Large vertebrates affect fire regimes in several ways: by consuming plant matter that would otherwise accumulate as fuel; by controlling and varying the density of vegetation; and by engineering the soil and litter layer. These processes can regulate the frequency, intensity and extent of fire. The evidence for these effects is strongest in environments with intermediate rainfall, warm temperatures and graminoid-dominated ground vegetation. Probably, extinction of Quaternary megafauna triggered increased biomass burning in many such environments. Recent and continuing declines of large vertebrates are likely to be significant contributors to changes in fire regimes and vegetation that are currently being experienced in many parts of the world. To date, rewilding projects that aim to restore large herbivores have paid little attention to the value of large animals in moderating fire regimes. Rewilding potentially offers a powerful tool for managing the risks of wildfire and its impacts on natural and human values.This article is part of the theme issue ‘Trophic rewilding: consequences for ecosystems under global change’. Most reIn many ecosystems fire is a natural recurrent disturbance that can promote habitat heterogeneity and maintain biodiversity through complex interactions with food webs . HoweverHere, we ask whether rewilding can contribute to reducing the risks and impacts of wildfire in fire-prone landscapes. The core of the paper is a systematic review of the evidence that vertebrates are able to control fire regimes and of the mechanisms involved. Having considered this evidence, we place it in a biogeographic framework to identify the biomes and climate conditions under which effects of vertebrates on fire regimes have been demonstrated. We conclude by translating this evidence to current opportunities for rewilding projects in parts of the world where natural environments and human communities face increasing threats from destructive wildfire.2.Vertebrates can influence natural fire regimes in several ways. First, herbivores limit fuel quantity by consuming and recycling plant matter that would otherwise accumulate as litter, and by reducing the density of vegetation . Second,3.(a)Many of the effects of herbivores on ecosystems are likely to increase with body size. This is because large herbivores are typically bulk feeders on low-quality plant material and thus consume a greater proportion of structural plant tissue than do small herbivores . FurtherHomo sapiens [Sporormiella, which are obligatorily associated with vertebrate herbivores and sporulate only on their dung [During the late Quaternary the largest herbivores vanished from most of the world's habitable continents and large islands in a wave of size-selective extinctions that followed the global expansion of modern  sapiens ,16. The  sapiens . Severaleir dung , to testeir dung . Dung fuSo far, studies at 14 sites have used dung fungi to track herbivore decline through time intervals spanning regional extinction of megafauna , while providing matching records of charcoal and pollen . At six of these sites the vegetation was forest or woodland before megafaunal extinction, and at these sites extinction was associated with large increases in charcoal. In several of these cases the temporal resolution of sampling was fine enough to show that charcoal increase followed dung-fungus decline on time-scales of decades or centuries –24. In sIn contrast, megafaunal extinction was associated with no apparent change in fire or vegetation at sites where the original vegetation was treeless tundra, steppe or arid grassland –31. The These are all observational studies that are subject to several uncertainties of interpretation. The most obvious is that we cannot test if increases in charcoal in past ecosystems truly signal higher flammability in response to relaxation of herbivory, or whether they had other causes such as rapid climate change or firing of the landscape by newly-arrived people . Also, charcoal concentrations in sediments are at best a crude measure of fire regimes . For exa2 conditions of the last glacial cycle—increases in fuel following loss of large herbivores may have been too small to cause observable changes in charcoal accumulation.Nonetheless, the evidence to date supports the hypothesis that past disappearances of large herbivores triggered increased fire severity or frequency, at least in places where abiotic conditions allowed development of woody vegetation. In such environments, increases in plant biomass due to relaxation of herbivore pressure may have been large enough to shift landscapes to states in which fire was more frequent or more intense, or both. In less productive environments, either too cool or too dry for development of woody vegetation cover—especially under the low-CO(b)There is overwhelming evidence that extant large herbivores reduce herbaceous fuel loads. For example, grazer exclusion in Hluhluwe iMfolozi Park in South Africa led to increased grass biomass ; in mixeWhile there are many studies demonstrating that vertebrate herbivores reduce fuel accumulation, there are relatively few which demonstrate that these reductions of fuel are sufficient to affect fire regimes. The available evidence is summarized in et al. [Cervus canadensis) had a sixth of the litter and standing dead biomass compared to unoccupied sites, and fire on those sites consumed less of these fuels and caused a smaller increase in bare ground [Artemisia) communities in Oregon found that consumption of fine fuel by fire was lower with moderate intensity pre-fire cattle grazing compared to ungrazed exclosures, resulting in reduced mortality of large perennial bunchgrasses [Bubalus bubalis), mortality of juvenile trees was three times higher in burnt than unburnt plots, but in the presence of buffaloes there was no difference in mortality between burnt and unburnt plots [Experimental studies showing effects of vertebrate herbivores on fire include Kimuyu et al. , who shoet al. . In grase ground . A long-hgrasses . A largent plots .Erica shrublands, cattle grazing resulted in slower post-fire fuel accumulation and discontinuous litter, and modelling suggested this would reduce fire intensity and rate of spread [Several different modelling approaches have been applied to the relationship of vertebrate herbivores and fire regimes. One class of studies uses modelled relationships between fuel loads and fire behaviour to predict changes in fire resulting from herbivore-caused alterations of fuel. In high-altitude Ethiopian f spread . In the f spread , while af spread . The spaf spread . Other af spread . New dynf spread , and couf spread .(c)The strongest evidence that vertebrate herbivores affect fuel loads and fire regimes comes from studies of large grazers, rather than browsers Scattered anecdotal observations suggest that animal trails and similar disturbances can function as firebreaks. The physical scale of these features can be substantial. For example, in a montane vegetation complex in Tasmania, 10% or more of the ground surface was covered by animal paths, mostly created by medium-sized macropods and wombats. Path surfaces were either bare ground or compressed leaf litter . The effSus scrofa) may disturb 27–54% of the forest floor [Bettongia penicillata), a marsupial that digs for truffles and invertebrates, found that its activity reduced surface litter loads in dry woodlands by 25%; this translated to a 74% reduction in flame height and a 33% reduction in the rate of fire spread predicted by a fire model [Isoodon obesulus) had a similar effect on fire predictions in remnant woodland in the city of Perth [Menura novaehollandiae) forage by turning over surface soil, burying leaf letter in the process. This activity reduced litter loads by 25% over nine months in plots where lyrebirds were free to forage, compared with plots from which they were excluded. Fire models predicted that this would cause up to twofold reduction of flame height, depending on weather [Leipoa ocellata) reduced fuel loads and model-predicted fire intensity in the vicinity of mounds [Vertebrates that dig for their food or otherwise disturb the litter layer can also affect fuel by burying plant litter or mixing it with soil. Some of these engineers have large physical effects on soil and litter. For example, in Switzerland, grubbing by wild boar (st floor . How sucof Perth . Superb  weather . Mound-bf mounds .4.a).In figure 1The geographical pattern of evidence for herbivore control of fire resembles a well-established pyrogeographic pattern of effects of fire on vegetation , in whicCeratotherium simum) create habitat heterogeneity and facilitate many other species .By removing fuel, large herbivores can to some extent replace fire as a dominant control of vegetation structure and density. This replacement is best understood in African savannahs. In savannahs with mean annual rainfall between approximately 500 and 800 mm, either fire or herbivory may be the dominant control on vegetation biomass . Notablyb). More research is needed in environments such as Mediterranean woodlands and shrublands and temperate forests worldwide.Geographically, studies of the effects of vertebrates on fire are concentrated in Africa, North America and Australia b. More r5.in situ recovery of native large herbivores (white rhinos and other grazers) from low levels enforced by past removals, and its role in management of conservation areas in southern Africa; intentional restoration of native large-herbivore communities in high-elevation woodlands in southwestern North America, where vulnerability to fire has recently increased due to increased fuel loads resulting from decline of domestic livestock, as well as climate change; and the effects on fire regimes of an invasive large herbivore, the swamp buffalo  in northern Australia that may be a partial ecological replacer for extinct megafauna, but with unwanted environmental impacts as well as beneficial effects on fire regimes.While trophic rewilding is potentially a powerful tool in the management of fire, its implementation will depend on many social, historical and environmental factors specific to particular landscapes. We illustrate some of these variations using detailed case studies of management of changing herbivore populations in relation to fire dynamics , detaileRewilding for management of fire could be especially important where recent changes in land use have increased the risk of dangerous wildfire. In much of the Mediterranean region, for example, original populations of large herbivores have long disappeared, but until recently fuel loads in managed savannah environments were controlled by domestic livestock ,71. WithLarge grazers may be especially important agents of fire control, given that they evidently have strong and consistent effects in reducing fuel loads and moderating the extent and impacts of fire in highly fire-prone environments such as savannahs. Grazers accomplish general reductions of fuel loads in biomes with a significant grass component, and they may also create and maintain open spaces with low fuel loads—grazing lawns—that can impede fire spread. Potentially, grazing lawns could be initiated by managers using mechanical removal of woody cover to stimulate localized grass growth and recurrent grazing. This has been attempted in the Kruger National Park, in response to concern that frequent large fires were creating undesirable habitat and limiting herbivore numbers . IronicaPinus and Eucalyptus forests, in strategic configurations likely to be effective in containing fires and protecting sensitive environments or human communities. Browsers could be valuable in reducing woody vegetation cover and creating habitat mosaics, but at the potential cost of increased grass and raised fire-danger; they should be complemented by grazers to prevent this. On the other hand, heavy grazing can lead to shrub encroachment [Bison bonasus) [In other situations, designed grazing lawns could be used to break up otherwise contiguous flammable vegetation, such as oachment , and in oachment . Mixed abonasus) ,88, coulbonasus) .The non-consumptive effects of ecosystem engineers on fuel loads may well be a valuable tool in rewilding for management of fire. This is especially true in Australia, because many of the species that are most active in digging for food and mixing litter with top soil are medium-sized marsupials and large rodents that have recently undergone severe declines caused by invasive predators . Some of6.Our review of the evidence makes it clear that vertebrates can have strong effects on fire regimes. Increases in fire frequency, severity and extent due to decline of large vertebrates probably began with extinction of Quaternary megafauna. Recent and continuing declines of large herbivores could well be contributing to increases in wildfire now being experienced in many parts of the world , includi


Consistent with previous studies, we observed that participating in health insurance significantly decreased the probability of unmet medical needs by 15 percentage points (p.p.) and that of incurring catastrophic out-of-pocket (OOP) health payments by 7 p.p. relative to no enrolment in the NHIS. Households living outside a 1-h radius to the nearest hospital had lower reductions in financial risk from excess OOP medical spending relative to households living closer (−5 p.p. vs −9 p.p.). We also find evidence that in Ghana, the scheme was highly pro-poor. Once insured, the poorest 40% of households experienced significantly larger improvements in medical utilization (18 p.p. vs. 8 p.p.) and substantively larger reductions in catastrophic OOP health expenditure (−10 p.p. vs. −6 p.p.) compared with that of the richest households. However, health insurance did not benefit vulnerable persons equally from financial risk. Once insured, poor, low-educated and self-employed households living far from hospitals had significantly lower reductions in catastrophic OOP medical spending compared with their counterparts living closer. Taken together, we show that enrolment in the NHIS is associated with improved financial protection but less so among geographically remote vulnerable groups. Efforts to boost not just insurance uptake but also health service delivery may be needed as a supplement for insurance schemes to accelerate progress towards UHC.A central pillar of universal health coverage (UHC) is to achieve financial protection from catastrophic health expenditure. There are concerns, however, that national health insurance programmes with premiums may not benefit impoverished groups. In 2003, Ghana became the first sub-Saharan African country to introduce a National Health Insurance Scheme (NHIS) with progressively structured premium charges. In this study, we test the impact of being insured on utilization and financial risk protection compared with no enrolment, using the 2012–13 Ghana Living Standards Survey ( Key MessagesIn Ghana, participation in the National Health Insurance Scheme (NHIS) increased the probability of meeting medical needs and decreased the probability of incurring catastrophic out-of-pocket health payments.We reveal significant inequalities in the benefits derived from the NHIS across sociogeographic subgroups and find evidence that though the poorest benefit most from health insurance, these benefits are curtailed among vulnerable groups living outside a 1-h radius to the nearest hospital.Our study reveals the extent to which the social benefit of public health insurance derives from geographic accessibility to essential health facilities and highlights the socioeconomic groups for whom distance to care matters most.From a policy point of view, we show that improving the geographic availability of quality health services is as important as promoting enrolment in national health insurance schemes in order to boost progress towards universal coverage in low- and middle-income countries. A strategic global health priority, universal health coverage (UHC), is widely recognized as the means to ensure that individuals do not suffer financial hardship when accessing quality health services , as wellSommers Ghana was the first sub-Saharan African (SSA) country to introduce a National Health Insurance Scheme (NHIS). Previous studies have assessed the catastrophic and impoverishment effects of OOP health payments prior to the introduction of the NHIS in Ghana  and examine the impact of the first NHIS in SSA in its first 10 years of implementation. We stratify population subgroups based on travel time to the nearest hospital and household socioeconomic characteristics to evaluate the effect of health insurance on financial risk protection and utilization among high-risk and vulnerable beneficiaries with and without limited geographic accessibility to care. We use probit models with region fixed effects, which were further tested using propensity score matching (PSM) and instrumental variable (IV) estimation methods to address potential selection bias into insurance. Using this sample, we test the hypothesis that the poorest benefit more from national health insurance schemes, but that this is attenuated for beneficiaries living in remote settings.To address these gaps, we draw on the 2012–13 Ghana Living Standards Survey data  per adult annually, varying according to region of residence. Vulnerable groups that qualify for exemptions include children under 18, adults over 70, pregnant women, individuals with disabilities and indigents. Every member must pay an initial processing fee towards a membership card  and a yearly renewal fee . Individuals who are not registered in the NHIS are obliged to make OOP payments every time they access health services, which may result in financial hardship.Mixed participation generates a differential ‘basis for benefit entitlement’: contributory for formal workers, discretionary for informal workers and non-contributory for vulnerable persons. Though coverage varies widely as a result, Ghana’s scheme type is not uncommon among LMICs experimenting with health financing reforms as part of broader UHC strategies (We use data from the sixth Ghana Living Standards Survey (GLSS-6). The details have been described elsewhere  but brieCatastrophic expenditure is a binary outcome variable indicating whether OOP health payments absorbed an excessive share of the household budget. OOP health payments consist of annual household level spending on both inpatient and outpatient services and all other reported spending directly related to the receipt of health services. We express OOP health payments as a ratio of total household non-food consumption , whereas to study financial risk protection, we restricted our sample to individuals whose households made any OOP health payments in the year of the survey (n = 25 971). To evaluate differences in the effect of health insurance across vulnerable subgroups, we disaggregated our sample across levels of household socioeconomic characteristics. These include household consumption expenditure, education and employment status of the household head. Across each subgroup, we tested whether travel time to care  influences the effect of insurance on utilization and financial risk protection.To examine the effect of health insurance on utilization, we restricted our study sample to individuals who reported being sick or injured in the 2 weeks prior to the survey  clusters of enrolled individuals might arise because residents in some geographical areas share higher quality of medical services, and (2) individuals living in a geographic area with a higher concentration of insured individuals may be influenced by the enrolment behaviour of their peers . The peeWe included cluster insurance rate in first-stage probit regressions and obtained the predicted probabilities of NHIS participation for each individual:To mitigate possible selection bias due to observable characteristics, a PSM estimator was calculated, using NHIS-affiliated individuals as the treatment group . We usedWe matched treated and control individuals based on covariates that may influence selection into insurance. For the utilization outcome equation, we matched individuals based on (1) demographic , (2) individual-level medical need (i.e. illness severity), (3) head-of-household , (4) household  and (5) geographic  characteristics. For the catastrophic expenditure outcome equation, we matched individuals based on (1) head-of-household , (2) household , (3) household-level medical need  and (4) geographic  characteristics. When conditioning on these covariates, the observed outcomes of uninsured units can be reasonably used to estimate the counterfactual outcome of insured units in the case of no treatment.t-tests for the covariates used to satisfy the balancing property offer evidence that the propensity scores were properly identified . When we disaggregate socioeconomic groups based on proximity to care, we find that vulnerable groups  benefit consistently less from health insurance when living outside a 1-h radius from the nearest hospital.P < 0.10). We observe larger reductions of catastrophic health expenditure among households headed by members with higher compared with that of the lower education  and among households headed by employed, compared with that of the self-employed members . When we disaggregate socioeconomic groups based on proximity to care, we consistently find that vulnerable groups who live farther than 1 h away from the nearest hospital benefit significantly less from the financial protection afforded by health insurance.t-tests (see We conducted a series of robustness and sensitivity tests on our PSM models by comparing relative effects across three alternative matching methods. In addition to NN without replacement, we applied kernel, radius and Mahalanobis matching. We verified the covariate balance graphically across matching procedures by comparing the standardized bias in matched and unmatched samples see . In addiests see  and estiWe also conducted simulation-based sensitivity analyses allowing us to assess whether the ATT estimates are robust to failures of unconfoundedness. All sensitivity analyses convey robustness of the matching estimate with respect to reasonable failures of the conditional independence assumption see . The comDetecting the conditions under which national health insurance systems offer protection to the insured and identifying the least protected beneficiaries is an important, albeit largely under-investigated area of research. Our findings show that participation in the NHIS increased the probability of meeting medical needs and decreased the probability of incurring catastrophic OOP health payments relative to no enrolment. We reveal significant effect differences across socioeconomic subgroups and find evidence that the poorest benefit most from health insurance, though these benefits are significantly curtailed among geographically remote vulnerable groups.We consistently find that poorer beneficiaries living outside a 1-h travel time to the nearest hospital benefit significantly less from the financially protective effect of health insurance. The fact that higher travel times are associated with utilization and financial protection penalties among vulnerable beneficiaries reveals an insightful decision-making mechanism. Poorer, less educated and precariously employed geographically remote households tend to forgo care, despite being insured, due to the time, difficulty and/or costs associated with reaching a health facility. For households faced by the disincentive of living far from a hospital, being enrolled in insurance is not a sufficiently effective incentive to utilize services even with the expectation of free care upon arrival.We show that being enrolled in the NHIS may still not be sufficient to ensure financial risk protection and access to health services among the most disenfranchized sociogeographic subgroups. They highlight that insurance schemes are unlikely to safeguard financial protection from catastrophic expenditure if higher-level healthcare facilities are not geographically accessible. Our findings are in line with a recent analysis of the Community-based Health Planning and Services initiative in Ghana, which underlined the importance of bridging geographical access to healthcare as a prerequisite to delivering on the promise of universal coverage (a priori degrees of vulnerability. Our results are also aligned with those obtained by previous studies on Ghana (Our findings are consistent with recent work by Moreover, the fact that households headed by less-educated members benefit less from the financially protective effect of health insurance indicates that navigating and securing the benefits of a national health insurance product is dependent upon the education level of beneficiaries. This partially reflects To ensure that the benefits of health insurance be experienced equitably across sociogeographic groups, UHC-driven policies should be enhanced with parallel improvements in transport infrastructure and focused expansion of the current hospital network to poorly serviced geographic areas. Our findings suggest that travel time is at least one of the decision-making components compelling insured individuals to seek or forgo needed healthcare. As such, we recommend the implementation of targeted health education interventions aiming to incentivize prompt care-seeking behaviour among geographically remote vulnerable groups. Our findings also indicate shortcomings concerning the implementation of policies meant to protect vulnerable people. In Ghana, vulnerable groups are exempted from paying enrolment premiums, however, the implementation of these policies is challenging. There may be important underlying conflicts between healthcare providers facing budget constraints and reimbursement uncertainty, and policies seeking to broaden access to care among vulnerable beneficiaries. Thus, implementation inefficiencies may be part of the explanation as to why some of the most vulnerable NHIS enrolees are least protected from financial hardship.These implications extend well-beyond Ghana, as other SSA countries with similar fiscal constraints are experimenting with hybrid health insurance schemes alike. Among them, Rwanda and Ethiopia have exemptions built-in their health financing structures aiming to target destitute groups. Our findings suggest that, although exemptions are part of the way forward, closer attention should be paid to long-term investments in road quality, supply network expansion and health education policies. Indeed, by targeting the junction of social, economic and geographic vulnerability, policymakers may be better able to identify a burdened high-risk group that is not yet benefitting from health insurance equitably despite the presence of well-intentioned exemptions.These findings should be viewed in light of the following limitations. First, although the comprehensive objectives that our work seeks to examine include access to promotive, preventive, curative, rehabilitative and palliative health services, we are able to assess the impact of health insurance on medical utilization focusing on curative care only. Second, though we consider UHC not as an end in and of itself but the means towards better health outcomes, our study assesses the effect of health insurance on improved health service use. Although there is a reason to believe that access to care leads to improved health outcomes, we do not directly measure the effect of the NHIS on these outcomes. Third, due to the data availability our study measures utilization 2 weeks prior to the survey and as such, offers a partial picture of utilization and a lower bound estimate of annual health service use. Fourth, the cross-sectional nature of our data has allowed us to capture annual OOP health expenditure at the time of the survey, which we have found to be sufficient to affect household financial well-being. However, it is possible that households incur recurrent catastrophic health expenditures, whose consequences may be more detrimental, and for which longitudinal data are needed.Overall, this study supports the UHC objective of the Ghanaian NHIS and offers valuable lessons to other LMICs seeking to broaden access to quality healthcare while lessening reliance on OOP payments. To our knowledge, our study is the first to investigate the effect of health insurance on utilization and financial risk protection across socioeconomic characteristics based on travel time to care. Our findings point to the need for developing more effective approaches to include vulnerable sociogeographic groups in nascent national health insurance systems and to ensure that they benefit equitably from utilization and financial protection. Finally, in an effort to identify the conditions under which health insurance offers protection to vulnerable beneficiaries, our study offers a novel contribution to the literature from a policy point of view. We reveal the extent to which the social benefit of health insurance derives from geographic accessibility to essential health facilities and highlight the socioeconomic groups for whom distance to care matters most.Ethical approval. No ethical approval was required for this study.czz093_Supplementary_DataClick here for additional data file.


Fetal growth is dependent upon utero-placental vascular supply of oxygen and nutrients from the mother and has been proposed to be compromised by vigorous intensity exercise in the third trimester. The aim of this systematic review was to investigate the effects of vigorous intensity exercise performed throughout pregnancy, on infant and maternal outcomes.Electronic searching of the PubMed, Medline, EMBASE, Cochrane Library, Web of Science and CINAHL databases was used to conduct the search up to November 2018. Study designs included in the systematic review were randomised control trials, quasi-experimental studies, cohort studies and case-control studies. The studies were required to include an intervention or report of pregnant women performing vigorous exercise during gestation, with a comparator group of either lower intensity exercise or standard care.n = 32,080) and five randomized control trials (n = 623) were included in the systematic review (n = 15), with 13 studies included in the meta-analysis. No significant difference existed in birthweight for infants of mothers who engaged in vigorous physical activity and those who lacked this exposure . Moreover, no significant increase existed in risk of small for gestational age , risk of low birth weight (< 2500 g)  or maternal weight gain . Women who engaged in vigorous physical activity had a small but significant increase in length of gestational age before delivery  and a small but significantly reduced risk of prematurity .Ten cohort studies  contains supplementary material, which is available to authorized users. Moderate intensity aerobic exercise throughout pregnancy is known to result in lower caesarean delivery rates, lower incidence of gestational diabetes and hypertensive disorders, decreased maternal weight gain, and improvements in antenatal and postnatal depression, and has not been found to negatively affect birth weight –3. HowevDecreases in utero-placental blood flow occurs during vigorous intensity exercise, and has been shown to result in fetal bradycardia . Physica2peak, it is recommended by the Canadian Guideline for Physical Activity throughout Pregnancy that women target a heart rate of between 142 and 169 bpm, depending on their age ). Those who participated in vigorous intensity exercise gave birth at an average of 39 + 4 weeks vs. 39 + 3 weeks in the control groups. In all studies, women who performed vigorous exercise were compared with those who did light or no exercise . Effect sizes were similar, but findings were not significant when exploring RCTs , prospective  and retrospective  studies separately, potentially due to the smaller number of studies and participants in each meta-analysis.A small but significant increase was observed in gestational age at delivery of babies of women who engaged in vigorous intensity exercise . These findings did not replicate when examining only the two RCTs  or when using only light intensity exercise as a comparison .Similarly, a small, but significant, reduced risk of prematurity existed in babies of mothers who engaged in vigorous physical activity . These findings were consistent across study design and comparison condition  of exercise sessions, that contributes to the lower birth weight reported in some of the studies. Bell, et al.  identifinot high-intensity (≥90% MHR). This is significant to highlight as a study in elite athletes showed normal fetal heart rate responses to an acute bout of exercise conducted at 23–29 weeks of gestation, until the intensity reached above 90% MHR [It is important to note that most studies in this systematic review reported intensities below 90% MHR . Indeed, according to the terminology reported in Norton, et al. , most st 90% MHR . Only tw 90% MHR , did rep 90% MHR , 41 and  90% MHR .Due to the difficulty in accurate assessment of MHR during pregnancy (as a result of haemodynamic changes), associations of exercise intensity with ratings of perceived exertion are recommended . The useIt has been recommended by the Canadian Guidelines Consensus Panel for Physical Activity Throughout Pregnancy that chronic high-intensity exercise, above the target heart rates recommended, is only undertaken in a monitored environment . Of the Moderate intensity exercise is well reported to reduce gestational weight gain in normal weight, overweight and obese pregnant women . HoweverThere are both strengths and limitations to this meta-analysis. The main strengths of the paper are that it is the first of its kind to pool the evidence for studies reporting vigorous intensity exercise specifically in the third trimester. Further, by including both randomized, cohort and case-control studies we have been able to capture the scope of evidence in this area. Indeed, by doing so we have been able to identify an important discrepancy in reporting of lower birth weight in RCTs compared with cohort and case-control studies. However, the heterogeneity of the research designs is also a limitation in synthesising the evidence . A randoFurther research on the effects of vigorous intensity exercise on maternal and infant outcomes is still needed, particularly in separating the benefits or detriments of high-intensity exercise versus high volume of exercise in the third trimester of pregnancy. Thorough documentation of adverse events should be prioritised, and future studies should also examine placenta function and growth in combination with birth weight. More evidence is needed on the impact of higher intensity on birth outcomes in elite athletes, who are the population likely to be exercising at > 90% MHR . It is aThe findings from this meta-analysis indicate that vigorous intensity exercise during the third trimester appears not to compromise birth outcomes for most low-risk pregnancies. Moreover, women undertaking vigorous intensity exercise had a significantly lower risk of prematurity. On the other hand, the meta-analysis did identify that RCTs showed a non-significant reduction in birth weight, which was not replicated in the cohort studies. However, this did not translate to a significantly increased risk of infants born small for gestational age. If the fetus is on the lower end of the birth weight chart, it may therefore be safer to suggest only moderate intensity exercise be undertaken in the third trimester. There was also no added benefit of vigorous intensity exercise over moderate intensity exercise or standard care on maternal weight gain in healthy weight women. However, vigorous intensity exercise did reduce maternal weight gain in overweight and obese pregnant women. Without a higher quality of evidence, any vigorous intensity exercise program during pregnancy should be individualised and conducted with guidance from an exercise professional and medical practitioner. Pregnant women should avoid exercising at a perceived exertion relative to ≥90% MHR, until further research can confirm its safety. The findings from this meta-analysis will help guide women and practitioners in prescribing vigorous intensity aerobic exercise throughout all trimesters of pregnancy.Additional file 1Systematic Review Search Terms (DOCX 15 kb)Additional file 2Exercise and Sports Science Australia position statement on physical activity and exercise intensity terminology. Reproduced with permission from Norton et al. .


A large proportion of offenders in the criminal justice system have alcohol-related problems. Therefore, it makes sense to implement alcohol screening and brief intervention programs for people in this setting, particularly for impaired driving offenders, who are likely to be alcohol dependent. Although most States mandate screening for impaired drivers, not much effort has been put forth to determine how the screening process could be improved and expanded to the entire criminal justice population. For example, more research is needed on the potential therapeutic benefit of the screening process and on how brief motivational interventions could be incorporated into this process to improve outcomes. To address this, more emphasis should be placed on developing and implementing national standards for screening programs in the criminal justice system, evaluating existing programs, and assuring that these programs provide adequate treatment services to offenders. Alcohol misuseThese observations indicate that a large number of people in the criminal justice system  have alcohol-use problems that should be addressed to prevent recidivism. This article describes the scope of alcohol problems among criminal justice populations, summarizes current knowledge about alcohol screening programs with these populations, and reviews the existing literature on the usefulness of these programs. Brief intervention approaches used in the criminal justice system also are discussed. The article concludes with recommendations for improving the alcoholism treatment services offered to clients in the criminal justice system.The association between alcohol use and criminal behavior is based at least in part on alcohol’s diverse physiological effects. In lower amounts, alcohol has a stimulating effect, acting both directly and indirectly on the brain’s pleasure center to induce a “high” that may motivate the drinker to consume more alcohol. As the person drinks more, however, alcohol begins to cause sedative and toxic effects, such as problems coordinating movements, longer reaction times, neurocognitive impairment , and perceptual distortion. In drinking drivers, impaired coordination and longer reaction times can contribute to traffic crashes; in other situations, cognitive impairment or perceptual distortions can increase the risk of other violent behaviors.Aggressive and impulsive behavior is more frequently associated with the stimulatory effects of alcohol. Moreover, alcohol intoxication is more likely to stimulate violent actions in people predisposed to aggressive behavior, such as those who are highly impulsive or who have antisocial personality disorder . Thus, aThe Scope of Alcohol Problems in the Criminal Justice System21.6 percent of victims of violent crimes thought or knew the offender had consumed alcohol; another 1.5 percent of the victims thought the offender had used either alcohol or another drug .40 percent of offenders on probation, in State prisons, or in local jails reported using alcohol at the time of their offense .There were 1.4 million DWI arrests in 2001, making DWI the number one crime, besides drug possession, for which Americans are arrested .18 percent of Federal prison inmates and about 25 percent of State prison inmates reported having experienced problems consistent with a history of alcohol abuse and dependence .29 percent of Federal and 40 percent of State prisoners reported a previous domestic violence dispute involving alcohol .About two-thirds of convicted DWI offenders are alcohol dependent .At least one-fourth of AOD-dependent offenders have lifetime histories of major depression or some form of bipolar disorder; 44 percent of inmates in a metropolitan jail had lifetime substance abuse disorders co-occurring with either depression or antisocial personality disorder .In a study of first-time DWI offenders interviewed 5 years after first being referred to screening following their DWI offense :85 percent of female and 91 percent of male DWI offenders had met the criteria for alcohol abuse or dependence at some time in their lives.32 percent of female and 38 percent of male offenders had met criteria for abuse of or dependence on another drug at some time in their lives.50 percent of women with an alcohol use disorder and 33 percent of men with an alcohol use disorder also had at least one psychiatric disorder (not drug-related), most commonly depression and post-traumatic stress disorder.Because increased alcohol availability is associated with higher alcohol consumption, communities where alcohol is readily available experience higher rates of alcohol-related crimes. Studies analyzing the effects of alcohol availability typically find that when access to alcohol is limited or inconvenient, or when alcohol is more expensive to purchase, the prevalence of alcohol-related problems  is reduced . These oBy the end of 2003, about 1.47 million people were incarcerated in U.S. Federal and State prisons, and an additional 4.85 million were on probation or parole ,b. Many No accurate statistics on the total number of criminal justice referrals to community-based alcoholism treatment programs are available. These referrals are numerous, however, constituting an estimated 40 to 50 percent of referrals to community-based programs . Other aThe main goal of screening criminal justice offenders is to identify those likely to have alcohol (or drug) use disorders. Another purpose of screening in some jurisdictions is to identify those who may benefit from sanctions, such as house arrest or electronic monitoring, that may restrict AOD use and reduce recidivism of individual offenders. In medical settings, short questionnaires can be used to screen people for alcohol use. In the criminal justice system, however, screening often incorporates procedures usually considered part of a more comprehensive assessment, such as more in-depth interviews, because offenders may be motivated to under-report their alcohol-related problems (see the section “Limitations of Current Screening Procedures”). Assessment in the criminal justice system typically involves examining the severity of a person’s AOD or mental health problems; this assessment then guides the development of a treatment plan. Together, screening and assessment aim to determine the need for further assessment, to ascertain which offenders need specialized treatment services, to match offenders’ treatment needs to appropriate services, and to determine the appropriate placement of offenders within different institutional units or facilities .There are no nationwide standards for whether or how non-DWI offenders should be screened for alcohol use. Similarly, the literature is largely silent on alcohol screening and brief interventions for people convicted of crimes other than DWI. Some drug court programs include screening components, but these have been neither well described nor well studied. Therefore, the remainder of this article mainly discusses the published literature that specifically addresses screening of DWI offenders.Most States mandate screening to evaluate the alcohol abuse problems of DWI offenders and to determine the offenders’ needs for further assessment and treatment . CurrentTesting—the use of self-report instruments  to evaluate the offender’s AOD use and related problems.Interviewing/assessment—face-to-face interactions between specially trained personnel and the offender to review the offender’s test results, further clarify the circumstances of the arrest, and identify family, medical, personal, or legal problems that may indicate a need for treatment.Referral and monitoring—referral of offenders for appropriate services, tracking their progress through the system, and assessing their compliance with court-mandated treatment.Ninety percent of screening programs surveyed report that they use both in-person interviews and self-report questionnaires . The mos2One factor limiting the effectiveness of current screening procedures may be the use of screening instruments that are not designed to evaluate offenders. Most instruments, such as the commonly used MAST, were developed in populations other than DWI offenders or other criminal justice populations and were not designed specifically for use in court-mandated screening . FurtherAnother potential limitation of current screening procedures is that all standardized instruments currently used for evaluating DWI offenders rely almost exclusively on self-reported information. Many offenders under-report their drinking, however, either unconsciously or because they want to avoid being labeled as having alcohol problems. Therefore, screening based on self-reports may underestimate the number of clients in need of intervention.Prompt and appropriate intervention after a DWI or other alcohol-related offense might offer offenders a unique opportunity to enter treatment without having to seek it on their own. Some offenders accept the need for screening and treatment, and several studies have demonstrated that clients who were ordered into alcoholism treatment by the criminal justice system showed reductions in alcohol use and illegal activities similar to clients who had entered treatment voluntarily . Other oAs a result of the limitations of current screening procedures and the coercive nature of court-ordered screening, offenders’ alcohol-related problems often are underdiagnosed. This is illustrated by a study that determined the rates of alcohol abuse and dependence in a population of 1,078 convicted DWI offenders . DiagnosOffenders may be screened at various stages of the judicial process, including at arrest or arraignment, at pretrial investigation, during interactions with court staff, or as a postsentence action. Just as laws are most likely to deter illegal behavior  if they are perceived to result in swift, certain, and severe sanctions e.g., , screeniWith the tight budgets in most communities and States, criminal justice systems faced with increasing numbers of incarcerated and nonincarcerated offenders and probationers are experiencing severe financial constraints. As a result, criminal justice systems are seeking to transfer much of the costs for alcohol screening and intervention to the offenders, particularly those who do not receive jail sentences for their offenses. For example, a survey of court processes for DWI screening revealed that only four States did not charge non-incarcerated offenders a fee for screening . Most DWFinancial constraints also affect the screening, assessment, and treatment of incarcerated offenders. Few correctional agencies have the financial resources to provide comprehensive assessment for all newly admitted inmates . Lack ofFederal regulations that serve to protect the confidentiality of patients receiving AOD abuse therapy also apply to criminal justice clients. Restrictions to prevent disclosure of information that would identify an offender as an alcohol abuser govern issues such as whether and how program staff may contact sources of information  or how the agencies responsible for the offender’s welfare communicate with each other about the offender’s assessment or treatment progress. Particularly when a team approach involving several agencies is used to screen and treat criminal justice clients, these confidentiality regulations can interfere with effective intervention.Information protected by Federal confidentiality regulations may be disclosed if the offender has signed a proper consent form. Obtaining the offender’s voluntary consent to information disclosure is the most commonly used method for allowing communication between the staff members of different agencies collaborating in the adjudication and care of AOD-abusing offenders .Research indicates that many offenders in the criminal justice system not only have alcohol use disorders but are likely to have other drug-related problems and mental illnesses as well. At least one-quarter of alcohol-dependent offenders have lifetime histories of major depression or some form of bipolar disorder. One study revealed that 44 percent of jailed inmates in a metropolitan jail had lifetime substance use disorders concomitant with either depression or antisocial personality disorder . High ra85 percent of the female and 91 percent of the male DWI offenders studied had met the criteria for an alcohol use disorder  at some time in their lives.32 percent of the female and 38 percent of the male offenders had met the criteria for abuse of or dependence on another drug at some time in their lives.Among the offenders with an alcohol use disorder, 50 percent of the women and 33 percent of the men also had at least one other psychiatric disorder (other than abuse of or dependence on another drug).The most commonly occurring comorbid disorders were depression and post-traumatic stress disorder.These findings indicate that criminal justice populations, including DWI offenders, should be evaluated for psychiatric problems commonly co-occurring with alcohol use disorders. This is especially important because studies in other populations have shown that alcohol-dependent patients with coexisting psychiatric disorders have worse treatment outcomes than patients without comorbid disorders .In various medical settings, brief interventions are recommended for patients who misuse alcohol and are at risk for dependence, but who are not alcohol dependent. These interventions typically:Involve four or fewer sessionsAre conducted in a nontreatment setting , andAre performed by health care providers and others who are not specialized in addiction treatment.One advantage of brief interventions is that they can be administered at a relatively low cost. For example, The process of screening DWI offenders for alcohol use disorders shares several of the characteristics of brief interventions. For example, screening usually involves one or two visits with the offenders. Therefore, screening itself is likely to have some impact on offenders’ drinking behavior. Consistent with this assumption, practitioners have recognized for more than 15 years that simply asking people about their drinking and its consequences can positively affect those people’s drinking patterns . ConsequScreening in the Criminal Justice SystemIn 2002, 40 percent of admissions to alcoholism treatment alone, and 34 percent of admissions to treatment programs for abuse of alcohol and other drugs, were accounted for by criminal justice/DWI referrals .Court-ordered screening misses many people with alcohol use disorders. In a study of 1,078 convicted offenders court-ordered to be screened for alcohol problems, lower proportions reported alcohol consumption patterns consistent with alcohol abuse or alcohol dependence at the initial screening than at a later voluntary screening .Limitations of screening procedures in the criminal justice system include:No screening instruments are available that have proven validity to assess both AOD use and the range of mental health problems found in criminal justice populations.No screening instruments are available specifically for criminal justice offenders.Current screening instruments rely almost exclusively on self report.Court-ordered screening is by definition coercive.Screening and treatment programs have limited financial resources; costs may be passed on to people being screened or treated who may be unable to pay.The appropriateness and efficacy of using brief interventions for offenders with AOD use disorders is undergoing evaluation. Such interventions, which typically consist of one to four treatment sessions and therefore are much shorter than traditional alcoholism treatment approaches, are increasingly being used in a variety of settings for clients with alcohol problems. Numerous types of brief interventions have been developed, ranging from advising clients to cut down on or quit drinking, to brief screening and feedback on results, motivational interventions, and contingency contracting. . These medications have been shown to be effective in preventing relapse when used in combination with psychosocial treatment such as brief motivational interventions. These pharmacotherapies are expensive, however, and their effectiveness has not been evaluated in criminal justice populations. Furthermore, compliance with taking the medications as prescribed may be low in these populations.In summary, not much is known about the effectiveness of various treatment approaches, particularly brief interventions, in criminal justice populations, including DWI offenders. Research conducted in other settings indicates that brief interventions can help patients reduce alcohol consumption and adverse consequences. Although these approaches should be effective with significant numbers of criminal justice clients, more studies are needed to establish their effectiveness with this group.A variety of other strategies that have proven useful for treating alcohol problems in the general population also may be appropriate for convicted DWI offenders. Because a significant proportion of these offenders meet diagnostic criteria for alcohol abuse or dependence, strategies found successful with people seeking treatment for their alcohol problem are likely to prove successful with significant segments of the DWI population. Therefore, alcoholism treatment for DWI populations can have a positive effect on public safety by reducing recidivism, as well as on public health by reducing the negative health and social consequences associated with excessive drinking.Alcohol abuse and dependence are highly prevalent among offenders in the criminal justice system, particularly among DWI offenders. For many of these people, screening and intervention could offer a valuable opportunity to reduce alcohol use and break the cycle of alcohol misuse and resulting criminal activities. However, a number of factors limit offenders’ access to comprehensive screening and treatment. In addition, the effectiveness of screening and the potential therapeutic effects of screening and brief intervention in these populations have not yet been evaluated adequately. Conducting this research is a challenge . Few judSo how can the availability of screening and brief intervention in the criminal justice system as well as their effectiveness in reducing recidivism be improved? Several options are possible:The procedures used to screen people entering the criminal justice system should be reviewed and retooled to better address the range of alcohol use and mental health problems found in criminal justice populations.More rigorous research studies should evaluate the effectiveness of screening and brief interventions for reducing recidivism among offenders. The current lack of evidence is particularly problematic considering the large number of people arrested each year for alcohol-related offenses and their high recidivism rates.More emphasis should be placed on understanding how different groups of alcohol-dependent offenders  fare within different screening and treatment modalities. When possible, research studies should include equivalent comparison groups.Despite the limitations of the existing research, investigators have gained sufficient knowledge regarding screening and brief interventions in the criminal justice system to recommend the following improvements to existing programs:A national strategy should be developed to improve and standardize current screening systems.Standards should be established for training and qualifying personnel conducting screening.Techniques of brief motivational interventions and similar evidence-based intervention approaches should be evaluated in criminal justice settings and incorporated into screening protocols. Given their effectiveness in other populations, it would be appropriate to include these approaches in education programs designed for DWI offenders who, based on screening results, are not alcohol dependent.Education programs designed for DWI offenders should include ongoing assessment for alcohol use disorders that may not have been identified during the initial screening or assessment. This is particularly important because alcohol-related problems often are underdiagnosed in these offenders.Less emphasis should be placed on self-reports and more emphasis on externally validated information  before making recommendations regarding possible interventions.Screening should address other drug use and mental health disorders that frequently co-occur with alcohol use disorders.Treatment services should be made accessible and affordable.Although these measures are associated with additional expense, the costs to society of failing to properly evaluate and treat alcohol abusers in the criminal justice system also are great. Tax dollars support law enforcement activities, the judicial system, and the costs of building and staffing jails and prisons. In contrast, appropriately delivered treatment costs much less than incarceration and can effectively change behavior and reduce re-arrests . Thus, d


The functional features of neural circuits are determined by a combination of properties that range in scale from projections systems across the whole brain to molecular interactions at the synapse. The burgeoning field of neurocartography seeks to map these relevant features of brain structure—spanning a volume ∼20 orders of magnitude—to determine how neural circuits perform computations supporting cognitive function and complex behavior. Recent technological breakthroughs in tissue sample preparation, high-throughput electron microscopy imaging, and automated image analyses have produced the first visualizations of all synaptic connections between neurons of invertebrate model systems. However, the sheer size of the central nervous system in mammals implies that reconstruction of the first full brain maps at synaptic scale may not be feasible for decades. In this review, we outline existing and emerging technologies for neurocartography that complement electron microscopy-based strategies and are beginning to derive some basic organizing principles of circuit hodology at the mesoscale, microscale, and nanoscale. Specifically, we discuss how a host of light microscopy techniques including array tomography have been utilized to determine both long-range and subcellular organizing principles of synaptic connectivity. In addition, we discuss how new techniques, such as two-photon serial tomography of the entire mouse brain, have become attractive approaches to dissect the potential connectivity of defined cell types. Ultimately, principles derived from these techniques promise to facilitate a conceptual understanding of how connectomes, and neurocartography in general, can be effectively utilized toward reaching a mechanistic understanding of circuit function. The mammalian brain is an impressive computational device, integrating external sensory stimuli with various internal states to select and implement adaptive behaviors. Can we provide a mechanistic explanation for how the brain performs each aspect of these computations? Just as the interactions of an atom can be understood by the orbital structure of its valence electrons, or the activity of an enzyme by the amino acid structures of its catalytic domain, one approach toward understanding the functions of the brain is to study the structural organization of its constituent circuits, cell types, and synapses. Only when we fully understand synaptic hodology and the dynamic interactions between circuit elements can we derive a mechanistic understanding of neural circuit computations .Since the foundational insight of the neuron doctrine made by Cajal over 100 years ago , we haveMuch progress has been made in the subsequent decades to address the simplifications made in these pioneering studies in order to paint a clearer picture of how the properties of the brain emerge from its constituent parts. The overarching goal of neurocartography is to approach a mechanistic understanding of brain function from a structural perspective by developing maps of the nervous system in terms of cell types, their synaptic connections, and the location of molecules that permit synaptic communication and plasticity . Just as3. The ultimate goal of neurocartography, to generate a connectome of the human brain, will need to contend with an estimated 86 billion neurons  section below for more discussion). In this way, smaller EM volumes can complement larger and more rapidly acquired LM volumes. Since the relevant spatial scale for neurocartography spans the mesoscale , the microscale , and the nanoscale , a variety of techniques will facilitate neurocartography see . Here, wIs there a canonical circuit motif that is repeated across all cortical structures, or does connectivity within circuits vary depending on their precise cortical area? How does the subset of synaptic inputs received by a neuron relate to the specific pattern of its axonal projections? Where exactly do circuits processing one modality of information converge with circuits processing information from a different modality? Mesoscale-level neurocartography approaches permit such questions to be examined in a cell-type specific manner over the entire extent of the mammalian brain.en passant boutons. Although the supposition that axonal-dendritic overlap is sufficient to describe connectivity patterns  frequenr’s rule , the spar’s rule .Of the methods that are capable of examining neurons and their potential connectivity across the whole mouse brain, one-photon microscopy  is widely used because of its availability and versatility. One-photon imaging is compatible with a large number of tissue preparations and permits multiple fluorophores to be imaged. These features dovetail nicely with the expansive toolkit of genetically encoded fluorophores  that canAn exciting alternative approach has recently been developed that circumvents some of these issues. Two-photon serial tomography, in which mouse brains are imaged on a high-speed two-photon microscope equipped with a vibratome slicer, permits fluorescently labeled structures to be mapped across the entire brain at high resolution  see Fig. In this1). The major advantage is the ability to unambiguously map out the regional projection targets of individual neurons, permitting experiments aimed at untangling how information is routed out of one circuit and into another at the level of single neurons. At the same time, one drawback of two-photon serial tomography, as it is implemented now, is that it neither reveals synaptic connections nor identifies the postsynaptic neuron classes. However, this approach could be combined with higher resolution, microscale approaches  to map synaptic connections between defined cell types across the brain.This is fundamentally different from the vast majority of tract-tracing experiments performed in the past, in which bulk injections led to a coarse description of the projection pathways in the brain. Already, this new approach has visualized populations of axon tracts of anatomically defined projection neurons , and beeNeurocartographic efforts at the microscale are focused on understanding the fundamental relationship between the dendritic organization of synaptic inputs and the computations performed by a neuron see . BecauseOne of the more versatile approaches for microscale neurocartography is AT, which was originally developed to probe the molecular phenotype of cortical synapses  see Fig. In the in utero electroporation), brain region (via virus injections), or cell-type  circumvents a major issue associated with conventional EM-based neurocartography, where the majority of the neuronal processes (dendrites or axons) that coarse through a given volume cannot be assigned to a particular cell type [though see , which is that synaptic connections are primarily inferred by the spatial colocalization of synaptic markers rather than defined by ultrastructure. This issue is compounded by the dependence of synaptic labeling on antibodies, which can be non-specific or only label a subset of synaptic structures. Correlative light-EM experiments, which should be performed for each antibody at each synapse type, have consistently demonstrated that AT can accurately identify bona fide synaptic connections with low false-positive rates .An alternative light-level approach for the analysis of synaptic connectivity avoids the issue of spatially resolving synaptic connections but rather defines them based on the functional complementation between two split, non-fluorescent GFP fragments; this approach is called GFP reconstitution across synaptic partners (GRASP) . In the The advantage of mGRASP is that synapses can be mapped across a large portion of a microcircuit including many neurons . Since tex vivo.Is there a spatial organization of spine size and associated strength of synapses along neuronal dendrites? Do synaptic strengths vary as a function of the afferent cell type providing the input? Do molecules that are important for synaptic function, such as neurotransmitter receptors and plasticity-related proteins, show equal expression at all synapses? These fundamental neurocartographic questions lie at the nanoscale, which for the last few decades has been almost exclusively investigated with traditional EM approaches  or with An emerging technology capable of simultaneously resolving cell-type specific connectivity and the nanoscale organization of synaptic proteins is expansion microscopy . ExpansiAlthough these light-level super-resolution technologies are powerful nanoscale approaches in their own right, they are still maturing and their optimal application to neurocartography is not yet entirely clear. In some cases, experiments can utilize separate but related datasets to place EM results within the context of ultrastructure  , 2009 capost hoc morphological reconstruction). In addition, the use of optical stimulation  enables the experimenter to distinguish between monosynaptic and polysynaptic inputs by bathing slices in a cocktail of sodium and potassium channel blockers that suppress endogenous action potential electrogenesis, meaning only ChR2-expressing axons can drive responses in the recorded neuron. Unlike AT or mGRASP, results from CRACM experiments can determine a number of important functional features. For example, CRACM maps can determine whether the synapses activated within a defined presynaptic class have high or low release probabilities, or if they contain a specific set of receptor subtypes compared to other synapses. However, the lack of a postsynaptic response to an optical stimulation cannot differentiate between a silent synapse or a lack of synaptic input. Combining techniques of activating and recording from neuronal subsets purely using optogenetic sensors and activators has given rise to all-optical electrophysiological approaches, which potentially can map connectivity across large numbers of defined sets of neurons in vitro or in vivo  has revealed a remarkable heterogeneity in how connectivity in the brain is organized. Recent results from a two-photon serial tomography approach using viral GFP expression to map the neocortical mesoscale projectome  have proResults from mesoscale, transsynaptic rabies mapping have suggested that cortical projections neurons have distinct sets of inputs, which differs from the broad input organization of the noradrenergic neuromodulatory system  [though + inputs; however, the authors found an unexpectedly large difference in the mGRASP+ excitatory input number on the apical and basal branches, arguing against random CA3-to-CA1 connectivity. The synapse numbers on these branches frequently differed from the predicted number of synapses based on a Poisson model, and inputs on single branches were found to cluster at a higher rate than predicted . These results suggest wiring mechanisms governing connectivity within the CA3-to-CA1 pyramidal cell pathway produce both branch-level and subbranch non-uniformities. Interestingly, such synaptic structure is not present in the same CA3 projection pathway onto CA1 interneurons. A recent study  wiring patterns are employed by distinct circuit elements and support specific cellular computations. This question has been addressed in the CA1 area of the hippocampus, in large part because of the interest in gaining mechanistic insight toward the mnemonic functions of the hippocampal formation. nt study  using thAn altogether different form of structured dendritic connectivity was found by using AT to examine inhibitory synaptic connections onto pyramidal cells within area CA1 . When thDo such structured connectivity patterns at the branch and subbranch levels generalize to other microcircuits in the brain? There is some limited evidence to suggest that they do; Neurocartography at the nanoscale has been dominated by studies using EM because of its inherent higher resolution  and abilMuch less LM nanoscale work has examined these features within the context of identified circuit connections . One approach toward this goal may be to obtain nanoscale data from EM experiments, then utilize light-level neurocartographic approaches in order to interpret the EM results within the context of previously identified neural circuitry. For example, several recent reports have found (using EM) that single axons can form multiple, “compound” synapses onto a target dendritic segment . Such coAn understanding of the structure-function relationship within the mammalian brain has remained elusive. This stems in large part because of the difficulties inherent in capturing the brain’s fine subsynaptic structures over volumes large enough to visualize whole brains, entire circuits, or even complete neurons. Significant advances in neurocartography have made remarkable progress, evidenced by the recent publication of the complete adult fruit fly brain imaged at synaptic resolution , a milesElectron microscopy reconstructions of tissue volumes from mammalian brains have gotten progressively larger yet remain far from the capability needed to produce a mesoscale map of the brain at nanoscale resolution. Two important reasons suggest this gap should only increase our excitement for the future of neurocartography. First, new light-level approaches that fill this gap continue to provide strong evidence that deconstruction of circuit connectivity is likely to be a fruitful avenue of research. Second, the pursuit of neurocartography continues to drive the development of creative new imaging technology and graph theoretical analyses that will enable the generalization of neurocartographic features across complex biological and artificial neural networks. As Sydney Brenner once said, “Progress in science depends on new techniques, new discoveries, and new ideas, probably in that order ,” the efEB and DH wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


From viruses to eukaryotes, genomic DNA filaments are confined in spaces of linear dimension much smaller than their contour lengths. In bacteriophages, the μm-long genome is stored in 50 nm-wide viral capsids and the corresponding packing density is so high that viral DNA filaments that have little chance to be entangled in solution (knotting probability <3%) become almost certainly knotted (>95% probability) once confined inside the capsid  showed that each chromosome occupies a compact region of the nucleus, termed territory  techniques have significantly extended our These and other advancements helped establish various results that foster the present discussion of genomic entanglement.Regarding inter-chromosome organization we recall that:the positioning of chromosome territories correlates significantly with sequence-dependent properties of the underlying DNA [most notably, gene density 〉 ~ ℓ−1 〉 ~ ℓ2/3  than the one expected (~ℓ−3/2) for equilibrated polymers  and unknotted  than at equilibrium. These heuristic conclusions are supported by various studies showing that the aforementioned scaling relationships obtained by FISH and Hi-C experiments can be ascribed to the topological constraints at play in solutions of unknotted and unlinked polymers (Khokhlov and Nechaev, These considerations appear particularly relevant for the structural modeling of eukaryotic genomes based on phenomenological data, such as spatial proximity constraints, which are typically too sparse to pin down even coarse-grained models of interphase chromosomes (Lieberman-Aiden et al., additionally be informed by the notion that interphase chromosomes must originate and eventually return to the separate and condensed mitotic state.A key question is whether such structural models should loci that were known to be significantly co-regulated. Importantly, topological constraints were accounted for by avoiding unphysical chain crossings during the steering process.Evidence presented in our earlier work help shed some light on the matter. With our co-workers, we considered a model system of six copies of human chromosome 19 in a cubic simulation box with periodic boundary conditions to explore the connection between coregulation and colocalization of genes (Di Stefano et al., Remarkably, and consistently with the gene kissing hypothesis Cavalli, , we founConversely, the same protocol applied to the same set of chains but initially prepared as generic self-avoiding random walks failed to give colocalization (Di Stefano et al., loci that corresponded to significantly enhanced entries of two independent Hi-C datasets (Dixon et al., Further elements come from the genome-wide structural modeling of human chromosomes of Di Stefano et al. . In thismicrophase separation of regions of different chromatin types (Jost et al., We note that the limitedly-entangled architecture of models of long eukaryotic chromosomes has emerged lately (Di Pierro et al., To some inevitable extent though, physical entanglements are still expected to arise in eukaryotic chromosomes.do occur in eukaryotic minichromosomes in vivo, for instance during transcription, due to transient accumulation of entanglement (Valdés et al., The recent work of Roca's lab showed that knots To conclude, we have discussed experimental evidence and general physical mechanisms based on polymer theory that consistently point to an unusually low degree of entanglement expected in long eukaryotic chromosomes. Such property, which is arguably essential for the capability of chromosomes to reconfigure as needed at various stages of the cell cycle, appears important for genomic modeling too.We argued that the structural modeling of long chromosomes can benefit, both for realism and computational efficiency, by starting off with disentangled self-avoiding chains, e.g., mitotic-like ones, because their plasticity makes it possible to accommodate a large number of phenomenological constraints in a physically-viable manner, i.e., without deformations involving intra- or inter-chain crossings.in vivo systems thanks to the action of topoisomerase enzymes. An important open question regards the extent to which these active mechanisms are involved in the shaping the overall intra- and inter-chromosome architecture. This point, we believe, can be significantly advanced in future studies with a tight synergy of experiments and models (Goloborodko et al., The latter are, of course, possible in All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent work towards this ambitious goal has provided new insights into the mechanisms governing life. With the fast-growing library of functional modules for synthetic cells, their classification and integration become increasingly important. We discuss strategies to reverse-engineer and recombine functional parts for synthetic eukaryotes, mimicking the characteristics of nature’s own prototype. Particularly, we focus on large outer compartments, complex endomembrane systems with organelles, and versatile cytoskeletons as hallmarks of eukaryotic life. Moreover, we identify microfluidics and DNA nanotechnology as two technologies that can integrate these functional modules into sophisticated multifunctional synthetic cells.With the ultimate aim to construct a living cell, bottom-up synthetic biology strives to reconstitute cellular phenomena  Bottom-up synthetic biology thrives in reverse-engineering a particular biological function using a minimal set of molecular components, like purified proteins.Recently, precision technologies, like microfluidics, have been used to recombine functional modules towards multifunctional synthetic cells. Synthetic biology can capitalize on a variety of pre-existing on-chip functions, which greatly increases the scope for complexity in the field.Advances in DNA nanotechnology gave rise to a diverse range of fully synthetic functional modules, like DNA-based ion channels or motors, which can replace some protein-based parts.Noteworthy progress has been made in achieving large and stable compartments, organelle-like multicompartment systems, and sophisticated cytoskeletal structures. These three features define the sections of this review as illustrated in Realizing the vision of a fully functional synthetic cell, however, will require strategies to recombine the mosaic of functional modules inside cell-like compartments. In this review, we explicitly highlight approaches that, in principle, allow for the stepwise integration of functional modules towards a multifunctional system (polymersomesgiant unilamellar vesicles (GUVs). Their key advantage lies in the use of lipids, which, as the building blocks of natural cell membranes, allow for the reconstitution of many cellular components without loss of functionality. GUVs are commonly produced by electroformation Typically tens of microns in size, eukaryotes are on average larger and enclose 100–10 000 times greater volumes compared to prokaryotic cells microfluidics has been used to produce GUVs on a chip by de-wetting of double-emulsion droplets  are examples of dynamic function-related membrane structures in eukaryotes. In synthetic compartments, however, the energetically favorable spherical shape remains predominant. Cells achieve membrane deformations inter alia via specialized membrane-inserting proteins. Reconstitution of such membrane-bending proteins, like epsin, into synthetic compartments revealed the concentrations required for membrane bending As a network of interlinking filaments extended throughout the cytoplasm, cytoskeletal elements share important roles in prokaryotes and eukaryotes, such as chromosome segregation and cytokinesis Filamentous actin (F-actin) polymerizes into polarized filaments, which favor growth at one end and shrinkage at the other. This ATP-driven process allows for rapid actomyosin cytoskeletal changes that generate forces for processes like motility or endocytosis. Minimal actin cytoskeleton systems have been reconstituted mainly in bulk. These works provided important insights related to the biophysical process of actin cytoskeleton formation and its functions. Imposing confinement, contractile actomyosin rings have been demonstrated in water-in-oil droplets in the presence of bundling factors The last step in the protrusion-based migration process is the myosin-based contraction of the actin network, combined with the detachment of the rear focal adhesions. Synthetic cells served as model systems to study how myosin builds tension in actin networks. Experiments reconstituting actin on the inside or on the outside of GUVs showed that cortex connectivity and membrane attachment govern the contraction outcome: cortices contract inward when weakly attached, whereas they contract towards the membrane when strongly attached Together with actin and intermediate filaments, microtubules form the eukaryotic cytoskeleton. They are capable of generating force via directional growth and serve as tracks for motor proteins, which carry organelles and other components to their target location. Compelling evidence exists that microtubules and molecular motors of the dynamin and kinesin families are involved in the tubulation of organelles, thereby establishing the architecture of eukaryotic cells.It has been shown that a minimal system consisting of GUVs and externally supplied microtubules and kinesin can produce membrane networks in the presence of ATP and GTP − without the aid of other proteins Deviating from natural systems, DNA-based motors that walk along a track are the most promising candidates to recapitulate intracellular transport in a purely synthetic manner. In these systems, movement is fuelled by the hybridization and subsequent hydrolysis of DNA base pairs between the DNA-based motor and its track. Long-range transport with rates up to 2 μm/min Recently, a DNA lattice has been absorbed onto the inner compartment membrane of a GUV In the past two decades, bottom-up synthetic biology has successfully reconstituted various phenomena occurring in living systems. Emphasis is now being put on strategies to integrate functional modules into multifunctional systems. Until recently, the lack of technologies for the sequential assembly and manipulation of synthetic cells was limiting the progress. This hurdle has largely been overcome thanks the introduction of precision technologies, like microfluidics, into the field. Previously developed functional modules can now, in principle, be assembled in a step-by-step manner with unprecedented control. These functional modules, however, may lose their activity when recombined due to undesired chemical or physical interactions or incompatible buffer conditions (see Outstanding Questions). Here, mimicking the architecture of eukaryotes with their multiple compartments and segregated reaction pathways holds the key to success. Engineering sophisticated membrane pores will then be necessary to allow for communication and selective exchange of reagents.Some cellular modules, however, will remain difficult to reconstitute. In this case, artificial mimics may be an adequate solution. Especially, the field of DNA nanotechnology offers new opportunities: as highlighted in this review, DNA-based constructs have successfully been used to program the assembly of cellular components and to achieve functionality that is normally provided by proteins. Additionally, the integration of synthetic designer proteins and computational tools for their structure prediction will increase the versatility of synthetic cells.Moving away from naturally occurring building blocks is conceptually interesting and offers a route towards truly synthetic systems, which has profound implications for the definition of life in itself. In some cases, however, it will remain difficult to match the efficiency and sophistication of engineering solutions offered by nature. Therefore, a combination approach using both natural and synthetic building blocks will likely be the best strategy. It will also be enriching to recombine bottom-up and top-down approaches to synthetic biology.Outstanding QuestionsWhat technologies, apart from microfluidics, allow for the sequential assembly of synthetic cells?Is there a universal ‘synthetic intracellular buffer’ that can be used for the assembly of all types of functional modules? Alternatively, can compartmentalization achieve the recombination of functional modules without loss of function?Which functional modules could or should be replaced with fully synthetic building blocks assembled, for example, using DNA nanotechnology? Can such hybrid systems perform new functions that are not found in nature?How will it be possible interface synthetic and natural cells and to create symbiotic cell communities? How can such systems be harnessed for applications, for example, in immunology or wound healing?How can synthetic cells be integrated into a compartment hierarchy analogous to the organelle – cell – tissue – organ structure of multicellular systems? Will it be possible to create tissues and eventually organs from synthetic cells?How can open-ended evolution be achieved in a synthetic cellular system?Will it be possible to achieve a fully synthetic cell which departs from nature’s own blueprint of cellular life and uses only artificial materials? What are the implications for the definition of cellular life?How can sophisticated hybrid cells be achieved, recombining building blocks from natural and synthetic cells? To give an example, will it be possible to boot a genome inside a synthetic compartment?Which ethical principles should be established for artificial forms of cellular life? At what point do they have to be applied?In the coming years, the integration of evolution into the conceptual toolbox of bottom-up synthetic biology will lead to exciting developments. So far, there is no study that has convincingly demonstrated open-ended evolution in a synthetic system. Additionally, a long-term goal is to integrate synthetic cells into a compartment hierarchy analogous to the organelle – cell – tissue – organ structure of multicellular systems. Here, it will be powerful to combine algorithmic self-assembly facilitated by DNA nanotechnology with microfluidic bioprinting of synthetic cells. After all, it was ultimately the emergence of multicellularity that gave rise to the mesmerizing diversity of life on Earth. We may thus be looking forward to witness future developments in this emerging field, when scientific curiosity continues to follow the intrinsic motivation of human endeavor – to discover the secret of life. Just like life will keep converging towards the best solutions, so will, no doubt, the myriad of approaches to synthetic biology.