of “Play Streets” bears recognition as an innovative approach that effectively addressed structural barriers and recreational space limitations. Three studies published after 2015 examined the physical activity effects from “Play Streets” in Colombia, San Francisco, and Belgium.,14,43 highlight the importance of assessing both, physical and social geographic factors. In a literature review, Mackenbach et al found perceptions of the geographic environments to be a key predictor of physical activity, particularly in disadvantaged neighborhoods. Similarly, in a systematic review of multilevel studies that examined the effects of the interaction between neighborhood socioeconomic status and geographic factors on individual health, Schüle and Bolte found that neighborhood socioeconomic status can mediate the association between built-in environments, and residents’ health. This review identified a variety of social factors such as social support, crime and safety, etc., which, in addition to neighborhood socioeconomic status influenced physical activity uptake  within the last 15 months of our review hints the emergence of a trend that favors socioecological approaches and transdisciplinary research expansion to integrate geographic information. This paradigm-shift previously observed in the cross-sectional literature Disparities that associated with physical activity interventions merit further investigation.Findings should be interpreted with caution given the gaps in the literature and the limitations in our review. Within the literature, a key limitation was the scarce number of articles that satisfied our inclusion criteria. To date, evidence of changes in physical activity following improvements in geographical environments is also insufficient. In addition, it is important to address the lack of consistency in measurements of physical activity and geographic environments, which precluded the elaboration of a meta-analyses. More studies that rely on objective measurements of both physical activity and the environment are needed. As a result of the limitations in the literature, we cannot conclusively determine whether measuring or modifying physical, social factors or both, influences changes in physical activity. Rigorous randomized-control trials are needed to reach these conclusions. In addition to rigorous randomized-control trials, evaluations that rely on natural experiments are also recommended. Moving forward, researchers should consider capitalizing in the increasing interest for developing physical-activity-friendly communities to examine whether changing the geographic environment yields improvements in physical activity. Another important gap is to evaluate changes in physical activity that may be attributable to the gentrification of poor neighborhoods across the populations’ social gradient. It has been suggested that without explicit measurement to limit disparities, physical activity interventions may exacerbate difference by benefiting the previously active populations. Without objective measurements of physical activity or validation assessments that identify and address misreporting issues in self-reported physical activity data, findings linking physical activity improvements to geographical factors must be interpreted with caution.Although this systematic review followed a rigorous protocol, there are important limitations in this review worth recognizing. Our findings are limited by the lack of validity in the self-reported physical activity data. Self-reported measurements of physical activity often differed greatly from objective measurements.Although the integration of international studies can enhance our review, the inclusion of studies from multiple countries also introduces limitations. The international variability in built-in environments and the differences in cultural norms that may influence self-reports of physical activity must be recognized. Considering the studies reviewed do not enable cross-cultural comparisons, corresponding multinational conclusions cannot introduce. Also, we limited our search to publications available in English. The overwhelming majority of studies included from developed countries may be attributable to this limitation, and not to the lack of interventions being conducted in middle- and lower-income countries.This review focused on primary research articles. It is possible that the exclusion of editorials, letters, commentaries, and policy briefs could have hindered the inclusion of physical activity intervention studies that implemented geographic information and were not also reported as primary research.Different strategies, such as the collaboration of multiple reviewers, and the quality review that focus on mostly objective assessments were implemented to limit the reviewer biases, yet recognizing lower quality papers can introduce reviewer bias despite our efforts. Finally, given the dearth of long-term data, it is plausible the physical activity improvements documented may have resulted from a novelty effect.Every place introduces barriers and opportunities for physical activity. The challenge of designing sustainable physical activity programs that encourage populations to be physically active, and that take advantage of the built in resources, inherently requires the use of geographic information. Evidence showed that supporting the creation and modification of the environment to address geographic barriers, specifically the availability of, and proximity to recreational spaces is associated with improvements in physical activity. These findings underscore the importance of using geographic information to identify the sources and location of physical and social factors in the environment that can influence physical activity uptake. However, more research is needed to identify the physical and social geographic factors that consistently influence changes in physical activity, and to determine if causal associations exist. Public health policy makers can benefit from the wide range of opportunities to measure, analyze, and modify physical and social geographic factors introduced by the intervention studies reviewed to promote long-lasting changes and physical active communities.The authors would like to thank Kevin E. Markes and Renee A. Pond for their assistance with this study.


Epithelioid hemangioendothelioma (EHE) is a rare soft-tissue sarcoma involving cells with histologic markers that suggest an endothelial origin. Around 90% of EHEs are caused by the fusion of Transcriptional Co-activator with a PDZ-motif (TAZ) with Calmodulin Binding Transcription Activator 1 (CAMTA1), a central nervous system-specific transcription activator. The 10% of EHEs that lack the TAZ–CAMTA1 fusion instead have a fusion of Yes-associated Protein (YAP) and Transcription Factor E3 (TFE3) genes (YAP-TFE3). YAP and TAZ are well-defined downstream effectors in the Hippo pathway that promote cell growth when translocated to the nucleus. The TAZ–CAMTA1 fusion transcript is insensitive to the Hippo inhibitory signals that normally prevent this process and thus constitutively activates the TAZ transcriptome. In EHE, this causes tumors to form in a variety of organs and tissue types, most commonly the liver, lung, and bone. Its clinical course is unpredictable and highly variable. TAZ activation is known to contribute to key aspects of the cancer phenotype, including metastasis and fibrosis, and increased expression of TAZ is thought to be causally related to the progression of many cancers, including breast, lung, and liver. Therefore, understanding TAZ biology and the molecular mechanisms by which it promotes unregulated cell proliferation will yield insights and possibly improved treatments for both EHE as well as much more common cancers. Experiments of nature are spontaneous phenomena that offer insights into otherwise mechanistically opaque events. They are usually both rare and extreme occurrences, two features that provide a basis for innovation and understanding. Such events have much in common with the ‘Black Swan’ occurrences described by Nassim Taleb as unexpected outlier events that can have a dramatic impact on our world-view . They caEpithelioid hemangioendothelioma (EHE), a rare, translocation-based, soft-tissue sarcoma, is one such experiment of nature that can provide meaningful insights into the connection of genotype to the clinical phenotypes of cancer. The underlying genetic alteration in most EHEs is a fusion of the WW Domain-containing Transcription Regulator Protein 1 gene (WWTR1) with Calmodulin Binding Transcription Activator 1 (CAMTA1 gene). In 2013, a second fusion event between the Yes-associated Protein (YAP) and Transcription Factor E3 (TFE3) genes (YAP-TFE3) that occurs in around 10% of all EHEs was identified . ResistaMallory first used the term hemangioendothelioma in 1908 to describe all endothelial proliferations . The disEHE usually presents in adulthood  with a (1.5:1) bias among females. Affected organs vary, with liver (21%), lung (12%), and bone (14%) being the most commonly reported sites, but involvement of the central nervous system, skin, and oral cavity have also been documented . It is aImaging, histology, and immunohistochemistry IHC are helpful in managing EHE, but are not always sufficient to accurately diagnose this rare vascular tumor. Therefore, diagnosing EHE can be a challenge and due to its rarity and variable clinical presentation it is often initially misdiagnosed. Laboratory abnormalities in blood counts are infrequently present and are rarely specific. Imaging is an essential tool in the detection of EHE with computed tomography (CT) and magnetic resonance imaging (MRI) being the most commonly used modalities. Some specific patterns on CT in patients with pulmonary epithelioid EHE include multiple pulmonary nodules, diffuse infiltrative pleural thickening, and multiple reticulo-nodular opacities. Similarly, some recurring patterns on CT/MRI in patients with hepatic EHE include multiple nodular lesions with calcifications and peripheral enhancement (lollipop sign).Histology and immunohistochemistry were key to diagnosing EHE before the responsible fusions were identified. Tumor cells typically show cords or nests of epithelioid endothelial cells in myxohyaline stroma; about 50% of tumors are associated with or seen as an outgrowth of a pre-existing vessel. The cell nucleus is typically bland although 25% of tumors exhibit some cytological atypia. Immunohistochemical confirmation of endothelial differentiation can be helpful in confirming the diagnosis, and EHE cells typically stain strongly positive for CD31/platelet endothelial cell adhesion molecule (PECAM-1), CD34 (cell surface glycoprotein), or erythroblast transformation-specific related gene (ERG) . The cliThe first clue about genetic drivers of EHE came in a study in 2001 that identified a chromosomal translocation involving chromosomes 1 and 3 p36.3;q25)) in EHE biopies . Two sub) in EHE The WWTR1–CAMATA1 gene fusion joins the amino terminus of WWTR1 with the carboxyl terminus of CAMTA1 and is under the transcriptional control of the WWTR1 promoter . TAZ, thSeveral variations of the WWTR1–CAMATA1 fusion with slightly different breakpoints have been identified  23,24,2,224,25. Inappropriate TAZ transcriptional activation and/or Hippo pathway dysregulation drives tumor development, progression, and metastasis in many cancer types ,6,7,8. TUnderstanding why EHE and the WWTR1–CAMATA1 fusion share this unique connection is an important step in understanding and curing this disease. If the only consequence of the fusion is constitutive TAZ-mediated transcription, then TAZ activation should be sufficient to promote EHE and the fusion should occur in other cancers where TAZ activation promotes tumorigenesis. The fact that this is not the case suggests that the CAMTA1 portion of the protein provides additional function beyond just recruiting the TAZ portion to the nucleus and driving transcription. Consistently, while there was significant overlap between the genes regulated by the fusion and TAZ in NIH3T3 cells, there were many genes that were unique to each. These differences in gene expression may account for some of the specificity of the fusion to EHE. Alternatively, it may be that the EHE cell of origin, which is currently unknown, is particularly susceptible to the fusion but not other alterations in the Hippo-YAP/TAZ pathway. Identification of the cell of origin is likely a critical step in understanding how the fusion drives disease formation and progression, and would also aid in the generation of EHE model systems, the lack of which hinders research efforts.Since the WWTR1–CAMATA1 fusion that drives EHE results in activation of a TAZ-like transcriptional program, it is not surprising that many clinical features of EHEs are also associated with TAZ activation in other cancer types and/or pathological processes. For example, in many diseased tissues TAZ activation plays important roles in driving fibrosis and can promote the differentiation of fibroblasts into highly contractile myofibroblasts ,63,64,65The extreme infrequency and highly variable rate of progression of EHE currently preclude establishing well-defined study designs and therefore guidelines for its treatment. Moreover, the unpredictable natural history of EHE makes the confident interpretation of clinical response to any medical therapy very challenging. For instance, it is difficult to ascribe benefit to a specific treatment in a disease that normally advances and remits intermittently with no clear pattern. The potential heuristic benefit of any trial is weak in a disease so rare that adequate powering to establish efficacy is nearly impossible. These factors also help explain the tendency for many physicians to choose a conservative, wait-and-watch approach to patients with indolent disease, using CT or MRI imaging at regular intervals to surveille disease progression. This also underscores the importance of an as yet unmet need for a robust biomarker of EHE activity to allow intervention at an early indication of progression.Despite the inherent limitations to medical progress in a very rare disease, a few decades of experience have taught us some things about treating EHE. Surgical excision of solitary lesions, especially those in the extremities, can be curative. Unfortunately, most patients present with systemic (metastatic) disease. In this case, the approach depends on the organ(s) involved and the accessibility of lesions to surgery or interventional procedures. For instance, multifocal liver disease, one of the most common presentations of EHE, is routinely treated with interventional radiologic procedures, including radiofrequency ablation, irreversible electroporation, or embolization. Several large-scale clinical series have also shown success with liver transplantation in EHE ,73. ThesCurrent approaches to metastatic EHE include various conventional cytotoxic and anti-angiogenic chemotherapy regimens, alone or in combination ,78,79,80Targeted therapy also shows promise in treating EHE. The PALETTE study was the first randomized phase III trial that showed efficacy of pazopanib in the treatment of sarcoma. Subsequently, a few case reports have documented therapeutic success of targeted agents. A case report evaluating pazopanib in metastatic pulmonary EHE, for instance, demonstrated stable disease over 24 months , while aUse of anti-angiogenic agents is rational given that EHE is a disease of unregulated proliferation in a cell with endothelial markers ,86. DrugLess-conventional approaches with some theoretical rationale that have yet to be used clinically include verteporfin, a porphyrin derivative approved for phototherapy of macular degeneration, which is thought to inhibit interaction of YAP, and presumably TAZ, with TEAD, thereby impairing transcription of downstream signals ,95,96,97An exciting future prospect for treatment is to specifically target and inhibit function of the disease-identifying fusion genes of WWTR1–CAMTA1 and YAP–TFE3. This would theoretically produce more durable outcomes than any standard or proposed medical regimens. Currently, the most promising approach is to target fusion–TEAD interaction. Given growing evidence showing that TAZ and YAP are important drivers of cancer development and progression, there is great enthusiasm for targeting YAP/TAZ–TEAD interaction generally, and this could potentially be very effective against EHE specifically. Although the fusion may be insensitive to Hippo pathway regulation, the list of Hippo-pathway-independent mechanisms of YAP/TAZ regulation continues to grow and several negative regulators of YAP and TAZ bind the WW domains ,92,107, One potential problem with this strategy is that TAZ and YAP play important roles in organ development as well as normal cell function and repair in adult tissues. Systemic YAP/TAZ or TEAD inhibition may therefore result in as yet unpredictable adverse side effects. Another potential approach is to attempt to target the CAMTA1 portion of the fusion. Indeed, as mentioned above, the CAMTA1 portion of the fusion is required for its oncogenic activity because it contributes the transactivation domain and NLS that are essential for function . Very liA third approach may be to target the genes regulated by the fusion that are responsible for EHE development and progression. Numerous TAZ target genes are known, and many have established roles in cancer . A more It is worth noting that inhibitors of the tankyrase class of PARP enzymes represent a potentially promising method of reducing YAP/TAZ activity in EHE. Zhao et al. showed that members of the angiomotin-like family of angiostatin binding proteins associate closely with YAP/TAZ and function effectively within the Hippo pathway to enhance their phosphorylation, thereby increasing cytoplasmic localization and degradation . ConversFinally, hypermethylation of Hippo-related promoters, such as LATS1 and LATS2 and MST1 and MST2, can contribute to tumor growth and aggressiveness in many cancers , includiStudy of the molecular biology of EHE will lead to a better understanding of mechanisms specific to YAP/TAZ-induced cell proliferation and, eventually, improved treatment of both this rare disease and many of the more common cancers. Chief among the remaining hurdles are: establishing robust model systems ; achieving a fine-grained dissection of the fusion’s function and regulation; and comprehensive elaboration of downstream targets. Genetic approaches hold great promise for treatment of EHE, a monogenic disorder. This includes, for instance, use of silencing RNAs specific to both the fusion transcript and its downstream effectors. Gene-editing strategies could also theoretically knock down fusion gene expression or potentially delete the underlying fusion from tumor cells altogether. Such advances could lead to potential therapies in a wide array of YAP/TAZ-driven cancers and thereby help many patients. Clearly, experiments of nature, no matter how rare, can reveal secrets with wide applicability.


The mean age at presentation is 14 years of age, whilst the median age of individuals who die of LQTS is 32 years, with men predominantly affected. Inherited LQTS is characterized by prolonged ventricular repolarization represented on the ECG by an increased corrected QT (QTc) interval. Three main genes account for approximately 90% of all genotyped cases of LQTS. The risk of arrhythmic events is determined by age, sex, QTc interval, a history of syncope, and genotype, and in acquired LQTS QTc prolongation secondary to certain medication further increases the risk of cardiac arrhythmias , whereas there is no significant gender-related difference among LQT2 and LQT3 carriers [The analysis of serial QTc interval measurements in a large cohort of children and adolescents with LQT1 and LQT2 demonstrated two main findings. Firstly, both LQT1 and LQT2 male patients show significant QTc interval shortening after the onset of puberty; in LQT2 male patients, this is preceded by a progressive QTc interval prolongation. Secondly, the age of 12 to 14 years is an important transitional period in which differences between males and females for both genotypes are seen, ages broadly corresponding with the onset of puberty [A recent analysis of the QTc interval in healthy individuals during childhood and adolescence  observed+ channel (Nav1.5). LQTS3 patients show increased risk of life-threatening ventricular arrhythmias, particularly after 40 years of age, consistent with ion channel abnormalities caused by aging [In older patients, aging is associated with increased prevalences of atrial and ventricular arrhythmias, reflecting disruption of the normal sequence of ion channel activation and inactivation generating the propagated cardiac action potential. In addition to these biochemical and electrophysiological changes, aging is associated with structural changes, particularly myocardial fibrosis. This is relevant for LQTS3 patients, who account for 5 to 10% of cases of congenital LQTS, whose LQT phenomenon is caused by a gain-of-function mutation in the SCN5A gene, which encodes the α subunit of the NaThe QTc interval in general increases with age, with the age-related increase is more evident in men than in women. This suggests that male sex does not afford protection against potentially fatal arrhythmias at older age and there is a need for an increased awareness of the QTc prior to the use of QTc prolonging drugs, e. g. psychotropics, and the need to evaluate the QTc after initiation of therapy . AdditioIn conclusion both inherited and acquired LQTS are influenced by age. The onset of puberty is an important transitional period for inherited LQTS, which affects QTc interval and clinical risk, probably in part secondary to sex hormone shifts. Individuals with inherited LQTS remain at higher risk for life-threatening cardiac events after age 40 years. Risk factors for SCD include age-specific factors, gender, clinical history, and the LQTS genotype. QTc generally increases with age, and age-related electrophysiological and structural changes may increase the risk of potentially life-threatening arrhythmic events. Consequently, clinicians treating individuals above age 40, with inherited or acquired QT prolongation need an increased awareness of their QTc prior to, and during, therapy with drugs that have the potential to prolong the QTc interval.


The thematic issue addresses the role of oxidative stress in the development of diabetes and its related complications. Diabetes is a growing pandemic of the 21st century that has reached 1 in every 11 people worldwide . ChronicIn the research article “Astragalus membranaceus and Panax notoginseng, the Novel Renoprotective Compound, Synergistically Protect against Podocyte Injury in Streptozotocin-Induced Diabetic Rats,” ever since the diabetic complications have been understood and oxidative injury has been established as a cause, many compounds/plant extracts that attenuate oxidative damage and cell death have been researched upon. Adding to this field, one article in this issue showed the renoprotective role of treatment with Astragalus membranaceus (AG) and Panax notoginseng (NG). Treatment significantly reduced albuminuria and improved renal histopathology and podocyte foot process effacement in STZ-induced diabetic rats. AG and NG synergistically attenuated the structural and functional abnormalities in the kidney, and in the future, it may provide treatment combination for diabetic nephropathy and other kidney diseases.The research article “Myocardial Ischemia and Diabetes Mellitus: Role of Oxidative Stress in the Connection between Cardiac Metabolism and Coronary Blood Flow” discusses the physiological and pathophysiological role of oxidative stress in myocardial metabolism and coronary blood flow (CBF), with particular attention to patients with diabetes mellitus. Ischemic heart disease (IHD) being one major current health risk to diabetic patients, the review summarizes the role of oxidative stress in coronary microvascular dysfunction and development of IHD. Oxidative stress further generates ROS, AGEs, and oxidized LDL causing coronary microvascular dysfunction. Therein inflammatory pathway is triggered and ion channel function is altered. The review suggests that balancing the oxidative stress and restoring the ion channel function may be exploited as therapeutic target in the treatment of IHD.in vivo and in vitro and explored the possible involvement of the nicotinamide adenine dinucleotide phosphate-oxidase-4/reactive oxygen species- (NOX-4/ROS-) activated p38 pathway. The BSF shows therapeutic potential in attenuating oxidative stress and apoptosis in podocytes in DN.In the research article “Effect of Baoshenfang Formula on Podocyte Injury via Inhibiting the NOX-4/ROS/p38 Pathway in Diabetic Nephropathy,” it is stated that more and more studies demonstrated that diabetic nephropathy is the leading cause of end-stage of renal diseases . PreviouHevea brasiliensis) appears to create tendencies as an angiogenic inducing tissue healing agent and as biomaterial, resulting from its structural qualities and its low cost when compared to conventional treatments. Therefore, the authors aim to summarize the results, methods, and scientific findings that certify or recommend the use of LBM as a new technique to be applied effectively in the treatment of wounds.In the research article “Evidence in Practice of Tissue Healing with Latex Biomembrane: Integrative Review,” wound healing is a perfectly coordinated cascade of cellular, molecular, and biochemical events which interact in tissue reconstitution. Chronic diseases such as pressure ulcers (PU) and diabetes mellitus (DM) are considered risk factors for wound healing. Latex biomembrane (LBM), a biocompatible material, derived from latex of rubber tree  is one of the most prevalent complications in diabetes. In the article from this issue, exendin-4, a glucagon-like peptide 1 receptor agonist (GLP-1RA), was shown beneficial against oxidative stress insults by activating superoxide dismutase in dorsal root ganglion neuron cell. Therefore, the authors suggest that GLP-1RA attenuates PDN by protecting insults on dorsal root ganglion neuron cell.In the research article “Effects of High-Fat Diet on eHSP72 and Extra-to-Intracellular HSP70 Levels in Mice Submitted to Exercise under Exposure to Fine Particulate Matter,” tha authors suggest that in type 2 diabetes, eHSP70 has been negatively correlated with iHSP70 in skeletal muscles and that leds to impaired glucose uptake. The elevated levels of eHSP70 are associated with insulin resistance in elderly population . In thisIn the research article “A New Way for Beta Cell Neogenesis: Transdifferentiation from Alpha Cells Induced by Glucagon-Like Peptide 1,” it is stated that current treatment modalities for diabetes include drug therapy or pancreatic islet transplantation which has its own limitations . One stuIn the research article “Advanced Glycation End Products Increase MDM2 Expression via Transcription Factor KLF5,” the authors state that increase in the level of AGEs has been associated with diabetic complications for a long time; however, their role in the overexpression of MDM2 and colon cancer development is an exciting finding by one of the papers in the current issue. The research highlights the activation of oncogenic pathway through activation of MDM causing Rb and p53 degradation, which may stimulate cell proliferation and metastasis. The concept of initiation of tumorigenesis or epithelial mesenchymal transition, in diabetics, via P53, AKT/mTOR, RAS signaling or Wnt signaling is a recent an open platform to explore.The editors anticipate this special issue to be of tremendous interest to the medical and scientific community. We hope researchers benefit in this issue to continuously make further progress in the understanding of the role of oxidative stress in the development of diabetes and its complications.


Ectopic calcification is an inappropriate biomineralization process occurring in soft tissues. Such calcifications usually consist of calcium phosphate salts, including hydroxyapatite. They sometimes contain calcium oxalates too as seen in cases of calcium nephrolithiasis.1. The clinical, biochemical and genetic aspects of the diseases responsible for nephrocalcinosis have now been clarified in detail, but there is still a paucity of information on the specific cellular events involved in this type of calcification process. The most likely explanation for the onset of interstitial nephrocalcinosis seems to be purely physicochemical, relating to a spontaneous Ca2PO4 crystallization in the interstitium as a result of it being oversaturated with calcium and phosphate2. Precisely how the renal cells are involved in the response to the influx of these potentially precipitating ions is still not clear. We were the first to suggest that nephrocalcinosis could be an osteogenic cell-driven process3, and our earlier studies were the first to produce evidence to indicate that human renal cells can undergo a process similar to that of vascular calcification4. We saw a phenomenon of spontaneous biomineralization occurring in primary renal cells from a patient with medullary sponge kidney (MSK)—a clinical condition associated to nephrolithiasis and medullary nephrocalcinosis—who had a GDNF gene mutation. When exposed to an osteogenic medium, an immortalized proximal tubule epithelial cell line from a normal adult human kidney (HK-2) with a silenced GDNF expression also proved better able to produce Ca2PO4 deposits than wild-type cells. This was due to the ratio of osteonectin (a pro-osteogenic factor) to osteopontin (an anti-osteogenic factor) being regulated differently, in favor of osteonectin4.Many in vitro and in vivo studies on the mechanisms behind calcium nephrolithiasis have revealed an association with medullary nephrocalcinosis, which involves the deposition of microscopic renal crystals in the tubular lumen  or the interstitium GDNF downregulation promoting the calcification process. We went on to investigate whether downregulated GDNF, which encodes the glial-derived neutrophic factor—a survival factor for many cell types, including renal cells5—could favor cell death.The core question that remained to be answered concerned which cellular mechanisms lead to 6. In advanced carotid atherosclerotic plaques, matrix vesicle-like structures derived from vascular smooth muscle cells (VSMCs) were found to contain high levels of BAX (a pro-apoptotic member of the BCL2 family), suggesting that they may be remnants of apoptotic cells7. Apoptotic VSMC-derived matrix vesicle-like structures can also concentrate and crystallize calcium, triggering calcification8. All these findings have paved the way to the theory that the formation of apoptotic bodies could initiate the ectopic calcification of some cells under certain conditions.It is common knowledge that pathological calcification is important in cell death phenomena. For instance, chondrocyte-derived apoptotic bodies might contribute to the calcification of articular cartilage9. To our knowledge, the role of cell death in the more common medullary nephrocalcinosis has yet to be explained.In the kidney, necrotic tubular cells have been associated with renal cortical nephrocalcinosis—a rare condition that is generally the result of a severe destruction of the cortex and any condition that causes acute and prolonged shock10, and that crystal size may have an influence. Sun et al.11 demonstrated that nano-sized crystals were the most likely to cause apoptosis, whereas micron-sized crystals caused necrosis. Lysosomes may internalize nano-sized crystals, and the resulting damage could trigger an apoptotic process. Nano-sized crystals can also pass through pores into the nucleus, where they can cause DNA cleavage into regular fragments—an important feature of apoptotic cell death. Various types of crystal can gain access to cells via a phagocytotic process, resulting in a caspase-independent cell death called necroptosis12.We know that calcium oxalate, calcium phosphate and other crystals can induce cell death, especially in renal proximal tubule cellsGDNF silencing triggered a caspase-independent cell death that strongly facilitated the formation of calcified nodules13. We know of several types of programmed cell death that do not involve any caspase activation14. The programmed cell death seen in our model would be classifiable as necroptosis because it had features coming somewhere in between apoptosis and necrosis. Since calcium phosphate aggregates were also found  in wild-type (WT) HK-2 cells cultured in osteogenic conditions, we replicated the previous experiments in WT HK-2 cells to clarify the relationship between biomineralization and apoptosis in normal tubular epithelial cells grown in an osteogenic medium. In our paper in Cell Death Discovery15, we demonstrated that HK-2 calcification with calcium (Ca) and phosphate (P) deposition was concentrated in multilayered cell nodules where the apoptotic process occurred, but the calcified cells showed the typical signs of caspase-dependent apoptosis. On monitoring the cells for two weeks, however, an apoptotic process began within 5 days of inducing osteogenesis, and the first small Ca and P crystals appeared. The importance of apoptosis in the process of HK-2 cell calcification was supported by the changes seen in BCL2 and BAX gene expression. In the osteogenic medium, BCL2 was less expressed than BAX from day 1 onward. This higher BAX/BCL2 ratio in the osteogenic medium than in the standard medium strongly suggests that apoptosis triggers HK-2 calcification before any Ca and P crystal deposition occurs. In fact, there was no sign of any Ca and P on von Kossa staining on the first day. Calcified deposits had become apparent by day 5, but only in or near areas where cells were apoptotic. This would indicate that early calcification is linked to HK-2 cell death, and that apoptotic areas provide the right milieu for this process—as in the case of vascular calcification. We hypothesized the following sequence of events for the calcification process we were seeing. Under osteogenic conditions, cells soon underwent apoptosis, and the subsequent release of apoptotic bodies allowed for mineral ions and/or calciprotein particles in the medium to accumulate. Then came an osteogenic-like process involving pro-osteogenic factors like Runx2, alkaline phosphatase and osteonectin upregulation, giving the impression that the osteogenic phenotype acquired by the surviving cells was a consequence rather than a cause of calcium phosphate deposition. The surviving renal tubular cells’ acquisition of an osteogenic-like phenotype could then help the process to continue (Fig. We demonstrated the fundamental role of cell death in the onset of renal tubular cell calcification in two in vitro models of nephrocalcinosis. The first involved GDNF-silenced HK-2 cells cultured in an osteogenic medium, a model revealing how


Aerobic glycolysis is an important metabolic adaptation of cancer cells. There is growing evidence that oxidative phosphorylation is also an active metabolic pathway in many tumors, including in high grade serous ovarian cancer. Metastasized ovarian tumors use fatty acids for their energy needs. There is also evidence of ovarian cancer stem cells privileging oxidative phosphorylation (OXPHOS) for their metabolic needs. Metformin and thiazolidinediones such as rosiglitazone restrict tumor growth by inhibiting specific steps in the mitochondrial electron transport chain. These observations suggest that strategies to interfere with oxidative phosphorylation should be considered for the treatment of ovarian tumors. Here, we review the literature that supports this hypothesis and describe potential agents and critical control points in the oxidative phosphorylation pathway that can be targeted using small molecule agents. In this review, we also discuss potential barriers that can reduce the efficacy of the inhibitors of oxidative phosphorylation. Metabolic adaptations allow tumors to maintain a highly proliferative state. Evidence in support of such adaptations was obtained nearly a century ago by Otto Warburg, and Carl and Gert Cori and their colleagues when they demonstrated an increased uptake of glucose by tumors as compared to normal tissues ,2. WarbuA rapidly growing body of evidence is demonstrating that an adaptation to aerobic glycolysis does not entail a complete shutdown of oxidative phosphorylation (OXPHOS) in tumors. Active electron transport occurs in cancer cells that trigger tumor recurrence and in cancer stem cells ,19,20. HOvarian cancer is classified into type I and II diseases ,22,23. CIn normal cells, glucose is metabolized through glycolysis, tricarboxylic acid cycle and OXPHOS to produce 34–38 molecules of ATP per molecule of glucose . In cancAn active glycolytic pathway supplies the biochemical precursors required for protein, nucleotide and lipid synthesis. This is an important reason why cancer cells limit glucose metabolism to glycolysis even when there is sufficient availability of oxygen. Ovarian tumors also show metabolic adaptation to aerobic glycolysis that allows them to maintain an increased proliferative capacity and survive under anchorage dependent conditions ,35,36. AThe metabolic shift to aerobic glycolysis provides the precursors for synthesis of proteins, nucleotides and lipids, at the expense of ATP. To compensate, cancer cells overexpress glucose transporters. As a result, there is an increased uptake of glucose that is catabolized through aerobic glycolysis. Serous ovarian tumors express high levels of glucose transporters GLUT1, GLUT3 and GLUT4, as compared to healthy and benign ovarian tissues ,42. IncrBRCA1 [The molecular mechanisms responsible for the metabolic reprogramming in cancer are under investigation. One mechanism is through the regulation of pyruvate kinase M2 isoform (PKM2) activity through its interactions with growth factor receptors . There iBRCA1  and mutaBRCA1 ), two geBRCA1 . Loss ofBRCA1 . All of While aerobic glycolysis is an important adaptation in HGSOC, OXPHOS is also an active pathway in cancer cells ,54,55,56Tumor cells that utilize aerobic glycolysis, coexist with cancer cells with active OXPHOS. A recent report by Yu et al  developeThere are, however, some indicators that active mitochondrial metabolism may have a favorable outcome. The Bioenergetic Cellular (BEC) index, a ratio β-F1ATPase (F1 portion of adenosine triphosphate synthase) to HSP60 (Heat Shock Protein 60) and GAPDH  expression, predicts the metabolic state of a cell . A highePerhaps the largest impact of OXPHOS is in the survival and proliferation of cancer initiating stem cells. Tumor initiating cells isolated from tumorigenic murine ovarian surface epithelial (MOSE) cells showed increased expression of glucose transporters and an overactive glycolytic pathway ,62. Howe+/CD117+ cancer stem cells isolated from the peritoneal fluid of HGSOC patients that had the ability to form tumors in mice, showed decreased levels of pyruvate dehydrogenase kinase (PDHK1) and increased expression of isocitrate dehydrogenase (IDH2) [+/CD117−) from HGSOC patients where the PDHK1 was upregulated and IDH2 was significantly lower. PDHK1 negatively regulates pyruvate dehydrogenase and as a result controls the amount of acetyl-CoA (Coenzyme A) available for the tricarboxylic acid cycle. The decrease in PDHK1 and increase in IDH2 in the CD44+/CD117+ ovarian cancer stem cell population are indicators of enhanced tricarboxylic acid cycle. The CD44+/CD117+ cancer stem cells produced higher levels of oxygen radicals and had enhanced OXPHOS than the non-stem cell (CD44+/CD117−) population. RAG2−/− mice implanted with HGSOC tumors when maintained on a diet supplemented with the glycolysis inhibitor, 2-deoxyglucose, instead of glucose, showed a decrease in tumor size. However, the surviving tumors from these mice were enriched in CD44+/CD117+ cancer stem cells [CD44e (IDH2) . This obem cells .There is also evidence that OXPHOS is an important pathway to target in chemoresistant tumors. Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial chaperone from the Hsp90 family . IncreasTumor cells produce high levels of lactic acid in the tumor microenvironment. While the lactic acid can be transported by cancer cells through monocarboxylic acid transporters (MCTs) and used to promote tumor proliferation, the effect of the acidic environment on the non-malignant cells is also an important factor to consider when determining the metabolic profile of the tumor. For example, the lactic acid in the tumor microenvironment can regulate the activity of immune cells infiltrating the tumor microenvironment (reviewed in ). Such crosstalk in the tumor microenvironment is not unidirectional as the tumor cells can also be affected by the metabolic status of the fibroblasts from the microenvironment. For example, Ras  mutations alter metabolism in cancer cells and increase the release of oxygen radicals . These rBRCA1 mutated breast cancers were treated with the anti-oxidant, N-acetyl cysteine [Stromal cells from the tumor microenvironment express low levels of caveolin 1 and high MCT4 allowing them to expel lactate into the tumor microenvironment ,69,70. Icysteine . Patholo2 are harvested in OXPHOS and transferred to molecular oxygen. Four multiprotein complexes located in the inner membrane of the mitochondria (Complexes I-IV) are required for electron transport . The quinone head group of CoQ10 participates in two electron redox reactions. Addition of one electron to CoQ10 yields semiquinone and further reduction of this intermediate leads to formation of ubiquinol. The electron transfer from NADH/FADH2 to ubiquinone occurs at the ubiquinone and ubiquinol binding sites, Q0 and Qi, in the mitochondrial complexes I-III. Electron transport from complex III to complex IV is aided by cytochrome C (Cyt C). In complex IV, the electrons are delivered to molecular oxygen to form water. High energy electrons from NADH and FADHransport . Electropion). The proton efflux from the matrix helps maintain a negative charge in the matrix and contributes to an electrical potential gradient (ΔΨm). The electromotive force generated through proton transport provides the energy necessary for the fifth mitochondrial complex, the ATP synthase, to convert ADP to ATP. The transfer of electrons is coupled with pumping of protons from the matrix to the intermembrane of the mitochondria. This transfer of protons leads to the maintenance of a proton gradient (ΔΨpion and ΔΨm) and transfer of electrons to oxygen and ATP synthesis can be developed as cancer therapeutics. While small molecules are likely the preferred agents to target OXPHOS, efforts are also underway to develop peptides that can specifically target this mitochondrial metabolic pathway . In theMetformin and proguanil are biguanides with complex I inhibitory activities . RegularMetformin inhibits complex I and thereby reduces ATP production. As a result of decreased ATP levels, AMPK is activated in cancer cells along with inhibition of mTORC1 . Millimolar concentrations of metformin are required to inhibit complex I activity and there remains an active question of whether such high levels of metformin can be achieved in solid tumors. Proguanil inhibits complex I activity in the malarial parasite and is therefore administered in conjunction with atovaquone, a complex III inhibitor. However, proguanil has limited effect on human complex I and is therefore not suitable for cancer therapy ,77. AnotA novel agent, IACS-0107059, that likely mimics the biguanide functional group has been investigated as therapy for acute myeloid leukemia ,80,81,82clinicaltrials.gov to test the effect of metformin in ovarian cancer patients. The majority of these trials are evaluating the combination of metformin with chemotherapy and are currently recruiting patients. Results from one trial (NCT01579812) showed that metformin was well tolerated. Ex vivo evaluation of the tumors showed significant decrease in viable cancer stem cell population. Additional studies are needed to determine if these positive benefits are due to the OXPHOS inhibitory effects of metformin. As demonstrated by Yu et al. [Eight clinical trials are currently posted in u et al. , monitorOxidative stress results from an imbalance between the processes responsible for generation and sequestration of reactive oxygen radicals (ROS) . Since tUnpublished results from our group are demonstrating that atovaquone should be investigated for treatment of HGSOC. The naphthoquinone unit of atovaquone engages in redox reactions and interferes with electron transport mediated by ubiquinone ,85,86. There are several naturally occurring and synthetic molecules that contain the quinone, naphthoquinone or anthroquinone head groups. Plumbagin and juglone are examples of such compounds. Treatment of HGSOC cells with plumbagin results in an immediate increase in intracellular oxygen radical flux . Plumbag2−, hydrogen peroxide H2O2 and hydroxyl radicals OH∙) that are formed due to incomplete transfer of electrons to molecular oxygen . H2O2, existing at nanomolar concentration in the cell, is the main ROS signaling molecule of the cell. It functions by oxidizing the thiolate anion (Cys-S−) of a cysteine to its sulfenic form (Cys-SO−). Oxidation of the cysteine thiol affects the formation of Inter- and Intramolecular disulfide bonds and has serious consequences on the biological properties of proteins [2O2 (as those observed during oxidative stress), the sulfenic form (Cys-SO−) is irreversibly oxidized to higher oxidized states of sulfinic (Cys-SO2−) and sulfonic (Cys-SO3−), which cannot be repaired through redox control and hence can cause significant cellular damage.Reduction of the superoxide anions by superoxide dismutases (SODs) yields hydrogen peroxide (Hproteins . This oxproteins . At abno∙) is the most reactive of the three ROS molecules described in this section. H2O2 reacts with metal cations  present in the cytosol in a reaction called Fenton reaction, to produce OH∙. Additionally, nitric oxide synthases (NOS) also produce OH∙ along with NO2∙ under limiting concentration of cofactors and co-substrates. OH∙ reacts indiscriminately with various substrates such as lipids, proteins and DNA and leads to genomic instability [∙ is abnormal and therefore, an indicator of high oxidative stress in the cell.The hydroxyl radical , activation of protein kinases and other signaling molecules, and decrease in the expression of survivin. Molecules such as plumbagin that increase intracellular oxygen radicals, are often thought to mediate pleiotropic effects that culminate in cancer cell death. However, it is important to consider that such molecules may also be specific in their ability to compete with ubiquinone and inhibit electron transport in the mitochondria and can therefore serve as important OXPHOS-targeting agents. Our studies with plumbagin clearly show that inhibition of electron transport results in a rapid increase in harmful oxygen radicals that cause significant cellular damage . With OXThere are elaborate antioxidant mechanisms to maintain steady state levels of oxygen radicals in all cells. Superoxide dismutase, catalase, peroxiredoxins, glutathione, glutathione reductase, thioredoxins and others form the network of anti-oxidant mechanisms that control oxidative stress. This network is controlled by a master regulatory transcription factor, Nrf-2. Cancer cells respond to inhibition of complex III by atovaquone, (unpublished observation) and plumbagin  by increUse of Nrf-2 inhibitors for cancer treatment also raises the possibility that such approaches may inhibit the natural protection against oxygen radicals in healthy tissues. Rational drug design, targeted delivery and specific drug formulations will be required to maximize the effect of Nrf-2 inhibitors in cancer cells while attenuating the side-effects of such drugs in healthy tissues.The extensive use of atovaquone has led to the realization that some malarial parasites have developed resistance to this drug through mutations in cytochrome B (Cyt B), an essential component of complex III ,99,100. Inhibition of glucose metabolism will result in significantly curtailing the ability of cancer cells to proliferate and modulate the tumor microenvironment through the release of lactic acid and other intermediates. OXPHOS pathway in tumors, cancer stem cells and the stromal and immune cells in the tumor microenvironment is recognized as a target for development of novel anti-cancer therapies. The multimeric complexes of the OXPHOS pathway are targets for small molecule inhibitors that can inhibit metabolism as well as induce oxidative damage and cancer cell death. OXPHOS inhibitors can be paired with immunologic and other therapies. While the development of novel OXPHOS inhibitors will be necessary, it will be important that these agents are specifically targeted to the cancer or the tumor microenvironment in order to reduce toxicity in healthy tissues. Finally, the use of OXPHOS inhibitors may result in Nrf-2 activation and mitochondrial adaptations that may pose as pathways that are typically not considered to contribute towards chemoresistance. Studies on atovaquone, proguanil, metformin have provided the foundation that will support the development of additional and more potent OXPHOS inhibitors for the treatment of HGSOC and other tumors.


Postpartum  psychosis (PP) is a rare, severe psychiatric disorder that affects women shortly after childbirth; risk is particularly high in individuals with a history of bipolar disorder or PP, but the underlying pathophysiology remains poorly understood. Emerging evidence suggests that immune system (dys)function plays an important role in disorder onset. On the basis of new findings from clinical and animal model studies, we hypothesise that the abundance and/or activity of regulatory T cells, and the efficacy of consequent (re)myelination processes in the brain mediated by CCN proteins, is perturbed in PP; this pathway may be modulated by risk and protective/treatment factors for the disorder, and identifying abnormalities within it could signpost novel predictive biomarkers and therapeutic targets. Postpartum psychosis (PP) is a rare, severe psychiatric disorder affecting some women shortly after childbirth.While the risk factors for PP are relatively well understood, our knowledge of the underlying pathophysiology is extremely poor; however, there is some evidence that immune system dysregulation may play a role.A recent study has shown that regulatory T cells (Tregs) may promote remyelination via the CCN family of proteins.Here, we argue, on the basis of emerging immunological, animal model, and neuroimaging findings, that PP risk may be elevated due to abnormalities in the Treg–CCN–(re)myelination axis and that risk and protective/treatment factors for the disorder may influence this axis. Postpartum psychosis (PP) is a severe psychiatric disorder affecting approximately one or two of every 1000 women shortly after childbirth (most often within 6 weeks); the main symptoms include hallucinations, delusions, cognitive disorganisation, and mood abnormalities The most intensively studied biological changes in PP have been those that occur in maternal reproductive hormones shortly after childbirth; however, no differences in absolute levels of oestrogen or progesterone have been identified in women who develop an episode of PP Key obstacles in identifying biological risk and protective factors for PP include low disorder prevalence, high between-patient symptom heterogeneity, lack of accessibility to patient brain tissue, and a historical lack of amenable model systems N-methyl-d-aspartic acid receptor Recent evidence has highlighted a potentially important role for the immune system in the onset of PP: women with the disorder have higher rates of autoimmune thyroid dysfunction and pre-eclampsia  regulatory T cells 2-type immunity , has been historically considered responsible for maternal tolerance toward fetal alloantigens to protect the foetus from maternal Th1 cell-mediated immunity and subsequent attack Two recent studies have reported that women with PP have lower levels of Th1, Th17, and Tregs Finally, monocyte and macrophage levels have also been found to be significantly higher in women with PP than in those without PP. Although Kumar and colleagues Collectively, the above-mentioned data support a model for PP whereby an imbalance between proinflammatory and immunosuppressive cells of the immune system in the postpartum period may be strongly associated, possibly causally, with the development of the disorder. It remains to be established whether, and how, peripheral immune activation might affect the maternal brain processes that underlie PP onset. However, data relevant to the pathophysiology of MS and other psychiatric conditions, discussed in the subsequent sections, suggest that an influence on myelination processes is possible.While gray and white matter abnormalities have been consistently reported in psychoses not related to the puerperium oligodendrocytes and facilitate partial remyelination, even in the presence of immune activation Studying white matter structure using sufficiently sensitive methods may be particularly informative in the context of PP, given evidence that maternal brain structure and associated physiology change substantially during pregnancy and into the postpartum period CCN protein family member CCN3 had been suggested as a candidate mediator of PP risk by our work in a novel pharmacological mouse model Ccn3 expression was upregulated in whole brain tissue from new mouse mothers acutely administered an inhibitor of steroid sulfatase  Ccn3 expression The recent publication of a study examining the cellular and molecular underpinnings of MS prompted us to consider previously unexplored mechanisms that may contribute to PP vulnerability. This new study elegantly demonstrated that Tregs can promote oligodendrocyte differentiation and (re)myelination within the CNS independently of inflammatory processes via secretion of the protein CCN3  Our pharmacological model also revealed increased brain gene expression for putative CCN protein interactors, including the pro-depressant CCN2(CTGF) CCN gene family members, including CCN3, are highly expressed throughout the cortex and the limbic system The CCN3 protein is present in the extracellular matrix, the cytoplasm, and the nucleus, and is involved in many diverse cellular processes including cell adhesion, migration, proliferation, differentiation, and survival CCN3 expression and/or with the greatest accessibility to Treg influx.The intriguing findings described above, together with emerging knowledge of immune system and brain structural alterations in PP, have allowed us to generate a novel and testable evidence-based hypothesis regarding PP risk that reconciles these lines of evidence. Generally, we propose that women at risk of PP are more likely than healthy individuals to display abnormalities in the immune system–CCN family (and interactors)–(re)myelination axis and that disruption of this axis is a core pathophysiological feature of PP. Specifically, we propose that a perturbation in Treg abundance/activity in women at risk of PP may be associated with altered expression of the CCN3 protein in the brain and subsequently with reduced levels of myelination in cortical and limbic brain regions exhibiting the highest If dysfunction of the aforementioned axis contributes significantly to PP pathophysiology, we might expect key risk and protective factors associated with the condition to impact upon it. Patients with bipolar disorder show consistent evidence for reduced numbers of circulating Tregs Interestingly, systemic kynurenine levels  fluctuate considerably across mammalian pregnancy and the postpartum period Finally, it is unclear how Tregs may mediate myelination processes in the brain in women at risk, or with a history, of PP. Theoretically, this could be via secretion of factors such as CCN3 into the peripheral circulation and subsequent uptake into the brain. Alternatively, Tregs may percolate into the brain via a permeabilised blood–brain barrier (perhaps facilitated by increased CCL2 expression) and/or via the choroid plexus to affect function in the nearby limbic system CCN family and DDR1 genes; levels of CCN3 (and CCN2) gene/protein expression in peripheral tissues such as serum, cerebrospinal fluid, and urine during pregnancy and the postpartum period; and the degree/integrity of myelination in relevant limbic and cortical structures.The precise mechanistic details by which Tregs might permeate the CNS, produce and secrete CCN3, and by which CCN3 may then influence oligodendrocyte-mediated (re)myelination processes remain to be clarified over the coming years. Identifying the effectors of these processes would allow a more focussed genetic, biochemical, and cellular investigation of the pathophysiology of PP. For example, genetic studies might focus upon specific candidate genes or pathways that are highlighted, negating the need for genome-wide multiple testing correction. A putative interactor with CCN3, discoidin domain receptor 1 (DDR1) in vivo or postmortem using imaging and/or histological methods, respectively. In adult mice, Treg depletion in combination with exposure to environmental stressors results in phenotypes of relevance to PP (notably increased anxiety-related behaviours) together with decreased hippocampal serotonin levels Ccn3 expression, hippocampal 5-hydroxytryptamine, and myelination levels in postpartum versus non-postpartum female mice, and versus control postpartum mice. Finally, gross manipulation of CCN3 levels in vivo  or in vitro in mixed neuron–oligodendrocyte cell cultures may be associated with perturbed myelination and corresponding PP-relevant behavioural phenotypes. Likewise, in the pharmacological and genetic mouse models that display abnormal maternal behaviour and altered Ccn3 expression Our hypothesis may also be tested in more experimentally tractable systems, such as rodent models in which detailed measures of myelination can be obtained We believe that the hypothesis described above provides a plausible and coherent central risk mechanism for PP, drawing together existing lines of evidence implicating risk and protective/treatment factors. The hypothesis makes specific and testable predictions across many clinical and experimental systems (see Outstanding Questions). Confirmation of abnormalities in the Treg–CCN3–myelin axis in PP might feasibly identify new avenues of treatment for PP  is a rare, severe, psychiatric disorder affecting some women shortly after childbirth; risk is known to be substantially elevated in women with a previous history of bipolar disorder, schizoaffective disorder, or PP, but the underlying pathophysiology is extremely poorly understood. Moreover, approximately half of the time, PP occurs in women with no previous psychiatric history.While the current treatments for PP are relatively efficacious, they do not target the cause of the disorder, they may be teratogenic if administered prophylactically during pregnancy, and they may have adverse side effects. There are currently no predictive markers available for PP; such markers would allow women at high risk to be identified early in pregnancy, thereby facilitating earlier, more individualised, and more effective clinical intervention.Here, we hypothesise, based upon emerging immunological, animal model, and neuroimaging data, that PP risk may be mediated by dysfunction of the regulatory T cell–CCN protein family–(re)myelination axis, a pathway that is also potentially aberrant in multiple sclerosis (MS); hence, we suggest that increased collaboration between clinicians and basic scientists studying PP and MS is warranted.The identification of abnormalities within the axis above in women at high risk of PP could ultimately allow the development of predictive biomarkers and more targeted treatment options with fewer side effects for this vulnerable group.Outstanding QuestionsHow do regulatory T cell (sub)populations differ between women who have experienced an episode of postpartum psychosis (or who are at high risk) and mothers who have not?CCN2, CCN3, and DDR1 genes differ between women who have experienced an episode of postpartum psychosis (or who are at high risk) and mothers who have not?Does the sequence of CCN2, CCN3, and DDR1 genes (and their associated proteins) differ between women who have experienced an episode of postpartum psychosis (or who are at high risk) and mothers who have not?Does the peripheral expression of Do markers of (re)myelination efficacy differ between women who have experienced an episode of postpartum psychosis (or who are at high risk) and mothers who have not? If so, do these changes show any degree of regional specificity in the brain?To what extent do between-group differences in regulatory T cell populations, the CCN system, and myelination markers correlate with one another?Do animal and cellular models with experimental manipulations of regulatory T cell number/function and/or CCN expression exhibit anatomical, neurochemical, and behavioural features relevant to postpartum psychosis?Might a better understanding of the molecular pathways mediating immune system-mediated remyelination pathways allow for better postpartum psychosis risk prediction ?Finally, our idea suggests that not only studies in inflammatory and autoimmune conditions previously associated with PP  but also studies investigating immune conditions associated with aberrant (re)myelination and neuropsychiatric phenotypes such as MS might be informative for understanding PP pathophysiology; as such, a greater degree of collaboration between clinicians and scientists working within these areas is warranted.