This review focuses on non-invasive endocrinology, which is a key component of amphibian conservation physiology. It enables rapid assessment of reproductive and stress hormones in free-living and captive populations. It also provides a direct physiological measure of population sensitivity to extreme environments and their sub-lethal impacts on reproduction, health and survival. Non-invasive endocrinology utilizes non-invasive biological samples  for the quantification of hormones in wildlife. Urinary-based enzyme immunoassay (EIA) and radio-immunoassay have enabled the rapid quantification of reproductive and stress hormones in amphibians (Anura: Amphibia). With minimal disturbance, these methods can be used to assess the ovarian and testicular endocrine functions as well as physiological stress in captive and free-living populations. Non-invasive endocrine monitoring has therefore greatly advanced our knowledge of the functioning of the stress endocrine system  and the reproductive endocrine system  in the amphibian physiological stress response, reproductive ecology, health and welfare, and survival. Biological  validation is necessary for obtaining the excretory lag time of hormone metabolites. Urinary-based EIA for the major reproductive hormones, estradiol and progesterone in females and testosterone in males, can be used to track the reproductive hormone profiles in relationship to reproductive behaviour and environmental data in free-living anurans. Urinary-based corticosterone metabolite EIA can be used to assess the sublethal impacts of biological stressors (such as invasive species and pathogenic diseases) as well as anthropogenic induced environmental stressors (e.g. extreme temperatures) on free-living populations. Non-invasive endocrine methods can also assist in the diagnosis of success or failure of captive breeding programmes by measuring the longitudinal patterns of changes in reproductive hormones and corticosterone within captive anurans and comparing the endocrine profiles with health records and reproductive behaviour. This review paper focuses on the reproductive and the stress endocrinology of anurans and demonstrates the uses of non-invasive endocrinology for advancing amphibian conservation physiology. It also provides key technical considerations for future research that will increase the accuracy and reliability of the data and the value of non-invasive endocrinology within the conceptual framework of conservation physiology. The Earth's biodiversity is experiencing pressure from multiple threatening processes, such as habitat loss, over-harvesting, global climate change, invasive species, and pathogenic diseases, that could be working in synergy to escalate the global extinction crisis . Conservin situ populations  and laboratory validation. It then explores, using field and laboratory-based examples, the contributions of non-invasive endocrinology to amphibian conservation physiology. The review also deliberates technical considerations, including strengths, limitations, and directions for future research that will increase the accuracy, sensitivity, and reliability of the non-invasive endocrine data. Finally, the review provides a conceptual framework that integrates non-invasive endocrinology with other fundamental research areas within conservation physiology.et al., 2007). For the purpose of this review, a stressor (or stress event) will be defined as a noxious stimulant, which exposes the amphibian to energetic costs outside of that required for predictable daily and seasonal requirements. The field of stress physiology was born in 1936, when Hans Selye characterized the stress response as a common set of generalized physiological responses that were experienced by all organisms exposed to a variety of environmental challenges, such as temperature change or exposure to noise. Several decades later, Comprehensive reviews related to the key concepts of the stress theories are available . Stress Stress responses can include a range of physiological systems and behaviours, but the common element is activation of the hypothalamo–pituitary–interrenal (HPI) axis in amphibians, which is homologous to the hypothalamo–pituitary–adrenal axis in higher vertebrates . The HPIin vitro that corticosterone and aldosterone were the primary steroids formed in incubates of the interrenal glands of the American bullfrog (Rana catesbeiana) by stimulating their production using mammalian ACTH (Rana pipiens (Schreber)] and the northern wood frog [Rana sylvatica (Le Conte)] larvae. In another study, R. pipiens. Interestingly, earlier studies have also reported that fully aquatic amphibians produce cortisol as their primary glucocorticoid, while terrestrial or semi-terrestrial amphibians produce corticosterone as their primary glucocorticoid (Rana esculenta) via a competitive protein-binding radio-immunoassay (RIA) method using baboon plasma as the source of corticosterone-binding globulin (CBG). Their study most possibly provided the first demonstration of a seasonal pattern of plasma corticosterone in anurans. Two years later, Dupont and colleagues provided the first evidence of circadian and circannual variation in corticosterone in mature male and female free-living R. esculenta (Bufo americanus). Their study also provided insights into the role of corticosterone in amphibian locomotion. Following on, Rana catesbeiana). Two years later, R. esculenta. Numerous studies based on the plasma RIA methods went on to explore the relationships between stress and reproduction in amphibians (see detailed reviews on this topic by 125I (60 days), 131I (8 days), and 3H  plasma corticosterone increased at 30 min and was higher at 3 h after capture (Ambystoma maculatum) and in the Alleghney dusky (Desmognathus ochrophaeus) after capture in studies conducted by Plethodon shermani), Ocoee (Desmognathus ocoee), and Allegheny dusky salamanders (Desmognathus ochrophaeus), as well as for Coqui frogs  physiological stress responses in laboratory model animals, such as rats and mice . In amph capture . Plasma Rhinella marina) that the magnitude of the short-term urinary corticosterone stress response was affected by the intensity and the duration of manual restraint  causes increased urinary corticosterone metabolite concentrations in anurans  between initial capture and blood sampling is available, because it is known that in birds the stress responses occur fairly quickly . In contHelogale parvula) in the Serengeti National Park to urinate on a rubber pad during the course of normal scent-marking. The result was hundreds of urine samples that were analysable for reproductive hormone metabolites, allowing elegant examinations of behavioural and endocrine mechanisms of reproductive suppression. The evaluation of reproductive hormone metabolites in either urine or faeces permits the long-term study of reproductive patterns in individuals, populations, or species, all without perturbing the animal , a non-traditional model of an explosive breeding anuran species, urinary testosterone metabolite levels increased when male toads in breeding condition were exposed to a repeated capture handling stressor. In another study, using non-breeding male toads, there was an inverse relationship between urinary testosterone and urinary corticosterone metabolites during exposure to a manual restraint stressor (Comprehensive reviews on the amphibian reproductive endocrine system and its interaction with the stress endocrine system are available . The pro2), or by comparing temporal excretion patterns of hormones (e.g. urinary estrone and progesterone) with external signs of reproductive status  using human chorionic gonadotrophin challenge. The results demonstrated ‘raise and return to baseline’ profiles in urinary testosterone metabolites in male frogs and urinary progesterone and estrone conjugate metabolite profiles in female frogs over 2–3 days, which reflected the experimental stimulation of the hypothalamo–pituitary–gonadal axis. Administration of ACTH mimics a natural interrenal stress response by causing a rapid rise in circulating corticosterone, followed by a return to baseline within a few hours. The same pattern should also occur in urine or faeces, with the onset of peak excretion being delayed by the species-specific excretory lag time (Litoria wilcoxii) and the Fijian ground frog (P. vitiana), using an ACTH challenge that demonstrated ‘raise and return to baseline’ profiles in urinary corticosterone within 2–3 days, thus reflecting the experimental stimulation of the HPI axis  of hormones in the blood circulation are bound to CBG or sex hormone-binding globulin. It is only the ‘free fraction’  that is usually considered to be the biologically active fraction . It has been suggested that CBG and/or sex hormone-binding globulin transport hormones to target tissues . The livPeripheral metabolism occurs mainly in the liver and to some extent in the kidneys, which delivers steady-state levels of plasma androgens and oestrogens. Oestrogens and androgens are broken down into the same class of metabolites by sulfatases, dehydrogenases, and glucuronide transferases to enhance their solubility and to facilitate their elimination. Steroid inactivation could also take place in target tissues, mainly upon completion of the specific biological response. As highlighted earlier, inactive androgens and oestrogens are mainly eliminated as urinary (mostly conjugated) metabolites. This conversion to hydrophilic compounds ensures their solubility in biological fluids at rather high concentrations  and the conjugated horseradish peroxidase label that were produced in rabbits using protocols that were standardized for a standard direct competitive EIA system by Laboratory validation and standardization of each assay system is an important step towards establishing a reliable non-invasive endocrinology laboratory. Parallelism determines whether the assay system is detecting the hormone metabolites of interest. The parallelism curve also gives the sample dilution factor based on the 50% binding point of the sample on the standard curve . It is excreted at a constant rate in individuals with normal kidney function and therefore provides a good index of the amount of time over which hormones have been metabolized into the urine regardless of the volume of the sample. Creatinine index, also known as the ‘Jaffe Reaction’ after Max Jaffe, has been used widely for correcting urinary hormone metabolite measures . Another method, using sterile pipette tips (200 μl) works reasonably well for large to medium sized species, such as the Stony Creek Frog , red-eyed tree frog  and for the Great Barred Frog . Typically, the sterile tip of the pipette is inserted at a very gentle pace inside (5 mm) the anuran's cloaca while the frog is manually held , and urine is obtained by capillary action. For medium to small sized species, such the Philoria species, a non-heparinized micro-capillary tube (20–50 μl) could be used for obtaining urine. Sigma-Aldrich (#P2174) micro-capillary tubes have been widely used for the purpose of collecting urine in small frog species, such as Pseudophryne and Crinia species . The time required for urine collection from initial capture to sampling is normally between 30 s and 5 min.Amphibians should always be handled by wearing new non-powdered gloves or disposable freezer bags for each individual to avoid the spread of chytrid fungus , this same measure was found to be 98% reliable for distinguishing between known males and females (Leiopelma pakeka). B. americanus) and boreal toads (Bufo boreas boreas). Their study reported that faecal testosterone metabolite concentrations provided the most useful measure of sex in the American toads  and used this as a physiological index, in combination with body condition, of habitat quality and fragmentation. Their study used manual shaking as a psychological stressor to test the acute stress hormone response of the toads, and they found that low body condition and elevated baseline urinary corticosterone levels were associated with low forest availability and high forest fragmentation within a 500 m radius around the ponds. They highlighted that non-invasive stress endocrine measures should be used as a complementary approach that can enable early detection of population health status within changing environments.Recently, non-invasive urinary corticosterone EIA was used to demonstrate the stress associated with transportation and captive transfer of the endangered Fijian ground frog (R. marina) in field conditions and suggested that toe clipping could have detrimental sublethal impacts on reproductive hormones and behaviour. This sort of data is important, especially when debates are on-going regarding the welfare of amphibians and whether toe clipping should be completely prohibited in countries that have huge amphibian diversity  and the Mallorcan midwife toad (Alytes muletensis). Their study demonstrated that tadpoles with high chytrid fungus loads had high concentrations of corticosterone metabolites. In another study, L. wilcoxii), that adult frogs with high chytrid fungus loads expressed much higher baseline urinary corticosterone metabolite levels in comparison to the frogs that were negative for the chytrid fungus.The use of non-invasive stress endocrinology is also gaining popularity for study of the complexities of amphibian decline, especially within the context of interactions between the host, the environment, and the pathogenic disease chytridiomycosis . RecentlM. fasciolatus), in an attempt to test whether sublethal high levels of stress are suppressing other key physiological systems, especially immunocompetence, and making high altitude amphibian populations more vulnerable to chytrid fungus . Results have shown that species living at higher elevations also have much higher levels of baseline urinary corticosterone metabolites in comparison to their counterparts living at lower elevations (sampling was done at the same time of the year). Thus, non-invasive reproductive and stress endocrinology provides amphibian ecologists with a useful tool to understand the physiological sensitivity of anurans to environmental stressors and their sublethal impacts on amphibian reproductive ecology and fitness. Overall, non-invasive endocrinology tools should be used to gain a holistic understanding of the factors associated with chytridiomycosis-driven amphibian declines.In Australia and globally, amphibian declines have shown a strong association with altitude, which is used as a climatic gradient by field ecologists . Non-invUrinary EIA has a number of advantages that make it an excellent tool for studying the anuran hypothalamo–pituitary–gonadal axis and HPI axis functions. Enzyme immunoassay techniques are relatively cheap, and hormone metabolites can be measured in small volumes (120 μl of anuran urine required per hormone assay). Anurans tend to urinate frequently and, once collected, urine can be preserved indefinitely by freezing at −20 or −80°C. Faecal sampling could be very difficult in field conditions due to the need to find discrete scat samples in the wild that can be linked to an individual; however, urinary methods do not have this problem. Furthermore, collection of faecal samples from an elusive species, such as a frog, would be time consuming and could require the capture and captivity of individuals.The polyclonal antibodies being employed in urinary-based EIAs detect the total content of the steroid metabolites of interest (a combination of free as well as conjugated forms). These are the metabolic end-products of biologically active hormones that take part in the endocrine response and are metabolized in the kidney prior to excretion. Therefore, it is important to test whether there are significant differences in the levels of conjugated metabolic end-products, which can also be affected by bacterial degradation in the gut and differences in metabolic rates between sexes and individuals .As highlighted in the latest review by It should be made pre-requisite that appropriate biological validation through exogenous hormone challenge  is done for each anuran species for which non-invasive reproductive or stress hormone methods are being developed for the first time. This will provide useful information regarding excretory lag times and also demonstrate that the assay system is in fact detecting the hormones of interest. Another consideration is the need for the quantification of creatinine references when measuring hormone metabolites in urine, which requires sufficient volume of sample, and this could be a limitation when working with tiny frogs. Creatinine is a useful urinary marker to estimate filtration rates, because the conjugated conjugated hormones possibly gain entry into the bladder through active secretion through the proximal tubule. Therefore, it is important for studies utilizing urinary hormone measurements to use creatinine assay as a reference value.Furthermore, the cross-reactivity of the antibody employed for non-invasive reproductive and stress hormone measurements should be readily available to confirm that the antibody cross-reacts only with metabolites of the original hormone. For example, cross-reactivity of the CJM06 anti-corticosterone antiserum was 100% with corticosterone, 14.25% with desoxycorticosterone, and 0.9% with tetrahydrocorticosterone . In my view, urinary measurements of reproductive and stress hormone metabolites in amphibians should not have issues such as the effect of diet because urinary measures do not require correction by the dry faecal mass. It is important to measure the plasma free hormone, because only 1–10% of the total hormone in the plasma possesses biological activity. According to the free hormone theory, while a hormone is bound to a binding globulin it is not biologically active . CorticoThis review clearly demonstrates that non-invasive endocrinology plays a fundamental role in advancing amphibian conservation physiology. As shown in the conceptual framework Fig. , this phNon-invasive endocrinology contributes directly to the advancement of amphibian conservation physiology. A non-invasive endocrinology approach can be used to evaluate the success or failure of captive breeding programmes by assessing the longitudinal changes in reproductive and stress hormone metabolites of captive individuals and comparing these hormone changes with the reproductive status and behaviour data. The EIA systems employed these days are readily available and inexpensive. The urine hormone analysis goes beyond captive populations. It is a useful tool for studying populations in the wild, particularly translocated individuals, reintroduced populations, or areas where some sort of management intervention has been implemented. Stress maintenance plays an integral role in the reproductive success of amphibians, because high stress levels could lead to reproductive failures both in the wild and in captive situations. Through the analysis of urine samples, this type of tool can provide invaluable management information about the physiological stress responses and reproductive status of diverse amphibian species.In conclusion, given that amphibian populations worldwide are declining rapidly as a result of disturbances to natural habitats, it will become essential to utilize non-invasive physiological measures of stress and reproductive health, because these physiological measures can provide objective and quantitative measures of the impacts of disturbance. Non-invasive physiological indices are particularly useful for the assessment of the relative impacts of disturbance, delineating the pressures that most need mitigation as well as tracking the effectiveness of such mitigation efforts. As demonstrated in this review, there are numerous ways by which this novel method is assisting in the conservation physiology of amphibians. Reproductive and stress hormone evaluation in field conditions could be merged with behavioural ecology studies to help improve our understanding of the complex interplay between stress and reproductive hormones and the environment in maintaining reproductive fitness and survival of threatened species. Ultimately, through regular technical updating of biological and laboratory validations, this non-invasive endocrine tool will provide a long-lasting contribution to conservation physiology.


Protecting water quality in forested regions is increasingly important as pressures from land-use, long-range transport of air pollutants, and climate change intensify. Maintaining forest industry without jeopardizing sustainability of surface water quality therefore requires new tools and approaches. Here, we show how forest management can be optimized by incorporating landscape sensitivity and hydrological connectivity into a framework that promotes the protection of water quality. We discuss how this approach can be operationalized into a hydromapping tool to support forestry operations that minimize water quality impacts. We specifically focus on how hydromapping can be used to support three fundamental aspects of land management planning including how to (i) locate areas where different forestry practices can be conducted with minimal water quality impact; (ii) guide the off-road driving of forestry machines to minimize soil damage; and (iii) optimize the design of riparian buffer zones. While this work has a boreal perspective, these concepts and approaches have broad-scale applicability. Instead, it is the hydrological connectivity between surface water and the biogeochemical sources of different solutes draining catchment soils that matters the most , under the assumption that topography and gravity control water movement, and that the surface of the water table follows the soil surface  locating areas where more intensive forestry practice can be conducted with minimal impact on water quality; (ii) minimizing driving damage to wet organic soils; and iii) optimizing the design of riparian buffer zones.The landscape sensitivity framework that we call hydromapping Fig.  is basedThe strong topographic control over hydrological pathways and biogeochemical properties of soils provides the conceptual basis for landscape management. With new computational techniques and high-resolution data, the spatial distribution of groundwater flow, and thus the biogeochemically active zones, can be obtained solely from catchment topography. Indeed, the emergence of high-quality DEMs has led to the development of multiple algorithms that predict hydrological pathways and water accumulation in landscapes -based maps are becoming available, providing DEMs with up to 1 m cell size. Although such small-scale accuracy of the DEM can substantially improve the hydrological models and consequently hydromapping, it also introduces new difficulties related to data management, computing requirements, and selection of the right scale for calculations  are wider than adjacent drier riparian sites, and often exceed standardized buffer widths (Kuglerová et al. Variable riparian buffer widths can be effectively designed using hydromapping (Kuglerová et al. The boreal forest comprise a mosaic of different soil characteristics, interspersed by scattered wetlands and lakes, and transected by numerous streams and rivers. At the same time, boreal forests in many parts of the world also represent managed semi-natural landscapes, with large-scale industrialized forestry (Rist et al. Maintaining or even increasing biomass production in boreal forests does not necessarily mean that other ecosystem services such as biodiversity will be unduly impacted, or that we will jeopardize either long-term soil sustainability or cause unacceptable deterioration of water quality. The state-of-the-art knowledge of water flow pathways in the landscape can lead the way to new tools that are based on mechanistic understanding of ecosystem functioning, and landscape heterogeneity. However, the legal protection and policy regarding forest management in connection with protecting small headwater streams will also partly depend on how small and temporary waters are defined by authorities (Acuna et al.


The capacity for strategic thinking about the payoff-relevant actions of conspecifics is not well understood across species. We use game theory to make predictions about choices and temporal dynamics in three abstract competitive situations with chimpanzee participants. Frequencies of chimpanzee choices are extremely close to equilibrium (accurate-guessing) predictions, and shift as payoffs change, just as equilibrium theory predicts. The chimpanzee choices are also closer to the equilibrium prediction, and more responsive to past history and payoff changes, than two samples of human choices from experiments in which humans were also initially uninformed about opponent payoffs and could not communicate verbally. The results are consistent with a tentative interpretation of game theory as explaining evolved behavior, with the additional hypothesis that chimpanzees may retain or practice a specialized capacity to adjust strategy choice during competition to perform at least as well as, or better than, humans have. Interaction among organisms, in which each organism's action influences the fitness reward of the other, is ubiquitous in biological life. However, the ability of different species to think about payoff-relevant actions of conspecifics and respond to action histories is not well understood. In this study, game theory is used to make predictions about choices and temporal dynamics in three abstract competitive situations with chimpanzee and human participants. We show that frequencies of chimpanzee choices are close to equilibrium game theory predictions, and shift as payoffs change, just as equilibrium (accurate-guessing) theory predicts. This is a surprising result. Equilibrium predictions assume mathematically that all organisms making choices correctly anticipate what others do. Dozens of studies with human subjects show substantial deviations from that idealized equilibrium state. However, chimpanzee choices in our data are also closer to the equilibrium prediction, and are more responsive to past history and to payoff changes, than human choices. These results show how game theory can be consistent with evolved behavior23Pan troglodytes), behave as they make choices in incentivized competitive strategic interactions in within-species competition. The interactions are referred to as “games”. Game theory is used to make predictions about possible behavioral outcomes. A chimpanzee-human comparison in repeated competitive games is inspired by the “cognitive tradeoff hypothesis”. This hypothesis is that cortical growth and specialization for distinctly human cognitive capacities (such as language and categorization) conceivably reduced more basic capacities, such as detailed perception and pattern recognition, useful for tracking opponent choice during competitionWe see how our closest genetic relatives, chimpanzees , in which players were not initially informed of their opponents' payoffs. The games are direct and competitive: Joint actions create one winner and one loser. Both players can either press Left or Right touch-panel buttons . There aGame theory offers a benchmark of optimal performance: Players acting individually earn the most if they guess accurately what others do, and if they choose strategies with maximal expected payoff given those guesses. If both players do so, their choices form a “Nash equilibrium” (NE), a pattern of play in which choices optimally anticipate what others are likely to do.The behavioral results answer two questions: (1) How close are the frequencies of Left and Right choices, P(L) and P(R), to the game theory NE predictions?; and (2) How predictably do current choices respond to changes in the opponent's history and to payoff changes?extremely counterintuitive: In this class of games, the payoffs of Matcher subjects change. However, their predicted behavior should not change across the games (they are predicted to choose P(R) = .50 in all three treatments). Instead, the behavior of the Mismatcher subjects should change, even though their payoffs do not change.The close match of chimpanzee choice frequencies to game theory predictions is generally closer than in most previous human experiments . IntriguWhy do the chimpanzees so closely approximate game-theoretic equilibrium? One clue is that working memory of experienced chimpanzees is surprisingly goodTwo empirical findings about response times (RTs) are notable.First, chimpanzee RTs are much faster than human RTs , about 6Second, Matcher RTs are faster than Mismatcher RTs. This difference could arise if Mismatcher choices either involve more complex calculations, or require override of an automatic motor response to match. The motor-override interpretation is consistent with recent evidence in human competitive games, showing that matching an opponent's physical action is automatic—but, remarkably, the difference disappears if both humans wear blindfoldsOur experiments created strategic interactions between pairs of conspecifics using a simple touch-panel protocol. The choice frequencies of food-motivated chimpanzees match game theory predictions as closely as in any species and comparable learning setting ever observed, even when changes in rewards predict highly counterintuitive changes in behavioral choice frequencies. The chimpanzees' choices, compared to the human choices, are closer to those predicted by equilibrium game theory.There are two broad hypotheses consistent with the facts that chimpanzee choices are much closer to game theory than human choices.The first hypothesis is that there is a species-based confound in the experimental protocols, and if this confound were eliminated the chimpanzee and human results would then be closer.are some cross-species confounds in these experiments. We do not think they fully account for direction or magnitude of the chimpanzee-human difference. However, closer matches of human and chimpanzee protocols are conceivable, and would be important to establish more conclusively the differences our data simply suggest.There less competitive behavior, away from equilibrium; and the difference between chimpanzees and humans does not go in that direction, and in fact goes in the opposite direction  see 8; . The leaThe second hypothesis is that chimpanzees actually are as good, or better, at competitive interaction and at adjusting toward equilibrium choices from experience than humans are. Note that this tentative conclusion certainly deserves further investigation since the best evidence of an underlying mechanism — from statistical models of learning — does exclude some unresponsive chimpanzee sessions; then the difference in learning rate between chimpanzees and humans is reasonably significant (p = .040) .physical advantages over humans in strength and speed, which have fitness value in a dominance-mediated social environment. In contrast, human society is relatively egalitarian and non-agonistic, and there is less of a fitness benefit to be gained from strength as a factor in intraspecific social interactionscognitive advantage for chimpanzees.Nonetheless, a chimpanzee cognitive match or advantage is plausible because chimpanzees have clear One cognitive advantage emerges in serial ordering tasks requiring memory of briefly exposed spatial displays of Arabic numerals15The relatively poor performance of humans, together with the conjectured importance of language for humans, raise issues about the relevance of those game theory experiments in which humans have traditionally been unable to talk to each other. If verbal communication is indeed key to human strategic interaction, it seems that external validity would be enhanced if one lets humans talk. Of course, this challenges classical game theory, which has generally struggled with how to model credibility of verbal communication  regularly practiced in development and into adulthood. However, it is notable that in our protocol humans are deliberately deprived of an extraordinary cognitive ability -- language. In competitive games language cannot increase group rewards. But in games requiring coordination and cooperation, where language is particularly useful in our ecology, the evolutive prediction is that humans will outperform other speciesDesign ; research (C.M.); new analyses ; analyzed data ; wrote paper .Supplementary Information


The Editorial on the Research TopicEndophenotypes for schizophrenia and mood disorders: implications from genetic, biochemical, cognitive, behavioral, and neuroimaging studiesSchizophrenia and mood disorders represent complex phenotypes that are characterized by multidimensional psychopathology and biological underpinnings. After many years of clinical observations, it has been recognized that there are several intermediate phenotypes between schizophrenia and mood disorders that share common clinical characteristics. This observation has provided grounds for the concept of endophenotypes, crossing traditional diagnostic boundaries and opening perspectives for better understanding of mental disorders. Indeed, the term endophenotype refers to a measurable construct that bridges a gap between phenotype expression and genetic variability. This intermediate phenotype construct must meet four characteristics: the association with illness in the population, heritability, state-independent manifestation (expression of the phenotype regardless of the illness activity), and cosegregation with illness in families . ConsequGrant describes various aspects of schizotypy and proposes that schizotypy itself might be a suitable endophenotype of schizophrenia. Following the definition of endophenotype developed by Gottesman and Gould   –4. ThereWang et al. found that functional alterations in a prefrontal–striatal–thalamic–cerebellar network and structural disturbances in the default mode network are common abnormalities across various psychotic disorders. Based on the data from B-SNIP study, Soh et al. performed joint independent component analysis of awake EEG frequency activity and MRI gray matter volumes in patients with psychotic disorders. Authors found a single component differentiating schizophrenia patients and healthy individuals. Specifically, they reported increased posterior alpha activity associated with decreased volume in inferior parietal lobe, supramarginal, parahippocampal gyrus, middle frontal, inferior temporal gyri, and increased volume of uncus and culmen in patients with schizophrenia.In two studies, the role of structural and functional brain alterations as potential endophenotypes of psychosis dimension has been investigated. In the large and multisite bipolar–schizophrenia network on intermediate phenotypes (B-SNIP) study, Kim et al. in their mini review proposed that phospholipase C-β1 (PLC-β1) hypofunction might be an endophenotype of schizophrenia. This enzyme is involved in phosphoinositide signaling, which is one of the main G-protein-related pathways in the central nervous system. It has been reported that PLC-β1−/− mice express several negative-like symptoms and cognitive deficits that resemble those observed in schizophrenia . In addophrenia . A recenophrenia . TherefoAlthough a number of candidate endophenotypes for schizophrenia and psychotic disorders have been proposed in this research topic, studies replicating these findings in large samples are required. In addition, future studies must look at these findings in a broader context of endophenotype criteria and verify how well they suit their original purpose – linking less complex disease phenotype to its genetic underpinnings. It is important to note that studies investigating endophenotypes should take into account that psychosis dimension is itself a heterogeneous construct. Indeed, psychotic symptoms associated with schizophrenia have different features compared to those observed in BD. For instance, most psychotic features associated with manic episodes tend to have a different, usually self-limited and self-remitting course. These symptoms appear in most severe episodes of BD. On the contrary, psychotic symptoms in schizophrenia have a more long-lasting course. This observation give rise to the conclusion that more objective and reliable endophenotypes offer a prospect of defining more etiologically homogenous subgroups of human psychopathology and thus facilitate our understanding of the basis and causal pathways of mental disorders. Finally, the existence of relevant endophenotypes should initiate discussion about reappraisal of the research diagnostic criteria beyond traditional diagnostic systems to enable better understanding of mechanisms underlying schizophrenia and mood disorders.All author listed have made 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.


Understanding how parasites adapt to changes in host resistance is crucial to evolutionary epidemiology. Experimental studies have demonstrated that parasites are more capable of adapting to gradual, rather than sudden changes in host phenotype, as the latter may require multiple mutations that are unlikely to arise simultaneously. A key, but as yet unexplored factor is precisely how interactions between mutations (epistasis) affect parasite evolution. Here, we investigate this phenomenon in the context of infectivity range, where parasites may experience selection to infect broader sets of genotypes. When epistasis is strongly positive, we find that parasites are unlikely to evolve broader infectivity ranges if hosts exhibit sudden, rather than gradual changes in phenotype, in close agreement with empirical observations. This is due to a low probability of fixing multiple mutations that individually confer no immediate advantage. When epistasis is weaker, parasites are more likely to evolve broader infectivity ranges if hosts make sudden changes in phenotype, which can be explained by a balance between mutation supply and selection. Thus, we demonstrate that both the rate of phenotypic change in hosts and the form of epistasis between mutations in parasites are crucial in shaping the evolution of infectivity range. Antagonistic coevolution between hosts and parasites can lead to directional selection for more effective defense and counter-defense mechanisms . In manyParasites frequently require multiple amino acid substitutions to infect a novel host, and the likelihood of several beneficial mutations occurring simultaneously or in quick succession is usually slim . In someEmpirical studies that demonstrate the importance of coevolution for the emergence of broad infectivity ranges have used host-parasite interactions that are governed by strong positive epistasis between infectivity mutations . This men biallelic loci in both host and parasite. Increasing the number of infectivity alleles improves infectivity to a wider range of host genotypes, and increasing the number of resistance alleles improves resistance to a wider range of parasite genotypes. We refer to this as a “symmetric” (SYM) interaction, because there is a one-to-one correspondence between resistance and infectivity alleles. We compare this scenario to an “asymmetric” (ASYM) form of genetic specificity, where interactions occur between a single locus in the host and n loci in the parasite (one-to-many). In this case, there are only two possible host genotypes (susceptible and resistant), and increasing the number of infectivity alleles can improve performance on the resistant host. Genotypes are represented by binary strings  and fitness costs  are captured by the following set of coupled ordinary differential equations: We translate this deterministic framework into a stochastic model by using the τ-leap method proposed by E, to be the maximum of this value over the course of a simulation . Thus, if k infectivity alleles at time t, then: We analyze the deterministic and stochastic versions of our model to evaluate how the previously described forms of genetic specificity (SYM and ASYM) and different types of epistasis (ψ) influence the evolution of broader infectivity ranges. In other words, we establish how these genetic factors affect the ability of parasites to accumulate infectivity alleles. At each time point, we measure the average proportion of parasite loci that contain an infectivity allele and define “peak infectivity range,” 5–109 are appropriate for microbial communities). Note that the mutation rates and the coefficient of density-dependent mortality are not used in the construction of the LHS, but are instead fixed by the base per-capita birth rate and/or carrying capacity, which are part of the LHS design. The parameters We measure the maximum value over the duration of each simulation as GFG frameworks can produce fluctuations in range e.g., , but theulations . The LHSFor the sake of brevity, here we only present results for parasite populations with three loci Using equation (3), we can derive the basic reproductive ratio, j should initially perform better than the wild type  provided In the absence of resistant hosts, this quantity is maximized when the parasite has no infectivity alleles. We can also derive the effective basic reproductive ratio, FigureNumerical analysis of the deterministic model revealed that infectivity range peaks when epistasis is positive ; Fig., but decThe pattern of parasite evolution in the stochastic SYM scenario was broadly similar to that described for the deterministic model: infectivity ranges peaked for strong positive epistasis er ψ Fig.A. Yet, uIn contrast, the stochastic version of the ASYM scenario produced markedly different outcomes to both the deterministic model and the SYM scenario Fig.B. SpecifE: 0.03). Notably, parasites that experienced very strong positive epistasis  to ensure that the differences between the two versions of the ASYM scenario were not attributable to faster evolution in the deterministic setting. However, longer simulations did not change the overall pattern of our results and only led to relatively minor quantitative differences . Thus, although a complete set of infectivity alleles may have been optimal for low values of ψ Figs., 3B, parAlthough broad infectivity ranges were much more common in the stochastic version of the SYM scenario than in the corresponding ASYM scenario for strong positive epistasis Our study was inspired by recent experiments where gradual changes in host phenotype provided optimal conditions for parasite evolution, presumably by facilitating the accumulation of multiple mutations in quick succession, whereas sudden changes in host phenotype prevented mutations from being fixed . HoweverWhen epistasis is strongly positive, parasites require a complete (or near-complete) set of mutations to overcome resistance. These are difficult to accumulate if the host makes sudden changes in phenotype  as individual mutations may carry an intrinsic fitness cost without conferring any benefits with regards to increased infectivity. If, however, hosts experience gradual changes in phenotype , then parasites may have access to hosts that are phenotypically intermediate between ancestral and future populations. Individual mutations may then confer an immediate fitness advantage, dramatically increasing the probability that multiple mutations will become fixed. Hence the symmetric (SYM) scenario in our model, which featured intermediate hosts, was much more favorable to the emergence of broad infectivity ranges under strong positive epistasis than the asymmetric (ASYM) scenario, which did not allow intermediate hosts to evolve. Conversely, when epistasis is negative there is a diminishing benefit associated with the acquisition of multiple infectivity alleles regardless of the potential presence of intermediate hosts. In other words, parasites can infect a reasonably broad set of hosts with a single mutation e.g.,  and the The results for the ASYM scenario can also be interpreted in terms of a balance between mutation supply and selection for broader infectivity ranges. If parasites perform poorly on current hosts, then selection for broader infectivity ranges is strong, but the mutation supply is constrained due to a lack of suitable hosts. This problem is accentuated when epistasis between infectivity alleles is strongly positive, as multiple mutations that may be costly in isolation are required before parasites can infect large numbers of hosts. Hence, strong positive epistasis results in a low mutation supply and broader infectivity ranges are unlikely to evolve. Conversely, if parasites perform well on the current host population, then the mutation supply is much greater, but selection for broader infectivity ranges is inevitably weaker. When epistasis is negative, parasites with few infectivity alleles can perform reasonably well on hosts that have invested in resistance; accumulating further (costly) infectivity alleles is unlikely to be beneficial under these circumstances, so again, broader infectivity ranges do not evolve. Between these extremes, parasites can infect sufficient hosts to maintain a modest supply of mutations while experiencing fairly strong selection for broader infectivity ranges. Hence, broader infectivity ranges are most likely to evolve in the stochastic ASYM scenario for intermediate values of ψ, which corresponds to weak positive epistasis.The discrepancies between the deterministic and stochastic versions of the ASYM scenario are striking, but were not attributable to different rates of evolution, as simulations that were allowed to run for much longer time periods produced very similar results . If hosts make sudden jumps in phenotype, then parasites that experience stochasticity and strong positive epistasis between infectivity alleles are likely to get stuck at a local fitness peak. In the deterministic model, parasites are able to explore the entire fitness landscape, which always allows the globally optimal phenotype to emerge. The contrasting outcomes in the deterministic and stochastic models highlight the need to compare different modeling approaches when studying host-parasite coevolution, as optimal phenotypes may not always emerge when demographic stochasticity is included see also . It is iOur results were robust to changes in a number of modeling assumptions, which indicates that our findings are likely to be quite general. Still, it is somewhat surprising that different forms of fitness costs  did not produce markedly different results, as different trade-off shapes are often associated with contrasting outcomes in studies of host and parasite evolution . This suPseudomonas fluorescens and the lytic phage Φ2, Escherichia coli. These mutations showed all-or-nothing  epistasis, but in contrast to studies on P. fluorescens, the acquisition of these mutations resulted from fitness benefits on a subpopulation of bacteria that had reverted to susceptibility. As such, broader infectivity ranges were again promoted by a gradual change in host phenotype, but this was facilitated by host polymorphism.The findings presented herein are consistent with recent empirical work showing that infectivity evolution tends to proceed faster in the presence of coevolving antagonists that exhibit gradual changes in phenotype . The hosSeveral studies have explored the evolution of broader infectivity ranges, using either explicit genetics . For example, hosts did not tend to become resistant in the stochastic SYM scenario when epistasis was negative, which explains why broader infectivity ranges did not evolve under these conditions. Host-parasite coevolution can lead to a variety of coevolutionary outcomes with respect to infectivity range, including competitive exclusion, stable polymorphism, and fluctuating selection . Indeed,Understanding the effects of epistasis in real biological systems represents a significant challenge for empiricists and we are not aware of many nonmicrobial systems where the strength of interactions between infectivity mutations have been accurately measured . Finding


We suggest this fits existing literature showing: (a) examples of exceptional creativity in individuals with mental illness, without an overall increase in creative achievement associated with “madness”; (b) increases in creative achievement among relatives of people with mental illness, or people with milder syndromes, for whom increased flexibility or stability is less disabling; and (c) effects of pharmacological manipulations, suggesting an inverted-U function resembling the Yerkes-Dodson Law, possibly linked to tonic and phasic dopaminergic transmission.Complexity theorists have suggested that production “on the edge of chaos” is important to self-organization and evolutionary change in thermodynamic systems, biology, and economics. We apply this heuristic to cognitive systems and neural network activation states, which can vary from an ordered (predictable) regime, to a chaotic (unpredictable) regime. Evolutionary cytoarchitectonic theory specifies complementary anatomical systems governing stability and flexibility. Psychopathology is associated with shifts in the regulation of stability and flexibility, and may yield both increased redundancy and increased entropy Creative works may be defined as combining novelty  and utility  within the domain from which they emerge   may be linked to increased creative achievement, but more severe illness is not. For example, children with pediatric bipolar disorder perform worse on tests of set-shifting or cognitive flexibility . But if the dynamics proceed too far in the direction of flexibility, entropy, and unpredictability, then cognitive products may be novel and unpredictable, but may be “over the edge of chaos,” and not be perceived as useful, valuable, or effective.These assumptions are consistent with the hypothesis that neural network dynamics associated with cognitive flexibility are linked to creative achievement because these dynamics generate activation states that are novel with respect to population averages, and favor performance on metrics that are tuned to The Edge of Chaos hypothesis may help understand the pathophysiology and treatments of mental illness, and suggest paths to augment creativity. We can assess positive traits of cognitive stability and flexibility (or disadvantageous traits: rigidity and lability), and determine their relations with cellular and neuromodulatory factors. For example, widespread dysconnectivity and DA dysregulation remain candidates in the pathophysiology and treatment of schizophrenia. To the extent that interventions impact these states, monitoring stability/flexibility may be beneficial in determining when treatments are managing chaotic (overly flexible) states but not causing cognitive rigidity. For example, measures of network stability/flexibility could help titrate treatments in schizophrenia to maximize freedom from positive symptoms while minimizing cognitive impairment, or titrate the DA and norepinephrine reuptake inhibition in Attention Deficit/Hyperactivity Disorder (ADHD) to maximize attentional control.Non-pharmacological treatments might someday be tuned to optimize flexibility and stability. For example, EEG neurofeedback strategies emphasizing cognitive stability or flexibility may enhance creativity (Gruzelier, There are further links of cognitive stability and flexibility to meditation practices referred to as focused attention and open monitoring, respectively (Lutz et al., In conclusion, we believe that understanding both mental illness and creative cognition from the perspective of neural network dynamics, and specifically the regulation of the stability and flexibility of cortical activation states, helps to clarify the relation between creativity and mental illness. We further believe understanding these cognitive dynamics may have profound implications for both understanding of pathophysiology of mental illness, the development of novel intervention strategies for those who are ill, and the enhancement of creative cognition more broadly across the population.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.


Transposable elements (TEs) are ubiquitous in eukaryotic genomes. Barbara McClintock’s famous notion of TEs acting as controlling elements modifying the genetic response of an organism upon exposure to stressful environments has since been solidly supported in a series of model organisms. This requires the TE activity response to possess an element of specificity and be targeted toward certain parts of the genome. We propose that a similar TE response is present in human cells, and that this stress response may drive the onset of human cancers. As such, TE-driven cancers may be viewed as an evolutionary by-product of organisms’ abilities to genetically adapt to environmental stress. Transposable elements (TEs) and their remnants constitute between half and two thirds of the human genome , and arede novo methylation during early development . And, importantly, that TE activity induced by external stresses should thus be regarded as an evolutionary adaptive mechanism.TE activity is readily reported in cancer cells  and is cGiven the functional diversity of human TEs, a global activation of the entire TE population would provide a crude measure to affect the genotype. Rather, one would imagine that the probability of TEs creating adaptive genotypes increases with specificity (different activity levels for different TEs) and/or targeting (TEs are predominantly inserted in certain regions of the genome). In the following, we briefly review examples of highly targeted TE activity from non-mammalian model organisms, and end by providing observations suggesting that stress-induced TE activity in mammalian cells can be both specific as well as targeted. Hence, environmental stimuli may trigger the formation of genetic changes through TE activity at all stages of cancers, including the very onset.All living organisms face an apparent paradox. On the one hand, their replication over time should be as exact as possible to maintain proper function, but on the other hand, organisms must evolve for their lineage to survive changing environments. When cells encounter changing environments  they may induce genomic changes potentially generating adaptive mutations.When multiple genes carry out overlapping functions, individual genes are allowed to diverge from their optimum without affecting the phenotypic outcome , allowinImportantly, the ability to generate genetic variability, termed evolvability, does not imply foresight or teleology . Errors Introducing genomic changes when facing environmental stress – as suggested by the TE pioneer Barbara McClintock  – makes Nicotiana tabacum, the Tnt1 retrotransposon is induced by abiotic stresses . However, we here address the different evolution and ecology of eukaryotic cells in single and multicellular organisms, which is essential for this discussion.In line with previous speculations , we haveWe have highlighted examples of mammalian stress-induced TE activity being both targeted and specific. Importantly, TE activity may not necessarily be limited to transposition but could consist of transcriptional activity only. Whether the ability of stress-inducible TE activity serves as an adaptive host response within our cells or it reflects an inherent ability of TEs of being activated upon changing environments is unknown. Perhaps, the innate properties that have allowed the exaptation of TEs in cellular stress responses  are the very same properties that underlie the ability to induce evolvability. This way, these properties would be selected for because of the former (heat shock) without the latter (evolvability) necessarily being adaptive. In organisms with a sequestered germ line, somatic TE insertions will not be passed on to future generations. Yet it is possible that somatic cells and tissues modify their hard-coded genetic information during an individual’s life span. The notion of the mammalian genome as a stable and inert code may largely result from our experimental inability to record changes between tissues and stages. Furthermore, somatic TE transposition may be limited to a few insertions in each cell making these hard to detect in healthy cells without the clonal amplification seen in cancer . AlthougIt is evident that unleashed TE activity  in neoplasm may induce further genetic changes accelerating cancer progression. However, the existence of stress-inducible TEs suggests that environmental stimuli alone may trigger the formation of genetic changes through TE activity at all stages of cancers, including the very onset. Similarly, it has recently been suggested that stress-induced TE activity may alter the neuronal genotypes in the human hippocampus, and that this could potentially be linked to neuronal disorders caused by severe stress . Importain vitro and of cancer cells in vivo could provide a rarely established link between the external environment and cancer genotypes in non-heritable cancers.The presented notion has several implications as different cellular stresses may elicit different TE responses in different cell types. Subsequently, different TE responses may underlie different cancers. This prompts for investigations not only into the genetic variability between cells in terms of TEs but also into the TE responses induced by cellular stress types. The advent of single-cell sequencing strategies  combinedThe 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.


A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in the transformation of malignant cells. Constitutive and abnormal activation of RTKs may occur in tumors either through hyperactivation of mutated RTKs or via functional upregulation by RTK-coding gene amplification. In several types of cancer prognosis and therapeutic responses were found to be associated with deregulated activation of one or more RTKs. Therefore, targeting various RTKs remains a significant challenge in the treatment of patients with diverse malignancies. However, a frequent issue with the use of RTK inhibitors is drug resistance. Autophagy activation during treatment with RTK inhibitors has been commonly observed as an obstacle to more efficacious therapy and has been associated with the limited efficacy of RTK inhibitors. In the present review, we discuss autophagy activation after the administration of RTK inhibitors and summarize the achievements of combination RTK/autophagy inhibitor therapy in overcoming the reported resistance to RTK inhibitors in a growing number of cancers. Receptor tyrosine kinases (RTKs) are transmembrane glycoproteins that participate in the transduction of external signals into cells . Due to Because the main role of RTKs is to regulate cell growth and survival, it is not surprising that their abnormal activity has been correlated with tumor development and progression . The conAutophagy is an important catabolic process that regulates the degradation and recycling of organelles and proteins within the cell. It participates in the regulation of general cellular homeostasis . Once auThe present review aims to discuss autophagy activation as a possible mechanism involved in impeding the cytotoxicity of RTK inhibitors. It will summarize troublesome resistance as frequent manifestation that arises when RTK inhibitors are used to treat different malignancies. Furthermore, it will postulate a rational for the use of a combination therapeutic strategy with autophagy inhibitors and RTK inhibitors to improve their success.Recent years have brought in evidence numerous reports that survey effectiveness of RTK inhibitors in the treatment of solid tumors. Initial enthusiasm for the RTK inhibitory treatment as principal targeted therapy waned when patients started to develop resistance to these inhibitors . At moleThe PI3K/AKT/mTOR is one of the most important signaling pathways that regulate autophagy , and at Ultimately, the studies that indicate the microRNAs (miRNAs) as the important intermediary of autophagy regulation in the eukaryotic cells are flourishing . These ~EGFR epidermal growth factor receptor, also known as ErbB1 [as ErbB1 , was theas ErbB1 , but it as ErbB1 , 38. Theas ErbB1 . Beside as ErbB1 , and theas ErbB1 . This obas ErbB1 .Sugita and colleagues  reportedThe efficacy of gefitinib in blocking the growth of breast cancer cells has also been tested. In these cells, the formation of autophagosomes was observed as an early event after treatment . The altConversely, an additional body of evidence has connected EGFR (ErbB1)-targeted treatments with the stimulation of autophagy, thereby helping tumor cells’ death escape . SimilarVEGFR, PDGFR, and c-Kit Many RTK inhibitors have more than one target. Sunitinib malate , a small molecule inhibitor, targets vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and stem cell tyrosine kinase receptor (c-Kit). After being implicated in the development of diverse malignancies, these RTKs have been targeted with sunitinib in metastatic renal cell carcinoma, imatinib-resistant gastrointestinal tumors, and pancreatic neuroendocrine tumors [e tumors . Importae tumors . In facte tumors .Sorafenib (Nexavar) is an inhibitor of the VEGFR, c-kit, PDGFR, and Raf/MEK/ERK signaling pathway . It has MET also known as c-Met or hepatocyte growth factor receptor (HGFR), is an RTK with deregulated function in certain types of cancer. Aberrant activation of MET during oncogenesis may occur due to MET gene overexpression or activating point mutations [LC3B and ATG7 mRNA levels. When they were used in combination with autophagic inhibitors (3-MA or CQ), a negative impact on cancer cell viability was achieved. The experimental evidence obtained from the use of MET inhibitors has suggested that the cyto-protective effect gained by activating autophagy might be a serious obstacle for effective therapy. Therefore, inhibiting autophagy could be an approach to guarantee significant improvements in the therapeutic strategy of inhibiting RTKs [utations . PHA6657ing RTKs .ALK Anaplastic lymphoma kinase dysfunction has been reported in several solid tumors, including neuroblastoma (NB). Its continuous activation in NB was reported to stem from point mutations or amplification of the ALK gene [ALK gene . RecentlALK gene . We founALK gene  in lung Altogether, the data summarized herein suggest that RTK inhibitors frequently cause the induction of autophagy, which plays a cyto-protective role that may impede their efficacy in cancer treatment. Moreover, they support anti-autophagy/RTK inhibitor combination therapy as an advanced approach for improving the efficacy of currently FDA (Food and Drug Administration) approved RTK inhibitors. This approach might provide significant benefits to patients with solid tumors in which at least one of the mentioned RTKs is deregulated. Thus, we conclude that it would be pertinent to conduct a more detailed examination of the use of this combination strategy Fig. c that haThe idea that targeting RTKs might be adopted as a suitable strategy for the clinical management of cancer has persisted since the moment it became clear that dysregulated RTK function is a frequent event in tumor cells . CurrentCurrently, available preclinical and clinical data indicate that autophagy inhibitors, given in combination with RTK inhibitors, might be a promising approach for treating cancer patients with deregulated RTKs. This approach might ensure not only higher efficacy of these inhibitors but also fewer toxic side effects, particularly in those patients who become resistant during drug treatment. By highlighting the increased efficacy of combination approaches with respect to a single RTK-targeted strategy, we would like to express our belief that this line of attack might be worth of intense investigation in all malignancies where resistance to RTK inhibitors is a problem. We believe that inhibiting autophagy in conjunction with RTK inhibitor treatment could give a better chance to improve the battle against solid tumors. It remains to determine whether combined therapeutic strategy might be considered in the future clinical practice, even before the resistance to RTK inhibitors is developed.


Biotic and abiotic stresses are the main problems affecting agricultural losses. Consequently, understanding the mechanisms underlying plant resistance or tolerance helps us to develop fruitful new agricultural strategies. These will allow us to face the challenges of producing food for a growing human population in a sustainable and environmentally friendly way.To compensate for their sessile life and face a broad range of biotic and abiotic stresses, plants have evolved a wide range of survival and adaptation strategies. Amongst them, higher plants are capable of inducing some stress “memory,” or “stress imprinting.” Bruce et al.  define sFollowing perception of microbe-associated molecular patterns (MAMPs), recognition of pathogen-derived effectors or colonization by beneficial microbes, priming can also be induced by treatment with some natural or synthetic compounds or even by wounding Conrath, . ThroughReactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals are inherent by-products of aerobic metabolism. ROS have not only the potential to cause oxidative damage by reacting with biomolecules, but it is widely accepted that they also have key roles as signaling molecules that contribute to control of plant development and to the sensing of the external environment  and hydrogen peroxide (H2O2)  is a water-soluble addition compound of vitamin K3, or pro-vitamin K. Menadione, previously thought to be synthetic, has been isolated from fungi and phanerogams  showed that MSB-pretreatment 24 h before inoculation with Leptophaeria maculans exhibited rings of necrotic mesophyll cells surrounding the invasive hyphae of L. maculans, after staining with aniline blue in lactophenol. In water pre-treated control plants, unobstructed L. maculans hyphal growth was observed at infection sites, with no visible host reaction  and cytochrome P450s . Interestingly, a recent MSB application has been patented for controlling Trioza erytreae and Diaphorina citri, the psyllid vectors carrying the genus Candidatus Liberibacter that are bacterial causal agents of the most serious citrus disease known as Huanglongbing (HLB)  and degradation  of proline, demonstrating that one of the mechanisms underlying this early adaptation to salt stress is an epigenetic mark (submitted for publication).Another effect of menadione on abiotic stresses is to induce tolerance to chilling stress in maize seedlings (Prasad et al., 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.


Tissue engineering is a rapidly advancing field that is likely to transform how medicine is practised in the near future. For hollow organs such as those found in the cardiovascular and respiratory systems or gastrointestinal tract, tissue engineering can provide replacement of the entire organ or provide restoration of function to specific regions. Larger tissue-engineered constructs often require biomaterial-based scaffold structures to provide support and structure for new tissue growth. Consideration must be given to the choice of material and manufacturing process to ensure the de novo tissue closely matches the mechanical and physiological properties of the native tissue. This review will discuss some of the approaches taken to date for fabricating hollow organ scaffolds and the selection of appropriate biomaterials. The increasingly ageing population represents a huge socioeconomic burden on healthcare systems worldwide. New therapies are urgently sought that can rejuvenate or restore function of physiological systems that no longer function correctly due to the cumulative effects of wear and tear. Organ transplantation offers a limited solution due to donor shortages and the need for lifelong immunosuppression . XenotraThere is therefore a clear need for alternative strategies aimed at producing curative treatments. Tissue engineering is a rapidly developing interdisciplinary field that involves both materials science and engineering with medical research to address clinical problems. The majority of tissue engineering strategies rely on the use of biocompatible materials to facilitate tissue regeneration. Scaffolds can be manufactured from different materials and by a variety of methods, depending on the intended application. Three-dimensional scaffolds have the ability to guide tissue regrowth, provide support, encourage cell adhesion and proliferation, be combined with biological agents to provide a sustained release of various factors or drugs, and support neovascularisation within an interconnected porous network –6. TissuThere are many different methods available to fabricate scaffolds for tissue engineering. The approach taken is often determined by the starting material, scale of the construct, and physiochemical and mechanical properties of the scaffold. Techniques that have been used to produce scaffolds suitable for hollow organ tissue engineering include electrospinning and extrusion methods that allow for the production of polymer fibres or meshes –12, therFor a material to be classed as a biomaterial, it must fulfil certain criteria. The material should be compatible with cells and tissues in the local milieu so as not to illicit a chronic inflammatory response. Other desirable properties may include biodegradation, porosity, specific mechanical properties and the ability to facilitate cell attachment , 17. SynCardiovascular diseases are within the top ten causes of death worldwide, with pathologies including myocardial infarction, stroke, congestive heart failure, peripheral arterial disease and vascular disease . The neeOne of the most popular cell types studied in bioengineering blood vessels is endothelial cells, which line arteries and veins and form capillaries. They control the function of the entire vessel by signalling to the surrounding smooth muscle tissue to response to changes in shear stress by adapting lumen diameter and wall thickness . Other cWhole blood vessels can be produced by various tissue engineering methods including 3D printing , 3D pattNew blood vessels have the ability to grow from the existing vessels throughout adult life in a process called angiogenesis. Angiogenesis is dependent on growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), as well as signalling and interactions between cell types such as endothelial cells and pericytes . PromotiBauters et al. (1995) showed that administering VEGF into a rabbit model of hind limb ischemia resulted in improved blood vessel formation compared to a control group after 30 days . UnfortuThe gastrointestinal (GI) tract consists of the oesophagus, stomach, small and large intestines and colon. It is a complex hollow organ system with diverse functions and structures that facilitate digestion, absorption of nutrients and excretion of waste from the body. Engineered GI tissue can be applied to repair damage caused by stomach cancer, inflamatory bowel disease, as well as replace sphincter tissue to cure faecal and urinary incontinence.l-lactic acid (PLLA) seeded with endothelial cells from the stomach. Maemura et al. (2008) implanted these constructs in an in vivo rat model. They found that the cells on the tissue-engineered scaffold had comparable secretory functions to a native stomach [Stomach cancer is the fourth most common fatal cancer in Europe . Gastrec stomach . The sam stomach . Another stomach . UnfortuInflammatory bowel disease (IBD) affects 1 in 250 people in the UK with the most prevalent examples being ulcerative colitis (UC) and Crohn’s disease. Both diseases are lifelong, with Crohn’s primarily affecting the small intestine and UC the large intestine . In the Faecal incontinence is a major public health issue with no curative treatment available  with 10 Shi et al. (2014) injected adipose-derived mesenchymal stem cells (AdMSCs) attached to silk fibrin microspheres into a model of urinary incontinence in rats. They found that there was long-term improvement (12 weeks) in the group administered cells attached to microspheres compared to the controls, as well as an improvement in leak pressure and decrease in lumen size . These mThere are many challenges in tracheal tissue engineering. Clinically, tracheal stenosis, tracheomalacia, trauma due to prolonged intubation and cancers can all result in irreversible damage to the trachea  with resThe most common cell types investigated for tracheal tissue engineering are chondrocytes and stem cells. The native trachea consists of cartilage rings, hence, why chondrocytes are often used as they produce collagen and aggrecan. Stem cells such as mesenchymal stem cells (MSCs), embryonic stem cells (ES) and induced pluripotent stem cells (iPS) have the ability to differentiate into many different cell lineages but, for this instance, are all used for epithelial cell regeneration. The cells are cultured with the scaffold to achieve re-epithelialisation similar to the native trachea.l-lactide-co-ε-caprolactone) (PLCL) and gelatin scaffold that was rigid and flexible. The scaffold was seeded with chondrocytes and functionalised with transforming growth factor beta 1 (TGF-β1) growth factor to further stimulate regeneration of the cartilage. The cellularised scaffold was implanted, and in vivo studies showed that regeneration of the cartilage similar to native cartilage had been achieved and the organ did not collapse under compression [Decellularised scaffolds are often considered for tracheal tissue engineering as they contain materials that form the ECM such as collagen, fibrin and hyaluronic acid , thus alpression . 3D prinpression . Engineepression . Based opression .Tissue engineering of hollow organ constructs is no longer in its infancy. Advancements in engineering allows for a better understanding of the behaviour of biomaterials and their fabrication in the context of hollow organ construction. Biomaterials play an important role in underpinning many of the tissue engineering technologies being developed for hollow organs. Selection of appropriate biomaterials with suitable physicochemical properties is imperative to the success of hollow organ scaffold structures. The fabrication processes available are continuing to evolve to allow the shape, size, porosity, topography and scalability of the scaffold to be controlled in order to meet the tissue requirements . Tissue


This article examines uncomfortable realities that the European hospital sector currently faces and the potential impact of wide-spread rationalization policies such as  payment reform and privatization.Review of relevant international literature.Based on the evidence we present, rationalization policies such as  payment reform and privatization will probably fall short in delivering better quality of care and lower growth in health expenses. Reasons can be sought in a mix of evidence on the effectiveness of these rationalization policies. Nevertheless, pressures for different business models will gradually continue to increase and it seems safe to assume that more value-added process business and facilitated network models will eventually emerge.The overall argument of this article holds important implications for future research: how can policymakers generate adequate leverage to introduce such changes without destroying necessary hospital capacity and the ability to produce quality healthcare. Clinically, hospitals still form a cornerstone of health care delivery systems, but important parts of (future) growth are now reserved for community care, outpatient surgery, and other outpatient services . MoreoveFinancially, hospitals expend a large part of healthcare’s fiscal appropriations and are a very ‘visible’ part of the healthcare budget. Many countries still enforce a central allocation for large hospital capital projects and most hospital payment schemes retain elements of global budgeting. Hospital expenditure alongside pharmaceutical costs form top priorities for current and additional cuts among budgetary decision makers . HoweverLastly and most relevantly for national health policymakers, when looked at from an organizational perspective, hospitals now form a mature industry, with the number of hospital beds having declined for a considerable period of time , and witThis article examines how changes in hospital structure and funding combine with tightening austerity measures to affect the process and outcome of hospital governance. It explores the fundamental tension between how hospitals have evolved and two main political strategies —payment reform and privatization— which have been advanced thus far to deal with financial austerity as well as the above-noted technological and epidemiological trends that shift healthcare provision away from hospitals. Section 2 describes why payment reform and privatization have become important aspects of policy strategies. Section 3 analyses further pressures on traditional hospital business models. Sections 4 (payment reform) and 5 (privatization) consider the evidence on these topics well as technical and non-technical rationales of moving in these directions. Section 6 covers several important methodological limitations. Section 7 concludes and summarizes the contribution of this paper.There exists a large body of literature that describes how hospitals gradually evolved from alms houses and nursing homes towards a combination of what currently are its core assets: emergency departments, operation theatres and (expensive) specialized equipment surrounded by ICU and CCU beds. Medium care nursing units and outpatient departments now are often literally situated as a second circle around these core assets. We do know from a large base of studies and analyses about the formidable veto powers and political influence of hospitals and their doctors , 21. HosSuch conflicts contributed to the shaping of gatekeeper systems in the UK and the Netherlands among other countries. Gatekeeping helped to guarantee that doctors in- and outside hospitals had their own patient base. The idea that gatekeeping is also a useful precondition for efficiency, which has been adopted by about half of EU health systems , only deDuring the era of the European welfare state, the increasingly prestigious and ever more expensive hospital sector led to a series of complicated funding issues. Maintenance of assets instead of expansion of capacity came to the fore. To distribute and fine tune existing resources, countries needed payment systems. For hospitals, this usually (but not exclusively) implied separate models between the funding for capital investments and the funding of current or operating costs . In geneTwo trends have become influential. The first includes a wave of reform in the running of public hospitals, starting with the application of various New Public Management models , 34 whicIn most European countries, private for-profit hospitals have grabbed a bigger share of the market, mostly by acquiring and re-structuring other hospital types. In most countries the private sector also is becoming increasingly consolidated into a few large groups . HospitaThe reform of payment schedules was a second trend and generally is debated in more technical terms if compared to issues of hospital privatization. Hospitals now are typically paid through some kind of activity-based costing system (diagnosis related groups or DRGs) in most OECD countries . The ideContemporary European hospitals operate a variety of different business models inside a single organization. For example diagnostics, office visits, elective surgery, emergency treatment or nursing and rehabilitation require different assets, human skills, and organizational procedures. However, since hospitals tend to deliver as many services as possible, these expansionist strategies inevitably increase the complexity of these organizations. Current ‘business school’ strategies for hospital redesign try to untangle such complexities and build more coherent groups of assets, such as focused factories or facilitated network organizations , 18, 36.Calls also exists for a fundamental redirection of the delivery system itself, which requires a paradigm shift and the adoption of patient-centric integrated care, improved hospital efficiency, and interventions in an optimal setting, either in hospitals, at home or in communities’ . InpatieOther than their very substantial size and bargaining power, hospitals hold few strong cards; increasing shares of their activities are performed on an outpatient basis, whether inside or outside the institution. The value of the activities undertaken in hospitals will depend ultimately on (i) the quality of the care provided and the lean implementation of such procedures, and (ii) on the impact of such care on patients’ health in comparative terms with primary care and ambulatory or hybrid modalities. The potential reach of such structural changes might be large; the traditional network advantages of hospital systems seem to decline. Preserving  organisations is not seen as a sustainable strategy to deliver value .Such statements align with Clayton Christensen’s contention  that hosRegarding value-added process business: ‘assembly lines’ of low cost, high frequency, low variation services still spread hesitantly in most countries. It is here where the for-profit sector is gaining most ground, although it is also accused of cherry-picking. In contrast to public and non-profit ownership types, for-profits also seem to have much more aggressively relied on traditional network efficiencies by building larger provider groups (back-office integration and standardization). Facilitated network organizations and e-health procedures hold potentially even more disruptive pressures. Such models rely on patient involvement and intensive communication between professionals. However, integrative models of outpatient departments and advanced primary care functions supplemented by new modalities, such as e-health and patient networks, often have difficulties accessing the healthcare markets and obtaining reimbursement due to their non-traditional character where scheduled appointments and treatments in physicians’ offices and clinics might no longer be needed.Christensen’s solution shop hospital stays important for complex treatments—neonatology, neurosurgery, many cancers etc. Despite their routine complaints about cherry picking by other providers, it is here where tertiary hospitals continue to have substantial leverage. ‘Palaces of complexity’ that handle high cost, high variation, and low frequency care are still needed in the distant and not so distant future. From that perspective, it is the less complex European community hospital that is under threat due to the growth of modern outpatient centers and facilitated networks, not the large academic providers such as Karolinska in Stockholm, Charité Hospitals in Berlin, or Guy’s and St. Thomas in London. Nevertheless, further growth of these new decentralized and often private provider models will necessarily change the relation between medicine and management. Other skills that relate to ICT and competencies of operational excellence will increase in relevance versus those that are needed for routine medical procedures and general consults at the doctor’s office.Hospitals need payment schemes. Such schemes may focus on beds, hospital stays, physician fees, global budgets, specific procedures and treatments, outlier payments and add-ons for certain expensive pharmaceuticals and treatments, or diagnostic-related payment. Modern healthcare systems register increasing amounts of data and more and more seek to pay for quality of care as the prime proxy for outcome of hospital treatments. Payment reform is framed as simply a technical effort to pay (higher amounts) for better outcomes and quality of care. Many countries are experimenting with paying for performance , paying for the entire continuum of care (bundling), and paying capitated models.Substantial numbers of policymakers hold high hopes that paying for quality will eventually solve adverse effects of the earlier reimbursement schemes. However, a number of fundamental problems still need to be resolved, such as the possible crowding out of intrinsic motivation and the use of rewards versus penalties . If thes‘Best-practice prices’ and ‘pay for performance’ (P4P) mechanisms of hospital care do require detailed information on input mixes, but also on quality, as well as on medical evidence and clinical agreement concerning what constitutes good practice both inside and outside the hospital—about which information is even more scarce . It is fAnother pressing demand will be the delivery of better-coordinated care for chronic disease patients. Reforming the structure of payment systems towards bundled payments has been one answer, although with inconclusive outcomes so far . One exaModels that combine risk-adjusted capitation, shared-savings and incentives for quality of care, such as the Alternative Quality Contract in Massachusetts and the Accountable Care Organization in Medicare, are hailed as the next solution that will combine cost control and better quality of care, typically with inclusion of indicators to improve population value (triple aim). Value-based contracting with weighted capitation funding defining ‘blocks’ of services—such as maternity, acute trauma care, care for the frail elderly and chronic care—that would stretch across a number of care settings and providers is an important challenge.Gesundes Kinzigtal, a small-scale experiment in southwest Germany claims substantial savings along improvements in quality of care [Europe holds some experiences of its own with these payment innovations.  of care . Capitat of care . It is a of care , 51, theHospital management in much of Europe typically decides on internal professional structures, on hiring and firing, on performance-related incentives, on day-to-day activity monitoring, on carrying out data collection for national/regional governments and other payers and stakeholders, on criteria to evaluate whether objectives have been achieved and on performance indicators (irrespective of whether or not they are later published). However, especially in public hospitals, politicians (directly by sitting on the boards or through appointed board members) continue to play a major strategic role: they set objectives, establish operational boundaries through planning and staffing-level requirements, negotiate and regulate staffing contracts and payment levels, and make final decisions about financial dependence. Hospitals are major local employers and thus politicians often have a firm electoral interest in the continuation of current activity and employment levels at these institutions. Not surprisingly, they tend to be hesitant to initiate major organizational change, usually preferring to add new layers of health care institutions crystallized under very different forms and functions in times of budgetary expansion. On the other hand, indebted public hospitals form large liabilities on public balance sheets.Some political actors view hospital privatisation  as an attractive option to handle this list of problems. Such discussions however come with strong non-technical and political rationales  and cannWhat do we actually know about the technical performance of private for-profit hospitals? In Europe, public, non-profit, and for-profit hospitals often co-exist. Economic theory tells us that enterprise models are more efficient if formal ownership controls equals effective control , and will attract for-profit entrants if decent returns on investment are probable. Hospital margins depend on high prices (e.g. due to market power and lack of price sensitivity) as well as on low costs . Kenneth Arrow pointed towards information asymmetry as an important market failure and a main rationale for the dominance of non-profit (and public) hospitals above for-profit competitors in well-functioning hospital markets. Others such as Pauly and Redisch  contend An account of the efficiency of for-profit hospitals still relies largely on peer-reviewed US studies. Systematic meta-reviews point towards the fact that for-profit hospitals generally (i) provide less community benefits, (ii) have somewhat worse mortality ratios in comparison with non-profit providers but better than public hospitals, and (iii) have few differences in cost-efficiency in comparison with other ownership types. Most studies illustrate that differences within hospital ownership types are far more important than differences between for-profits and public or non-profit hospitals .However, such sobering results come with caveats from a European perspective. The fact that for-profits provide less uncompensated care is less relevant in European countries that generally reimburse all necessary hospital services. Longitudinal studies can also come to other conclusions than cross-sectional research. Herr  points tThus most technical evidence on the efficiency of for-profit ownership seems to be inconclusive. But what about the ‘winners’ and ‘losers’ of for-profit hospital margins and cost structures versus other hospital types? A consistent picture exists among the available evidence, both in the US as well as Europe . On top This review necessarily has several methodological caveats and limitations. It does not describe in depth the wide range of external factors that may be shaping the future development of the hospital sector. An increasingly international healthcare workforce market and different workforce planning assumptions in nation states could impact hospitals’ ability to operate efficiently. If occupancy rates are currently above 85 %, as they are in many European hospitals, declining occupancy rates might also improve bed availability in emergency cases and thus medical outcomes . Due to There are other methodological limitations that should be noted as well. Hospitals are not always comparable institutions and focus on different competencies and specializations, running from small community hospitals to large university clinics. Publicly operated hospitals can vary considerably from directly managed units through to near-independently-managed entities. The range of hospital ownership arrangements across Europe varies from publicly owned  to various types of private not-for-profit (religious and/or voluntary) or private for-profit companies . As a coThis article examines the uncomfortable realities that the European hospital sector currently faces and the potential impact of wide-spread rationalization policies such as  payment reform and privatization. Based on the evidence we present, such policies will probably fall short in delivering better quality of care and lower growth in health expenses. Reasons can be sought in a mix of evidence on the effectiveness of these rationalization policies, which probably will reflect important non-technical and political rationales that in practice pursue other goals, such as protecting vested interests of the hospitals as well as many difficult-to-handle technical complexities that guide these policy instruments.The above conclusions suggest decidedly modest expectations for the solutions proposed by business school professors such as Michael Porter and Clayton Christensen. To be sure, medical professions’ top performers will continue to need academic and tertiary care hospitals for their solution shop as they seek to explore important new frontiers in patient care. Nevertheless, pressures for different business models will gradually continue to increase and it seems safe to assume that more value-added process business and facilitated network models will eventually emerge, although it is uncertain when such developments will disrupt the business model of the hospital as we know it today. Our overall argument holds important implications for future research: how can policymakers generate adequate leverage to introduce such changes without destroying necessary hospital capacity and the ability to produce quality healthcare?Up to a certain extent, European countries do seem receptive towards hospital consolidation and privatization. In many countries, private hospital groups are at the forefront of this consolidation process to build national or even pan-national networks of hospitals. Privatization might increase the responsiveness of healthcare systems, especially of ‘contractable’ services and improve the performance of the worst providers, if acquired. These might be important prerequisites for necessary changes. However privatization does not seem to provide an answer to the need for savings simply for being too volume- and price- driven, although short-term operating budget savings might occur due to the selling of public assets .Policy learning is needed for real change that can tackle the challenges which cannot be solved within the current hospital landscape and current relations between medicine and management. Europe forms an ongoing natural laboratory test that melts distinctions between political, managerial and administrative realms as well as between policy formulation and implementation in a crowded arena. Both in the public and private health sectors, hospitals are increasingly seen as transitory stepping stones with blurred boundaries, but this transition is still unclear at the moment in terms of destination. Most countries do not have active purchasers of care with the capacity to make refined, value-based contracting arrangements with providers. Only when the purchasing function in European healthcare has been strengthened will the patterns of change in hospital care show identifiable trends.In the meantime, outcomes from the present combination of organizational pressures and financial restriction will as always differ in line with normative, organizational and cultural issues. Similar solutions may well work out differently across Europe due to these contextual differences. A general pattern however can perhaps be expected: policymakers and public budget holders will continue to face a tough time as they try to respond to being boxed in by hospital spending on the one hand and revenue austerity on the other. Such challenges used to be handled by top-down freezing of budgets, although with few directions about where to seek efficiency savings. More of both might be needed.