Incorporation of global climate change (GCC) effects into assessments of chemical risk and injury requires integrated examinations of chemical and nonchemical stressors. Environmental variables altered by GCC  can influence the toxicokinetics of chemical absorption, distribution, metabolism, and excretion as well as toxicodynamic interactions between chemicals and target molecules. In addition, GCC challenges processes critical for coping with the external environment , leaving organisms sensitive to even slight perturbations by chemicals when pushed to the limits of their physiological tolerance range. In simplest terms, GCC can make organisms more sensitive to chemical stressors, while alternatively, exposure to chemicals can make organisms more sensitive to GCC stressors. One challenge is to identify potential interactions between nonchemical and chemical stressors affecting key physiological processes in an organism. We employed adverse outcome pathways, constructs depicting linkages between mechanism-based molecular initiating events and impacts on individuals or populations, to assess how chemical- and climate-specific variables interact to lead to adverse outcomes. Case examples are presented for prospective scenarios, hypothesizing potential chemical–GCC interactions, and retrospective scenarios, proposing mechanisms for demonstrated chemical–climate interactions in natural populations. Understanding GCC interactions along adverse outcome pathways facilitates extrapolation between species or other levels of organization, development of hypotheses and focal areas for further research, and improved inputs for risk and resource injury assessments. Environ. Toxicol. Chem. 2013;32:32–48. © 2012 SETAC A variety of environmental variables influenced by global climate change (GCC) can directly or indirectly affect the health of organisms. These variables include temperature, salinity, pH, and penetration of ultraviolet (UV) radiation in aquatic environments. Global climate change is causing increases in the severity and frequency of droughts and extreme precipitation events, as well as regional-scale declines in air quality  in terrestrial systems Risk assessments for toxic chemicals historically have relied on apical, whole-organism end points directly related to key demographic processes such as survival, growth, and reproduction. Data reflecting mechanistic aspects of biological effects of toxic chemicals—such as altered gene or protein expressions, metabolite profiles, and histopathology—typically have received little or no direct use in either human or ecological risk assessment. Yet, mechanistic information can help to address fundamental uncertainties inherent to current risk-assessment approaches, including those related to chemical effect extrapolations across species, endpoints and chemical structures, as well as variations in risk outcomes across settings A major impediment to the use of mechanistic data in risk assessments has been an inability to clearly translate information collected at lower levels of biological organization  into endpoint alterations that are meaningful to risk assessment, namely, effects on individuals and populations. To address this limitation, Ankley et al. In the present analysis, we describe adaptations of the AOP concept and by extension mechanistic data as tools to help identify potential adverse effects elicited by GCC and chemical toxicant exposures. The ultimate goal of this effort is to develop assessment approaches and tools that enable predictions of when and how significant interactions between chemical and nonchemical stressors might be expected to occur, rather than solely relying on collections of empirical data for different combinations of stressors. One important dichotomy involves toxicant-induced changes that alter the ability of an organism to respond to GCC stressors (toxicant-induced climate susceptibility [TICS]), in contrast to climate-induced toxicant sensitivities (CITS), the scenario where GCC affects the toxicity of chemicals. Such CITS scenarios produce altered or enhanced toxicity of chemicals under climate change conditions that might not be predicted in controlled laboratory testing or among natural populations residing within or at the edge of their physiological tolerance ranges. The former scenario (TICS) involves alterations caused by toxic chemicals that impact the ability of an organism to acclimate to a stress introduced by GCC through various biochemical, physiological, or behavioral responses. These responses can be metabolic , structural , or homeostatic , and can trigger associated behavioral changes  are a class of compounds whose environmental concentrations, through pyrogenic events such as forest fires, are projected to increase as a consequence of GCC Almost 30 years ago researchers noted that toxicity of the PAH anthracene to fish could be increased by an order of magnitude or more through simultaneous exposure to sunlight Our example concerning PAT has several noteworthy characteristics from the standpoint of assessment of interactions between chemicals and variables influenced by GCC. First, this example clearly shows how information and models derived from knowledge of toxicity mechanisms could be used to make predictions of risk under changing environmental conditions. Both key determinants of toxicity, PAH exposure and UV intensity, could vary as a function of GCC, likely in a very site-specific manner, so it is critical to have tools capable of simultaneously assessing the possible consequences of alterations in either or both parameters. Based on past research, enough is known about the physiochemical and biological mechanisms of PAT that robust existing models could be utilized for the purpose of assessing different risk scenarios associated with GCC. This example also provides an illustration of a possible adverse response that in some situations could well be decreased by GCC. Specifically, in situations where PAH exposure is unchanged but UV penetration in aquatic systems decreases due to increased precipitation and surface runoff, the potential for PAT decreases.Beyond the accessibility or availability of chemical and nonchemical stressors to organisms, environmental alterations with GCC have the potential to alter the biological disposition (or toxicokinetics) of chemicals that can in turn influence internal exposure sites, concentrations, and durations. Toxicokinetic modifications can lead to changes in organism-level responses and eventually produce population-level impacts. In our model AOP, this would be captured by alterations in absorption, distribution, metabolism, and excretion (ADME) of toxicants . UnderstSome broad observations can be made regarding potential climate effects on the uptake and disposition of chemical contaminants Lota lota) over a 30-year collection period Persistent organic pollutants, such as PCBs, are problematic in that they can migrate thousands of miles from their original point of release to high-latitude ecosystems. For example, the Mackenzie River basin in northwestern Canada has been experiencing substantial warming trends and declining snow cover. While atmospheric concentrations of PCBs have generally declined in the Arctic with elimination and reductions in use, increasing trends of contamination by more highly chlorinated PCBs have been measured in Mackenzie River sediments and the predatory arctic fish burbot (Oncorhynchus mykiss) receiving dietary exposures Increasing trends of PCBs in biota are important because environmental conditions being altered by GCC can also influence the biological disposition of these compounds. Several studies have shown that fish metabolize PCBs by cytochrome P-450 (CYP) mixed-function oxidases to more toxic, hydroxylated PCBs (OH-PCBs). For example, the role of temperature in the disposition of PCBs has been examined in juvenile rainbow trout . In these poikilothermic organisms, increased chemical uptake and enhanced metabolic transformation to more toxic oxon metabolites generally accompany increased temperatures. These adverse interactions have been demonstrated, for example, in 96-h midge lethality studies with methyl parathion and chlorpyrifos, where acute toxicity increased as temperatures increased from 10 to 30°C due to increased uptake and likely increased metabolic activation of the parent OPs to their more toxic oxon forms Effects of temperature on pesticide toxicity can be demonstrated by contrasting the toxicity of organophosphorus insecticides (OPs) and pyrethroids in the midge  Daphnia magna) Rana sp.) Palemonetes sp.) Conversely, some type 2 pyrethroids have demonstrated increasing toxicity with higher ambient temperatures, including in cockroaches (Hypomesus transpacificus]), and the fact that these areas are increasingly impacted by anthropogenic activities, including substantial point and nonpoint discharges of chemicals into the system.Sea-level rise linked to thermal expansions, reduced snow cover, and the accelerated melting of glaciers, ice caps, and polar ice sheets is projected to increase the salinity of estuarine and coastal freshwater habitats Oncorhynchus kisutch) acclimated to different salinity conditions and subsequently exposed to the OP phorate in 96-h acute toxicity tests. These fish demonstrated a 30-fold increase in acute toxicity to phorate when acclimated to 32 g/L salinity, compared with those acclimated at <0.5 g/L salinity O. mykiss) to aldicarb and elevated salinity increased acetylcholinesterase inhibition, toxicity of the pesticide, and microsomal production of the aldicarb sulfoxide Morone saxatilis × chrysops), a species that expresses FMOs that are nonresponsive to salinity induction, salinity did not affect acetylcholinesterase activity, toxicity, or aldicarb sulfoxide formation. Coincubations of microsomes with competitive CYP inhibitors had no impact on sulfoxide metabolite formation, suggesting that CYPs were not contributing to the salinity-induced sulfoxide formation. Extension of these findings to assessments of other anticholinesterase compounds that require thioether oxidation  has demonstrated similar increases in toxicity with increases in salinity The relevance of increased salinity on the toxicity of chemicals has been demonstrated in coho salmon  resulting from increased temperature and salinity conditions illustrate an AOP whose initiating event, metabolic activation enhanced by climate-modified temperature or salinity conditions, ends with increased lethality at the organism level. Prospective scenarios might suggest that other documented effects of pesticide toxicity in fish, such as interference with olfaction or behavior In both ADME alteration scenarios , interactions occur due to CITS scenarios. The idea of using a prospective approach is also highlighted in both ADME examples because the underlying mechanism is fairly well understood. It may be feasible for risk assessors to make similar types of predictions for other interactions between GCC stressors and other toxicants.In addition to alterations in chemical uptake and disposition by climate-associated ecosystem changes, the endocrine systems of biota are important targets of chemical toxicants. Because hormone systems are highly integrated into the life functions of organisms, toxicant disruptions of these systems can lead to many specific and nonspecific responses depending on the life stage and condition of the organism at the time of exposure. The vertebrate endocrine system is also highly sensitive to environmental cues, such as precipitation, temperature, and food availability, all of which are being altered by GCC. The combined role of the endocrine system in maintaining internal homeostasis and responding to the external environment makes it a sensitive and important target of toxicant and GCC interactions. The following three case studies provide examples to demonstrate some of these endocrine disruptor and GCC interactions, with a focus on potential perturbations of the thyroid and gonadal endocrine systems.Thyroid system functioning can be impaired by exposures to numerous environmental contaminants, including PCBs, polybrominated diphenyl ether (PBDE) flame retardants, PAHs, organochlorine and OP pesticides, metals, sex steroids, and pharmaceuticals. Exposures to thyroid-disrupting chemicals may impair the ability of vertebrates to adequately respond to GCC .Exposures to thyroid-disrupting chemicals in aquatic breeding amphibians are of special concern because metamorphic transitioning from water-based tadpoles to semiaquatic or terrestrial juveniles depends primarily on programmed secretions of thyroid hormones. Water availability and temperature are key variables affecting the timing of larval transitioning, with evidence of accelerated metamorphosis in amphibian tadpoles subjected to drying conditions and elevated water temperatures Thyroid hormones are involved in many important biological processes, particularly those mediating growth, development, reproduction, and metabolism. A substantial body of evidence has identified toxicological mechanisms by which contaminants can disrupt thyroid regulation and thyroid-dependent physiological processes. Several informative reviews have been undertaken that may serve as a reference Specifically, some of the increasingly well-understood molecular initiating events associated with thyroid-toxic contaminant exposures provide an opportunity to use an AOP approach in a prospective analysis to formulate hypotheses to determine and test whether thyroid toxicants could alter organism resilience to GCC-linked water shortages and temperature elevations. The biosynthesis and regulation of thyroid hormones  are under negative feedback control by the central hypothalamic–pituitary–thyroid axis. Contaminants can perturb the thyroid system at a variety of points along the hypothalamic–pituitary–thyroid axis. Perchlorate, a propellant used by the military, aerospace, and industrial sectors, is a well-documented thyroid disruptor that impairs iodide uptake by the sodium iodide symporter of thyroid epithelial cells A variety of compounds have also been shown to alter the transport, metabolism, and elimination of thyroid hormones. For example, OH-PCBs and OH-PBDEs are biotransformation products of phase I metabolism that have been shown to impair thyroid hormone homeostasis by competitively binding to plasma thyroid hormone transport proteins The AOP construct in For example, although not represented in the AOP, plasticity in metamorphic timing has important effects on overall fitness. Amphibian tadpoles that undergo metamorphosis earlier in development as an acclimative response to GCC may bear a fitness cost associated with this rapid metamorphosis in the form of smaller juvenile size. This smaller size has been shown to confer increased sensitivity to predation and reduced fecundity at first reproduction Gonadal hormone systems, responsive to both environmental cues and contaminant perturbations, provide examples of how chemical and nonchemical stressors can combine to impact reproductive performance and success, highly relevant organismal and population-level adverse outcomes. The hypothalamic–pituitary–gonadal (HPG) axis controls virtually all aspects of reproduction and sexual development in vertebrates. Normal function of the HPG axis is modulated by a variety of external stimuli such as food availability, photoperiod, behavioral interactions, and, particularly in poikilotherms such as fish, temperature Over the past decade, a substantial amount of research has focused on environmental contaminants, including a number of pesticides, drugs, and industrial chemicals, which have been shown to affect reproduction and development through interactions with the HPG axis. Large-scale screening programs in several countries, including the United States, are designed specifically to identify chemicals that affect HPG functioning through various mechanisms and pathways Given that a variety of environmentally relevant chemicals affect HPG functioning in fish and that the HPG axis is controlled, in part, by temperature, it is highly plausible that there would be biologically meaningful interactions between the two types of stressors. Unfortunately, little experimental work has been done to systematically explore this hypothesis. Studies have documented that temperature can affect the induction of vitellogenin (egg yolk protein) by exogenous estrogens in several different fish species Specifically, data collection relative to chemical–temperature interactions can be guided by knowledge of relevant molecular targets and associated AOPs in the fish HPG axis. A large amount of work has been done with HPG-active chemicals in fish, and AOPs have been described for reproductive effects of estrogen receptor agonists and inhibitors of vitellogenesis in females Many aquatic environments are subjected to widespread hypoxia  and anoxia . It is an increasingly urgent global problem that has caused species declines and major ecosystem changes. Hypoxia has been reported across more than 400 aquatic systems, covering thousands of square kilometers, including expansive areas of the Baltic Sea, Gulf of Mexico, and Chesapeake Bay Climate change is projected to worsen hypoxia by increasing nutrient runoff in regions subjected to increasing and extreme rainfall and by increasing oceanic oxygen stratification and warming Hypoxia alone has been shown to act like an endocrine disruptor in aquatic species While there is substantial evidence that hypoxia impairs the reproduction and development of fish In addition to hypoxia alone, GCC-linked increases of some pollutants  and remobilization of others  coupled with worsening hypoxia may impair the ability of organisms to respond to these dual environmental stressors. Exposures to contaminants such as PCDDs, PCDFs, planar PCBs, and PAHs elicit biological responses that are triggered by binding to the aryl hydrocarbon receptor (ahR). Functioning of the ahR pathway and its underlying mechanisms have been well described in vertebrates and include adaptive signaling upregulating xenobiotic metabolizing enzymes; toxic signaling causing adverse effects from high-affinity ligands, notably TCDD; and developmental signaling contributing to normal development of some tissues and organs On activation, ahR proteins form heterodimers with ahR nucleotranslocator proteins. The formation of these ahR/ahR nucleotranslocator complexes is regulated by ligand (or xenobiotic) binding. This complex can then bind to dioxin response elements upstream of gene coding regions, leading to altered gene expression a]pyrene). Consistent with 2,3,7,8-TCDD/hypoxia studies, PAH-ahR agonist exposures in the presence of hypoxia reduced CYP1A activity in zebrafish embryos but did not increase teratogenicity Studies in zebrafish larvae and cell cultures suggest that hypoxia may reduce ahR signaling and inhibit 2,3,7,8-TCDD induction of CYP1A  The situation presented by hypoxia serves as an example of the complexity of interactions between changing environmental conditions under GCC and the array of interactions of chemical and nonchemical stressors on biota. This case demonstrates that worsening hypoxia coupled with increasing pollution under GCC could act at multiple levels of biological organization and by multiple toxicological pathways, leading importantly to impaired reproduction and development of fish. Hypoxia itself can impair the HPG system, potentially eliciting impacts on reproduction and development, and TCDD-like contaminants could contribute to these impacts, albeit by very different mechanisms. A prospective AOP would be greatly beneficial in examining these complex interactions because it could help to identify and translate the range of interconnected mechanisms by which hypoxia and pollutants might interact to culminate in potential impacts on reproduction and development. This type of AOP construct could then be used to identify natural populations that might be subjected to these complex interactions for further risk analysis. It could also help to identify data gaps and other chemical contaminants that might act by similar pathways. To this end, a fish vitellogenin model has been used successfully in the field to identify mechanisms underlying hypoxia-induced endocrine disruption At the highest levels of biological organization, chemical and GCC exposures can influence the interactive dynamics of individuals, populations, and communities. For example, modifications of food-web dynamics by climate change can produce community-level changes that can impact how individual animals are exposed to contaminants. Prospective AOP analyses can help to predict and interpret potential ramifications of these complex GCC and pollutant interactions observed in the field.Ursus maritimus) in the western Hudson Bay (WHB) region of Canada occupy the extreme southern range for this species, where GCC effects have been strongest. Dependent on the Hudson Bay sea ice for movement, mating, and feeding, WHB polar bears have suffered declining body condition, birth rates, and survival rates over the past 20 years. Measured declines have been concurrent with sea-ice breakup occurring three weeks earlier in the summer As previously described, warming air and sea temperatures in Arctic and subarctic regions are among the strongest and best-documented climate change indicators Erignathus barbatus) and smaller ringed seals (Pusa hispida). Smaller female bears feed primarily on ringed seals Phoca groenlandica and Phoca vitulina, respectively) Polar bears feed on seals that inhabit both pack ice and open water. Fatty acid dietary tracers from polar bear adipose tissues demonstrate that males feed on larger pack ice–inhabiting bearded seals  contaminants in polar bear adipose tissue. Concentrations of total PBDEs and β-hexachlorocyclohexane increased, as did DDT and DDT metabolite clearance, from 1991 through 2007 n = 8) from the time the bears came ashore to emergence from their dens with new cubs, with average body mass loss of 43% of their prefast body weight During ice-free periods, when polar bears are forced ashore and prey are scarce, fasting leads to substantial body fat mobilization and body mass loss in polar bears, with WHB females fasting up to eight months through the denning and early cub-rearing periods The seasonal increases of OHCs in fasting polar bears are notable as they bring systemic concentrations to levels similar to those of the more contaminated polar bears of the Svalbard Islands (Norway) and eastern Greenland In the present case study, effects of climate in elevating OHC concentrations (CITS) could lead to a TICS scenario due to resulting endocrine, immune, and neurodevelopment perturbations that could potentially hinder acclimatization to GCC stressors. The use of a prospective AOP approach allows for prediction of potential cause-and-effect relationships resulting from observed field data. These predictions can be used by risk assessors in the interpretation of existing findings in polar bears and in predicting the nature and magnitude of anticipated effects in more northerly populations of polar bears, where sea-ice retreat has not yet reached the extremes that confront the most southern WHB populations. In this complex case study where CITS leads to TICS, we also see the role that AOPs can play in integrating community-level GCC perturbations In addition to building prospective and theoretical AOP scenarios based on empirical findings in the fields of climate change and ecotoxicology, risk and resource injury assessors have recently been confronted with adverse outcomes in wild populations without straightforward explanatory pathways. When this occurs, working backward from adverse outcomes through mechanistic pathways can help to formulate hypotheses to identify causative factors (and discard unlikely scenarios). Development of cause-and-effect relationships that explain GCC–toxicant interactions in a specific population can provide models and hypotheses that can be applied to other populations faced with similar combinations of stressors.Tachycineta bicolor). Following a report that mercury had reduced production of tree swallow nestlings during a drought but not during the previous year of normal weather Field studies have recently illustrated how ambient temperature and a contaminant interact as costressors on reproduction in tree swallows  and a pathway for acclimation to elevated heat conditions (hyperthermia) in tree swallow nestlings. An AOP was constructed that provides a starting point for a retrospective analysis of causation in the combinatory toxicity of weather and mercury in tree swallows .N-methyl-d-aspartic acid receptor calcium channels on adjacent neurons. This and other ROS-generated membrane-disruption mechanisms lead to flooding of calcium into sensitive neuronal cell structures, where it disrupts mitochondria and causes neuronal cell death. The ramifications of these effects, in addition to direct interference of MeHg with mitochondrial oxidative phosphorylation components, are an increase in cellular occurrence of ROS leading to lipid peroxidation, DNA and protein oxidation, mitochondrial injury, and cell death. In the avian nervous system, such effects may lead to behavioral dysfunction, manifest in decreased/lethargic foraging effort and reduced incubation times in nesting adult birds Work with live animals and in vitro studies provide insights into the mechanisms of MeHg toxicity  with little input from adult birds .In the absence of parental effects on nestling survival, a retrospective assessment of CITS and TICS pathways within the AOP provides insight into potential interactive mechanisms. Were extreme heat events affecting MeHg toxicity (CITS), it is likely they would impact either the disposition of MeHg or its interaction with molecular targets . Young nThe alternative pathway, where toxic effects of MeHg interfere with nestling acclimation to hyperthermic conditions (TICS), is also plausible as developing nestlings are particularly sensitive to MeHg toxicity A fruitful next step in unraveling the mechanism of mercury–GCC interactions would be a mercury dosing study varying temperature and MeHg treatments to examine the effects of mercury and temperature on nestling bird survival to one week of age. Testing of TICS mechanisms might prove expeditious as thermolytic behaviors such as gaping mouth, panting, and elevated respiratory rates, or the lack thereof due to MeHg, would provide easily accessible evidence of hindered acclimation mechanisms. Alternatively, increased ROS occurrence in thermally stressed and MeHg-dosed nestlings would be evidence that temperature was accelerating the ROS generation pathways. It would not be out of the question if both types of effects were observed, with a temporal progression of heat-induced increases of ROS generation leading to greater neurodegeneration and a loss of thermolytic capacity. The AOP in Whichever pathway or combination of pathways is responsible for climate–stressor interactions in the tree swallows, the ramifications are important for conservation and species protection. If mercury impairs the developing thermoregulatory system, leading to higher mortality of young nestlings during unseasonable heat, and the identified mechanism is conserved across bird species, risk managers would have a strong basis for predicting the effects of GCC on temperate songbirds with predicted exposure to mercury. If, instead, temperature exacerbated MeHg-induced ROS generation, neurotoxicity, or other toxic responses, there could be wide repercussions for the range of other compounds that work via similar mechanisms of toxic action . In either case, if mitigation or habitat triage were called for, reducing environmental mercury concentrations and/or availability would be, in the long run, the most important approaches, with conservation resources applied selectively to populations residing at the edge of their physiological temperature tolerance range.These case studies demonstrate, at a mechanistic level, how GCC can interact with known toxic chemicals to change anticipated exposure–effect outcomes. In the process, they illustrate essential concepts for understanding the combined stresses of GCC and chemical contaminants. What stands out as an important lesson is that environmental toxicologists cannot ignore the increasing influences of climate change when developing effects inputs for chemical and contaminant assessments. It would be equally unwise to forecast the effects of GCC without considering the interaction between climatic variables and contaminants that can alter current risk outcomes. This is not to suggest that all chemical interactions with all species will be altered by GCC, but rather that, in risk and injury assessment scenarios, careful consideration should be given as to whether inclusion of GCC as a costressor is necessary to avoid erroneous estimates of impacts in study systems or defined populations Recognizing the importance of potential climate–chemical interactions in assessing risk is the crucial first step, and a path forward using AOPs is suggested. Through AOPs, the stepwise progression from exposure to chemical–receptor interaction to molecular, cellular, tissue, organ, and organismal responses can be tracked. The influence of changing climate variables can be tested, extrapolated, or predicted at critical points along the AOP, providing data on CITS. Similarly, an understanding of biological processes necessary for acclimation to GCC effects allows anticipation of how chemicals might perturb the acclimation process, leading to an understanding of TICS. As toxicity mechanisms are often shared by wide chemical classes and climate acclimation mechanisms are shared across taxonomically similar organisms, established climate–chemical AOPs provide a means by which important climate–chemical interactions can be characterized and applied across broad chemical classes and species groups of concern.Building on pathway approaches, the case studies demonstrate the significance of the timing, duration, and overlap of exposures to chemical and climate stressors. Of similar importance is the recognition that sublethal alterations due to acclimation to the dual stresses of GCC and contaminant exposures can negatively impact an organism's fitness and survival. To answer questions of how climate and chemicals might interact as costressors, prospective scenarios can be built based on known or theoretical AOPs that incorporate climate modifications or, alternatively, on climate acclimation or adaptation processes that incorporate toxicological modifications. Conversely, when adverse outcomes with suspected climate and chemical interactions are observed, a retrospective analysis can be used to identify knowledge gaps and propose and test hypotheses that might identify the mechanistic basis for the interaction. Once clarified, the findings from both prospective and retrospective assessments can be used to identify and improve our ability to predict impacts occurring in the field.Although the case studies addressed nonhuman impact assessments, the methods described will work equally well in GCC–chemical interaction scenarios with humans. Increases in human mortality from cardiovascular system– and respiratory system–related complications during heat waves are well established, suggesting one might work retrospectively through the physiological processes involved to identify the knowledge gaps and propose hypotheses and experiments that may explain the role of chemicals in morbidity and mortality due to combined temperature and contaminant exposures. Although extensive research along these lines has not been carried out, heat waves will clearly be a more frequent event as the result of GCC, and assessing risk of human deaths in major cities is going to be an important part of the planning and risk-assessment processes What is clear from the examples provided here is that a better understanding of how toxicological mechanisms interact with climate-induced stressors will provide a solid platform for improved effect assessments for both humans and wildlife. In chemical risk assessments for human health, retrospective AOP approaches can provide the biological plausibility that will inform predictions of changed patterns of disease as a result of GCC. Epidemiologists can use empirical data from existing and past associations between disease and climate to make projections about the human health implications of GCC, tying them to toxicological mechanisms through AOP scenarios. For environmental managers, AOP constructs and toxicological mechanisms allow prediction of GCC–contaminant interactions, which can facilitate refocusing and prioritization of regulatory efforts, species protection plans, research agendas, and conservation planning.


Reproductive health among cancer survivors is an important quality of life issue. Certain cancer therapies have known fertility risks. There is an existing cohort of adolescents and young adults (AYA) cancer survivors that, seen less frequently in clinical care settings than active patients, are likely not having discussions of fertility and other reproductive health issues. A survivor or healthcare provider can easily assume that the window of opportunity for fertility preservation has passed, however emerging research has shown this may not be the case. Recent data demonstrates a close relationship between fertility and other late effects to conclude that ongoing assessment during survivorship is warranted. Some fertility preservation procedures have also been shown to mitigate common late effects. This review explores the link between late effects from treatment and common comorbidities from infertility, which may exacerbate these late effects. This review also highlights the relevance of fertility discussions in the AYA survivorship population. Reproductive potential and other reproductive health concerns among cancer survivors is an important quality of life issue. Certain cancer therapies, particularly alkylating chemotherapy agents and radiation, have known fertility risks. Fertility issues within oncology have ignited a robust research focus that explores physiologic and psychological late effects, advancing technologies that reduce the risk of becoming infertile in the future, and broader reproductive health concerns such as appropriate contraception and the HPV vaccine –4. SustaThere are heterogeneous reports exploring the rates of discussion of fertility. Ranging from 34 to 70% –11, whetAdolescents and young adults cancer survivors have distinct developmental needs. While many adolescents without a history of cancer spend these formative years affirming their unique identity, survivors are on a journey toward “normal,” as they have experienced significant disruption in identity formation . EngaginWith attention to fertility gaining more attention in pediatric oncology settings, discussions are improving . That isThis AYA survivorship cohort is growing  but survThere are likely misconceptions regarding AYA cancer survivors’ quality of life concerns. There is an overshadowing focus on survivors’ disease status and the assumption that cancer survivors are less interested in sex and reproduction than their healthy peers. In reality, research indicates adolescents with a chronic illness are at least as sexually active as their healthy counterparts , 21. TheThere are unique relationships among infertility and the late effects of cardiotoxicity, bone health, and other endocrine disorders. It is important for healthcare providers (HCP) to be aware of the potential for infertility to exacerbate these late effects. At the same time, HCP must also be aware of the risk for infertility symptoms to go untreated due to a focus on other common physiological late effects. We discuss late effects that HCP routinely assess, and the similarity of these symptoms within infertile persons that may go overlooked.It is well documented that survivors are at risk for cardiovascular disease and mortality due to treatment alone –24. FemaInfertility may aggravate these existing cardiovascular impairments. Females who experience premature ovarian failure (POF) as a result of treatment are at risk of cardiovascular disease due to the early loss of estrogen , 30. EstBone health may also be affected by both infertility and cancer treatment, and should be regularly assessed at follow-up visits. Reduction in bone health quality from treatment can occur in a variety of ways. Many commonly utilized chemotherapies, such as corticosteroids, antimetabolites, anthracyclines, and alkylating agents have been shown to adversely impact bone mineral density (BMD) through creating an imbalance in bone metabolism with increased bone resorption and decreased bone formation –38. ThesGonads in both males and females secrete hormones that facilitate bone metabolism . There iIn males, there is a unique, reciprocal relationship between bone health and fertility. Evidence for this can be found in adults with liver cancer; in those with impaired gonadal function and osteoporosis, liver transplantation was found to have restorative action on both gonad function and bone health . ConversPerhaps the strongest relationship warranting close assessment is that of infertility and endocrine late effects. Up to 50% of survivors have reported effects such as hypopituitarism, precocious puberty, and adrenal insufficiency . EndocriIn females, chemotherapy and radiation have varying risks on the reproductive organs as well as the endocrine system that regulates hormones within these organs. Females are particularly at risk of infertility due to cancer treatment as females are born with a finite number of non-replicating oocytes. Furthermore, luteinizing hormones stimulate theca cells to produce a steroid hormone that is converted to sex hormones in the granulosa cell which is stimulated by follicular stimulating hormone (FSH) . This prMen may experience comparable menopausal symptoms during andropause such as osteoporosis and sexual dysfunction as a result of a dramatic decrease in testosterone . AndropaMale and female cancer survivors can be burdened with emotional, social, and psychological consequences as a result of infertility. AYA survivors have experienced a dramatic acceleration of life’s challenges, beginning with confronting their own mortality. Survivorship comes with a new set of complications, negotiations, and coping skills. Adolescent cancer patients have been shown to have long-term goals that include childbearing and parenthood, as well as informational needs for fertility preservation . A diagnOther barriers may prevent the assessment of reproductive potential aside from solely physician awareness. Males may be uniquely overlooked regarding the psychosocial effects of survivorship. Male AYA survivors have been noted to have issues with body image and appearance, masculinity and sexuality, cynical about planning for future parenthood, and afraid of transmitting cancer to a partner . PerceptThe existing AYA survivorship cohort seen for follow-up care is in a unique transitional period, not only the transition of patient to survivor, but also the normative developmental transition of adolescent to adult. Past goals are now seen as capable of becoming actualized in adulthood, including goals for parenthood; an unknown fertility status may heighten distress and anxiety . ZebrackFertility can be compromised in different ways for males and females depending on the initial disease site, stage, age of patient, therapeutic treatment regimen, and condition of the patient. Alkylating agents within some chemotherapies do not target specific cells, and therefore affect all cells, especially those that divide rapidly such as cancer cells in the bone marrow. Other cellular activity can be impacted such as also oogoniums in pre-pubertal females, primary oocytes, and sperm cells, as well as theca and granulose cells in ovarian follicles that mature oocytes –69. BecaMale survivors who did not undergo a fertility-sparing procedure before treatment also have options post-treatment if fertility is impaired. Due to the rapid regeneration of sperm, recovery of reproductive potential in males after treatment with non-akylating chemotherapeutic agents is higher than with alkylating agents . NaysmitIn addition to achieving pregnancy, some fertility preservation options may offset other late effects experienced as a result of treatment. Recent experimental trials have examined the benefits of re-implantation of ovarian tissue in females for purposes other than achieving a pregnancy. Transplantation of cryopreserved ovarian tissue has shown to be a potential method for recovery of ovarian function , 79, whiIt is important to keep up with the fast-paced technologies of fertility treatments, as it is to stay abreast of the latest treatment regimens. Active treatment is being re-shaped by the push to discuss fertility preservation options prior to treatment, which is a result of the needs expressed by the ever-expanding, vocal survivorship cohort that exists today. What’s now needed is a way to bridge these two worlds and inform survivors that the window of opportunity for future parenthood, reversing POF and other late affects may not be gone. Long-term follow-up guidelines should incorporate the benefits of discussing fertility and fertility preservation options so that HCP can ensure comprehensive care and long-term quality of life.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.


To investigate socioeconomic inequalities in a comprehensive set of health indicators among persons with spinal cord injury in a wealthy country, Switzerland.Observational cross-sectional data from 1549 participants of the Swiss Spinal Cord Injury Cohort Study (SwiSCI), aged over 16 years, and living in Switzerland were analyzed. Socioeconomic circumstances were operationalized by years of formal education, net equivalent household income and financial hardship. Health indicators including secondary conditions, comorbidities, pain, mental health, participation and quality of life were used as outcomes. Associations between socioeconomic circumstances and health indicators were evaluated using ordinal regressions.Financial hardship was consistently associated with more secondary conditions , comorbidities  and pain , whereas mental health , participation  and quality of life  were reduced. Persons with higher education reported better mental health  and higher quality of life ; other health indicators were not associated with education. Household income was not related to any of the studied health indicators when models were controlled for financial hardship.Suffering from financial hardship goes along with significant reductions in physical health, functioning and quality of life, even in a wealthy country with comprehensive social and health policies. An inverse association of socioeconomic circumstances with various health indicators is one of the most robust findings of social-epidemiological research Previous studies on health inequalities in persons with disabilities have addressed the concept of ‘health’ by using particular health indicators such as functional limitations In this study, we set out to tackle these shortcomings by investigating social inequalities in health among adults with a severe physical impairment, namely spinal cord injury (SCI). SCI has a far-reaching impact on a person's functioning and health as affected persons suffer from a loss of sensory and motor function below the lesion level The aim of this study is to investigate social inequalities in a comprehensive set of health indicators in persons with SCI. We therefore analyze the association between socioeconomic circumstances  and various health indicators including secondary conditions, comorbidities, pain, mental health, participation, and quality of life.We analyzed cross-sectional data from the population-based community survey of the Swiss Spinal Cord Injury Cohort Study (SwiSCI) Socioeconomic circumstances: Level of education, net equivalence household income and perceived financial hardship were chosen as three indicators of individual-level socioeconomic circumstances. Education was classified according to the International Standard Classification of Education as total years of formal education, combining school and vocational training Health conditions & body functions: To assess the prevalence of health problems in addition to SCI, a sum score of four dichotomous items on comorbidities  based on the Self-Administered Comorbidity Questionnaire Activity & participation was measured using the 11-item subscale on participation restrictions of the Utrecht Scale for Evaluation of Rehabilitation-Participation (USER-participation) Quality of life was assessed by a single item rated on a 5-point Likert-scale ranging from 0 (‘very poor’) to 4 (‘very good’) Control variables: Age, gender, lesion characteristics , aetiology (traumatic vs. non-traumatic), and social support were included as potential confounders in multivariable analyses. Severity of disability had been linked to diverse health indicators Following descriptive analysis of the study population, we explored unadjusted bivariate associations between socioeconomic circumstances and health indicators. Given the fact that dependent variables were not normally distributed, we used a nonparametric ANOVA to evaluate differences between groups  and a test for trend across ordered groups according to Cuzick Next, a set of regression analyses was applied to evaluate associations. As effect sizes of ordinal regressions are easier to interpret as compared to models for continuous dependent variables, ordinal logistic regression models were applied for all outcomes. Ordinal logistic regressions are extensions of logistic regressions In the main data analyses, we adjusted for both unit and item nonresponse Item nonresponse, which refers to the failure of survey respondents to answer a specific question, was addressed using multiple imputation (MI). We used MI by chained equations (MICE)To assess the risk of bias due to list-wise exclusion of cases with missing data in health indicators, a sensitivity analysis was performed comparing four scenarios:imputed data for socioeconomic circumstances/controls, but only full cases in health indicators (as displayed in the main results),only full cases in all variables,imputed data for socioeconomic circumstances/controls and replacing missings in health indicators by ‘best case’ ,imputed data for socioeconomic circumstances/controls and replacing missings in health indicators by ‘worst case’ .Results of the sensitivity analysis are displayed in –39, persons indicated on average 10.6 points. Nearly three out of four persons did not report a comorbidity and the overall pain intensity was somewhat over 3 in the total sample. On a total score from 0–100, mental health scores were on average around 72 and participation around 70, with higher scores indicating better mental health and less participation restrictions. Nearly 60% of the total population rated their quality of life as at least good.Sample characteristics are displayed in Results from bivariate analysis are displayed in Results from multivariable analysis are presented in This study of a large cohort of Swiss men and women with SCI demonstrates consistent associations of financial hardship with all health indicators under study. Experiencing severe financial difficulties goes along with more health problems as well as reduced functioning and quality of life. This holds true even for those reporting only some financial difficulties. Interestingly, the links with established indicators of social inequalities are weaker in case of education or non-existent in case of household income when adjusting for perceived financial hardship.In light of these inconsistent associations, the question arises why financial hardship exerts such strong and consistent effects on health. One possible argument is that financial difficulties aggravate people's burden of everyday life as they restrict the access to relevant resources and generate feelings of relative deprivation with subsequent stress reactions The link between education and health was restricted to mental health and quality of life when financial hardship was considered in the same model and was therefore less consistent as compared to the one observed with financial hardship. In case of income, none of the associations was significant after adjustment for all other influence factors and potential confounders. Whether these findings can be replicated or whether they reflect imprecise measures in populations with disabilities needs to be analysed in further studies. For instance, as education was measured by a summary index of school and vocational training, it may not adequately reflect the socioeconomic circumstances of persons with SCI, particularly as many of them underwent long-standing vocational re-training. A similar point can be made for household income. First, household income only moderately correlates with financial hardship (Pearson correlation coefficient −0.335) thus indicating somewhat different economic facts. Second, net equivalence household income may not adequately take into account the economic burden of people with disabilities, given their regular additional health care expenditures or other fixed costs related to the disabling condition .Yet, in line with our main finding of a consistent association of financial hardship with health, a recent cohort study demonstrated a strong effect of financial hardship on mortality, above and beyond the one produced by education and income This study has several limitations. First, findings are based on cross-sectional data, which does not allow to draw conclusions on causal associations. Second, our sample may not be fully representative of persons with an SCI living in Switzerland. Although several SCI-clinics and the national association for persons with SCI have been involved in recruitment Despite these limitations, our study has several strengths. The study population is based on a large community sample rather than on some selected group, and data meet high quality standards. We analyse a comprehensive set of health indicators thus drawing a detailed picture on health problems, functioning and quality of life in persons with SCI. Moreover, we conducted additional sensitivity analyses to correct for item and unit nonresponse. Given the small number of aberrations see , we concIn this study on the association of socioeconomic circumstances with health in persons with disabilities, financial hardship was the only indicator consistently related to reduced health. This indicator may reflect material as well as psychosocial adversity in people with disabilities. As this association was evident in Switzerland, one of the wealthiest countries worldwide, even stronger effects of financial hardship on health might be expected in less developed countries. If supported by further research, our results may have implications for more equitable resource allocation.Appendix S1Details of the imputation modeling.(DOCX)Click here for additional data file.Table S1Sensitivity analyses of associations between socioeconomic circumstances and health indicators, odds ratios of ordinal regressions and  using different scenarios to handle missing values, weighted (W) and unweighted (U) results.(DOCX)Click here for additional data file.


Animal models of chronic pain are widely used to investigate basic mechanisms of chronic pain and to evaluate potential novel drugs for treating chronic pain. Among the different criteria used to measure chronic pain, behavioral responses are commonly used as the end point measurements. However, not all chronic pain conditions can be easily measured by behavioral responses such as the headache, phantom pain and pain related to spinal cord injury. Here I propose that cortical indexes, that indicate neuronal plastic changes in pain-related cortical areas, can be used as endpoint measurements for chronic pain. Such cortical indexes are not only useful for those chronic pain conditions where a suitable animal model is lacking, but also serve as additional screening methods for potential drugs to treat chronic pain in humans. These cortical indexes are activity-dependent immediate early genes, electrophysiological identified plastic changes and biochemical assays of signaling proteins. It can be used to evaluate novel analgesic compounds that may act at peripheral or spinal sites. I hope that these new cortical endpoint measurements will facilitate our search for new, and more effective, pain medicines, and help to reduce false lead drug targets. Pain has at least two different major forms: acute pain that is short-lasting, and often serves as protective and learning signals. It is also called physiological pain in part due to its important physiological functions. The second form is chronic pain that is long-lasting pain caused by injury to tissue or nerve systems. Although it also informs animal or patients the location of the injured area, the long-term component of chronic pain is not physiological critical, and it causes cognitive impairment, emotional sufferings, loss of sleep and mood disorders. Thus, it is also called pathological pain.Due to the lack of understanding of cellular and molecular mechanisms of pain mechanisms; the black box experimental approach for studying pain has been widely used by basic researchers and drug developers Figure . Among mThe progress made in human brain imaging has significantly improved our understanding of chronic pain. Brain imaging in conscious humans allow us to evaluate roles of various cortical areas in pain, and brain activation by painful stimuli can be evaluated and compared with patient's psychological reports of pain and emotional feelings . RecentlWhile animal models of chronic pain have greatly facilitated our understanding of basic pain mechanisms, there are still many major problems that cannot be solved using animal behavioral models. Many forms of clinical pain cannot be mimicked in animal models. For example, it is very difficult to measure 'phantom' pain in amputated animals; and it is impossible to measure nociceptive withdrawal responses in animals with spinal cord injuries. Mimicking headaches and chronic back pain in animals is also proved to be extremely difficult, since there is not a clear behavioral motor response related to such pain that can be measured. Furthermore, even in many pain conditions that the measurement in animal behavioral responses to pain are possible, the pain index is often affected by unwanted side effects on motor or pre-motor functions. For example, in case of animal models of chemotherapy induced neuropathic pain, the same chemicals also caused injury to motor neurons/nerves. Defects in motor functions directly affect the measurement of nociceptive responses, if only motor responses are used as pain index such as withdrawal thresholds or latencies. Finally, most of potential analgesic drugs also act on motor neurons non-selectively. There is great need to develop additional non-behavioral measurements for chronic pain.Unlike animal experiments, human brain imaging using functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) have greatly increased basic neuronal mechanisms for chronic pain, especially at the brain areas. The imaging in conscious patients provides the unique opportunity to investigate if any abnormal neuronal activities may be related to chronic pain in patients -7. ProgrIn basic research area, behavioral studies have been commonly used for measuring endpoint of chronic pain. Most of pharmaceutical drug discovery is focused on two major areas: target proteins and the behavioral endpoints, at least to my knowledge. The behavioral evaluations in animal models of chronic pain are used as the end point for preclinical studies. Despite many limitations of behavioral tests , these 'analgesic' indexes are commonly used, because it is economic, fast, and easily understood by laypersons such as private investors.The failure to provide consistent non-behavioral endpoints for chronic pain also complicates drug discovery; and too many potential lead drugs work well in animal models of chronic pain. In any pain-related conferences held in recent years, you often hear much more positive drug target proteins than negative ones. Furthermore, the failure to require solid basic scientific evidence by the drug regulator encourages the cheap- and short-cut experimental approaches used by private investors and drug developers. Furthermore, many of these 'analgesic drugs' failed to be translated into clinical drugs, in part due to the fact that only behavioral endpoints were used in many of these preclinical studies.It is easy to tell humans are different from animals in term of reporting pain, although humans complain about pain for just for pain. Previous studies reported that animal vocalization activities may be used to measure pain or especially spontaneous pain -13. HoweRecent studies of neural correlates of social exclusion by neuroimaging study found that the ACC is more active during exclusion rather than during inclusion , suggestHere I would like to propose the use of cortical plasticity as an endpoint measurement for chronic pain, including biochemical, electrophysiological and imaging measurements Table . Such noActivity-dependent immediate early genes are known to be activated by neuronal activity in the central nervous system. Peripheral noxious stimuli or injury triggered activation of c-Fos and phosphorylation of cAMP response element binding protein (pCREB) in the spinal cord dorsal horn neurons ,8. Such Activation of IEGs is not limited at the spinal cord level. Along the somatosensory pathways, activation of IEGS have also been reported in the thalamus, and related cortical areas. Among several pain-related cortical areas, activation of IEGs in cortical neurons after the injury has been reported in the ACC and IC. Using c-Fos, NGFI-A , and pCREB, neurons in the ACC are known to be activated by peripheral noxious stimuli, injury or amputation -24. Wei Using gene knockout mice, it has been shown that the amount of IEG activation may be related to behavioral pain phenotypes. In adenylyl cyclase subtype 1 (AC1) or AC1 and AC8 knockout mice, it has been shown that chronic inflammatory pain and neuropathic pain were significantly reduced. Consistently, pCREB immunoreactivity induced by hindpaw inflammation was also reduced in AC1, AC8, or AC1&8 double knockout (DKO) mice. Activation of IEGs is also noted in other cortical areas such as IC . SimilarIn addition to assay baseline neuronal changes after the injury, some of these makers can be also used to evaluate allodynic responses in animals with the injury. In animals at two weeks after the injury, most of IEGs activities return to baseline level in the ACC. Non-noxious stimuli that usually did not induce any obvious behavioral response and gene expression in the ACC triggered remarkable IEG activation within the ACC [see ] Figure. SimilarGlutamate is the major excitatory fast transmitter in the cortical areas including the ACC . PostsynSimilar changes are found in ACC neurons in animal models of inflammation induced by complete Freund's adjuvant (CFA) . CFA injPaired-pulse facilitation (PPF) is a transient form of plasticity commonly used as a measure of presynaptic function, in which the response to the second stimulus is enhanced as a result of residual calcium in the presynaptic terminal after the first stimulus ,36. In cIn addition to the use of PPF in the ACC after peripheral nerve injury, AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in ACC neurons in the presence of 0.5 μM tetrodotoxin (TTX) were also found to be affected. After peripheral nerve injury, there was an obvious increase of mEPSC frequency in ACC neurons compared with that of control group. Furthermore, there was significant difference in the amplitude of mEPSCs between the two groups, indicating postsynaptic changes in AMPA receptor mediated responses (see below). By contrast, no significant change in mEPSCs amplitudes is detected in animal model of inflammation . HoweverIn a recent study, the use of c-Fos transgenic mice allows one to selectively record pain-trigger cortical cells . The com2+ permeable and inwardly rectifying [AMPA receptors without GluR2 are Cactifying -40. Inwactifying . To idenctifying . Consistctifying . These rSimilar rectification of the AMPA receptor mediated responses in ACC neurons of rats has been reported after peripheral inflammation with hindpaw CFA injection .Evidence for the involvement of cortical NMDA NR2B receptor in chronic pain first comes from the NMDA NB2B forebrain overexpressed 'smart' mice ,43. In tIn an animal model of arthritic pain, it has been reported that inflammation increased phosphorylation but not up-regulation of NMDA NR1 proteins in the central amygdale . In paraWhat is likely to be the synaptic mechanism responsible for the analgesic effects produced by ZIP in neuropathic pain? PKMζ can potentiate postsynaptically the amplitude of AMPA receptor-mediated EPSCs ,47. BecaThe trafficking of AMPA receptor subunits has been proposed to contribute to synaptic plasticity underlying hyperalgesia -50. We iIn rats with CFA inflammation, Bie et al reported that AMPA GluR1 in the synapsome preparation of the ACC neurons from rats with CFA injection showed significant increases, suggesting that synaptic AMPA GluR1 is significantly increased .The phosphorylation of GluR1 subunit of AMPA receptors is critical for synaptic expression of the receptors, their channel properties, and synaptic plasticity -53. We tWe examined whether, in the ACC, peripheral nerve injury causes changes in PKMζ . Behavioδ and C fibers, evoked field EPSPs were recorded in the ACC. Digit amputation at the contralateral hindpaw causes a rapid and long-lasting enhancement (more than 120 min) of sensory responses  that amygdala neurons showed differential sensitization (measured by spike activities) to sensory afferent inputs in a model of arthritic pain .In addition to spike recordings, recent studies using whole-cell patch-clamp recordings from adult ACC neurons found that many of ACC neurons are responsive to peripheral nociceptive stimulation. Whole-cell patch-clamp recording offer better sensitivity for detecting possible changes in chronic pain, and evaluate the possible analgesic effects of drugs .Recent several studies have reported brain activation in awake rats using the imaging technique . In thisIt is known that injury triggers molecular and cellular changes in different parts of the brains. Depending on the specific regions of the brain, these neurobiological changes may contribute to different aspects of pain, such as learning and memory, anxiety, unpleasantness and attention. Although some of these changes can be observed at behavioral levels , some are difficult to be studied at behavioral level. One good example to use the cortical endpoints is to evaluate the effects of new drugs in spinal cord injury induced pain. It is commonly reported in patients with spinal cord injury that they suffer long-lasting neuropathic pain caused by the injury. However, in animal models of spinal cord injury, it is impossible to evaluate the drug's analgesic effect using behavioral tests. Using the proposed cortical endpoints, I propose that these cortical endpoints can be used. For example, activation of various IEGs in the pain-related cortex such as ACC can be used to evaluate early activation, and measuring glutamate mediated responses and biochemical markers can be used to evaluate enhanced transmission during chronic pain induced by spinal cord injury. The drugs that inhibited or erased cortical makers may be potentially useful for treating spinal cord injury related chronic pain in patients.The same endpoints can be used to evaluate drugs that acting at peripheral or spinal mechanisms. In additional behavioral responses, cortical endpoints can be measured before and after drug application. Drugs that reduce peripheral or spinal sensitization or potentiation should reduce activation of cortical markers. The cortical measurement will help to determine if behavioral inhibition is due to pure inhibition of motor neurons that are required for sensitized withdrawal responses in chronic pain conditions. There are some limitations of using these proposed cortical indexes to study 'pain' in animals. It is important to perform additional experiments to distinguish possible changes in these cortical indexes that are important for pain-related memory but not pain itself. Considering that cortical neurons are often activated in different situations, it is not an easy task if the injury is also located within the brain. The use of selective gene-manipulated mice or pharmacological inhibitors may help to address some of these concerns.In summary, recent basic neurobiological investigations of physiological and pathological mechanisms of pain and chronic pain provide critical information for our understanding of long-term plastic changes in cortical areas. These cortical changes that persisted during the course of chronic pain can be used a valuable index to measure 'pain' in animals. The combined use of these neurobiological indexes, together with or without behavioral motor withdrawal responses, will greatly facilitate our searching for new drugs, and also help us to understand why some of current pain medicine does not work in some clinical conditions.The author declares that they have no competing interests.MZ designed and finished the manuscript.


Neuromodulation in sensory perception serves important functions such as regulation of signal to noise ratio, attention, and modulation of learning and memory. Neuromodulators in specific sensory areas often have highly similar cellular, but distinct behavioral effects. To address this issue, we here review the function and role of two neuromodulators, acetylcholine (Ach) and noradrenaline (NE) for olfactory sensory processing in the adult main olfactory bulb. We first describe specific bulbar sensory computations, review cellular effects of each modulator and then address their specific roles in bulbar sensory processing. We finally put these data in a behavioral and computational perspective. Neuromodulators such as noradrenaline (NE), acetylcholine (ACh) and serotonin (5HT) serve important functions in sensory perception. Sensory perception, as much as other brain functions, needs to be regulated according to task demands, features of the sensory environment such as e.g., signal-to-noise ratio, and the animal's physiological state. Classically, each of these neuromodulatory systems has been linked to specific functions such as improvement of neuronal signal to noise ratios, attentional processes, general arousal, learning and memory  that innervates primarily glomerular and granule cell layers of the bulb (Heimer et al., In vitro data from adult rodent OB shows that activation of α1 and α2 receptors have opposing effects on granule cells: α2 activation decreases spontaneous IPSCs while α1 activation increases spontaneous IPSCs. Due to the fact that α2 receptors have a higher affinity for NE than α1 receptors, α2 effects dominate at very low concentrations of NE (Nai et al., In vivo recordings show that stimulation of the LC makes mitral cells more responsive to subthreshold olfactory nerve shocks (Jiang et al., in vivo studies using glutamatergic activation of LC neurons (Okutani et al., The OB receives a dense noradrenergic projection from LC that terminates in all but the most superficial layers. NE fibers preferentially target the granule cell layer, and to a lesser extent the mitral cell layer (McLean et al., In summary, NE and ACh exert some common—depolarization of mitral and granule cells—and some differential—depolarization of PG cells—effects on bulbar networks. At the glomerular level, excitation of PG cells affects glomerular computations such as decorrelation of odor representations and concentration invariant representations—effects suggested to be modulated by cholinergic activation of nicotinic receptors (Linster and Cleland, in vivo recordings (Mandairon et al., Distributed patterns of activity in response to chemical stimuli are transmitted to the olfactory bulb via OSN axons that terminate in the glomeruli of its input layer. It is clear from recent investigations that the perceptual qualities of odorants can be predicted, to a limited degree, from the patterns of activation that they evoke at the olfactory bulb input layer (Linster and Hasselmo, Contrast enhancement in the glomerular layer is achieved by dendrodentritic interactions between PG and mitral cells Figure . Both ceOdor receptive fields, shaped by the glomerular computations described above, are then processed in the deeper bulbar layers, in which mitral cell spiking activity is further modulated by local interneurons before being conveyed to higher-order processing networks. In these deeper layers it is assumed that mitral cell odor receptive fields, i.e., whether and to what extent a mitral cell responds to an odorant, are already determined and that mainly spike timing is modulated (Cleland and Linster, Interestingly, in our previous behavioral experiments, modulation of muscarinic receptors was not effective by itself but did enhance the effect of nicotinic receptor modulation on olfactory discrimination (Mandairon et al., In summary, in deeper layers of the bulb, cholinergic inputs activate the mitral-granule cell feedback loop, thereby possibly increasing synchrony among odor-responsive mitral cells Figure . Increasin vivo data shows that α1 receptor activation modulates the response of mitral cells to weak olfactory nerve stimulation, rendering them more sensitive to near-threshold stimuli (Jiang et al., Noradrenergic inputs act on α1 receptors on mitral cells as well as α1 and α2 receptors on granule cells. Brain slice and Overall, activation of NE receptors on mitral and granule cells changes the excitability of mitral cells in a non-linear dose dependent relationship that is reflected in the behavioral response Figure . The recSignal-to-noise ratio in sensory systems can be defined as the ratio between the number of spikes evoked by a sensory input and the total number of spikes during a comparable time frame (Linster and Hasselmo, While the cellular effects of ACh and NE on bulbar cells can look similar on the surface, they differ in specific details of channels modulated and precise effects on neural behavior. In the glomelular layer, ACh acting on nicotinic receptors is strongly implicated in regulating periglomerular inhibitory microcircuits performing contrast enhancement and receptive field decorrelation (Linster and Cleland, in vivo, behavioral and modeling approaches.Central and modulatory inputs to primary sensory structures allow for flexibility and regulation of sensory processing according to behavioral demands. The olfactory bulb, for example, receives more efferent than afferent inputs (Shipley and Ennis, We have reviewed the role and function of two major neuromodulators, ACh and NE, for modulation of main olfactory bulb processing in adult rodents and early olfactory perception. Each modulator affects bulbar processing through a variety of cellular mechanisms acting on multiple circuits within the OB. Presumably, each modulator has a specific functional role, although their experimentally observed effects overlap substantially, making a clear dissociation difficult. Although these two neuromodulatory systems may exhibit differences in activation dynamics during specific behaviors, both systems are typically at least somewhat engaged during the waking state. Ultimately, it will be important to elucidate interactions between these neuromodulatory systems, either by direct projections between them or local effects, as well as additive effects. Furthermore, neuromodulatory systems project broadly throughout the central nervous system and target numerous structures beyond early sensory areas. Another key question that remains to be addressed is to understand how neuromodulatory inputs regulate interactions between early and higher-order sensory and cognitive networks. Indeed, it has been shown computationally that manipulating even subtle features of OB output can lead to dramatic changes in the response of higher-order processing networks (Linster and Cleland, 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.


We have developed a novel combined experimental/modeling approach to determine the mechanical properties of primary cilia. Specifically, we developed a large rotation formulation of cilia bending with fluid flow that accounts for rotation at the base of the cilium, the initial shape of the cilium and fluid drag at high deflection angles. We also have obtained three dimensional configurations of primary cilia in their deformed configuration at 3Hz acquired with high-speed confocal microscopy. We found a wide variety of previously unreported bending shapes and behaviors. Cilia appear to deflect in smooth bending shapes, rigid-body rotation, and in a hinged or kinked pattern. This suggests that both the axoneme and basal body anchorage are important to understanding deflection patterns. We also analyzed post-flow relaxation patterns. These results indicate that with removal of force, cilia slowly return to their initial configuration. However, they also can return to a different configuration with “overcorrection” observed that suggests adaptation of mechanical properties. Results from our combined experimental and theoretical approach suggest that the average flexural rigidity of primary cilia might be higher than previously reported. In addition our findings indicate the mechanics of primary cilia are potentially non-linear, richly varied, and mechanisms may exist to alter their mechanical behavior.


This paper summarizes current knowledge about electrical impedance tomography (EIT) and its present and possible applications in clinical practice in pediatric respiratory medicine. EIT is a relatively new technique based on real-time monitoring of bioimpedance. Its possible application in clinical practice related to ventilation and perfusion monitoring in children has gaine increasing attention in recent years. Most of the currently published data is based on studies performed on small and heterogenous groups of patients. Thus the results need to be corroborated in future well-designed clinical trials. Firstly a short theoretical overview summarizing physical principles and main advantages and disadvantages is provided. It is followed by a review of the current data regarding EIT application in ventilation distribution monitoring in healthy individuals. Finally the most important studies utilizing EIT in ventilation and perfusion monitoring in critically ill newborns and children are outlined. Electrical impedance tomography (EIT) is a relatively new, noninvasive monitoring technique. Its usefulness in lung function monitoring in adults has been examined in numerous studies. The increasing number of publications in pediatric population in recent years shows a wide range of clinical applications of this method. Unlike conventional radiography or computed tomography (CT), EIT is radiation-free and enables continuous real-time monitoring of the lung function. The current knowledge about the application of EIT in clinical practice in children will be reviewed in this paper.Z) represent well-ventilated segments; whereas low ΔZ corresponds with low regional ventilation. Taking the concept one step further, the idea of ΔfEIT [The theoretical aspects of electrical impedance tomography were described over 30 years ago . The phyThe precise quantification of absolute impedance values is challenging and requires taking many factors into account, including specific information about the shape of the chest. Thus EIT is more suitable for assessing dynamic impedance changes in time. Substantial effort has been made in order to quantify information obtained during EIT monitoring, which resulted in several numerical indices of EIT results that have been described in detail in a recent review .EIT has been extensively validated as a reliable ventilation distribution monitoring technique in both healthy and lung-injured animal models, as well as in humans. Validation data confirms that EIT is a highly reproducible method and that impedance changes reflect changes in regional ventilation accurately –9.Electrical impedance tomography has several potential disadvantages. Most importantly, the relatively low spatial resolution precludes using it to obtain morphological information, as computed tomography and magnetic resonance both offer substantially higher resolution. EIT is also more suitable for observing dynamic changes in time in one particular patient, rather than for interindividual comparison. The other technical issue is associated with feasibility of electrodes placement, especially in extremely prematurely born children.The pulmonary function testing in children, neonates, and infants in particular poses considerable technical difficulties due to the inability of these patients to cooperate. EIT offers a promising alternative tool that can be used to monitor regional ventilation distribution over extended periods of time, with no need of sedation or cooperation of the patient.Brown et al. examined the dynamics of total lung resistivity in the first months of life in healthy newborns using EIT . They coThe body position is known to change ventilation distribution in both adults and children. According to the long-existing paradigm, based on studies performed over 20 years ago , 12, nonThe study by Frerichs et al.  reportedA study published by Schibler et al. in 2009  defies tAnother study published by the same group  evaluateIn recent years, a growing number of studies try to address the problem of feasibility and usefulness of EIT as a noninvasive monitoring method in newborns and infants with different respiratory and other pathologies. Those studies widely contribute to gaining more extensive knowledge about respiratory pathophysiology.The study published in 2001 by Frerichs et al.  was one Several studies were aimed at establishing the influence of different therapeutic procedures on regional ventilation distribution in critically ill children. The paper published by Wolf et al. in 2007  assessedZ) reflecting increase of lung compliance and ventilation, respectively.Surfactant treatment efficacy in prematurely born children with respiratory distress syndrome (RDS) poses a very promising target for application of EIT as a monitoring tool. Until recently, the effectiveness of surfactant treatment was assessed based on patient's clinical improvement, with very little insight into direct effect of surfactant on regional and global ventilation and respiratory mechanics. This issue was addressed in a paper by Miedema et al. . The autOne of the most important aspects of EIT application in clinical practice is describing the regional inhomogeneity in lung ventilation. Even though pneumothorax seems to be a plausible condition in this context, there is only one casuistic publication that reported using impedance monitoring in a newborn with pneumothorax . MiedemaEIT-guided ventilatory support is the major hope and ultimate goal associated with this modality. Adequate ventilation settings adjustment is crucial for minimizing the risk of volutrauma and barotraumas but ensures sufficient ventilation on the other hand. It has become clear that global parameters such as arterial blood gases or lung compliance are insufficient in ventilation efficacy and safety monitoring. Recently published paper  proves tAnother possible EIT application that was recently described is the endotracheal tube position assessment . Based oSignificant efforts have been made to assess the feasibility of EIT-based lung perfusion monitoring, as this could improve our understanding of ventilation-perfusion mismatch and its role in respiratory pathophysiology. The current state of knowledge about this issue was summarized in recently published reviews , 30. We Electrical impedance tomography is a very promising technique for noninvasive, radiationfree monitoring of lung function. It is widely applicable and safe for the patient, allowing real time continuous monitoring of mechanical properties of the lungs over extended periods of time. Even though the physical principle of EIT is thirty years old, we have seen increasing number of publications suggesting its possible application in clinical practice in the recent literature. This is mainly due to constantly improving quality of hardware and data processing modalities. However most of these studies were based on small heterogeneous groups of patients. Thus, larger, well-designed trials are necessary to assess the role of electrical impedance tomography in everyday clinical practice.


Aging is the single most important risk factor in human disease in developed countries but when it comes to research on prevention or cures, aging is seldom taken into account. Nevertheless if aging is a significant contributor to age-related conditions, we would hope that an understanding of aging mechanisms could prompt the design of rational therapies. Moreover, if aging causes multiple diseases then it is reasonable to think that pharmacological agents that slow aging could be also effective in preventing or slowing a wide spectrum of diseases.Caenorhabditis elegans (C. elegans) is influenced by hundreds of genes and metabolic processes including an insulin signaling-like (ISL) pathway that regulates the FOXO-like transcription factor DAF-16 [C. elegans [Considerable progress has been made in understanding the genetic influences on lifespan in simple metazoans. In particular, the longevity of the nematode  elegans , 3. ThisProtein aggregation is a hallmark of aging and several age-related pathologies, collectively known as conformational diseases (CD) . This siThe general picture that that has emerged is that conformationally-altered proteins escape the surveillance of repair and degradation systems, form aggregates, and this process contributes to aging; aging could be therefore a manifestation of a loss in protein homeostasis. This then prompts the question: to what extent could chemical modulation of protein aggregation alter the rate of aging? Furthermore, would such an intervention influence disease pathology?C. elegans model. We were then able to directly assess the degree to which protein aggregation influences normal aging rates [In a recent publication, we addressed this issue by identifying small molecules able to slow protein aggregation in the ng rates . We undeSeveral models of protein aggregation of the type observed in neurodegenerative diseases have been developed in C. elegans. Elegant work from the laboratories of Richard Morimoto  and ChriA large body of literature demonstrates that ThT binds to amyloid-like aggregating protein . This suThese results indicate that the protein homeostatic network can be manipulated by using small molecules to suppress age-related disease pathologies and highlights some important concepts for a novel approach to the discovery of new drugs with potential to modulate aging and improve the conditions of conformational diseases.C. elegans models of neurodegenerative disease and aging. Perhaps prevention and cures of age-related diseases will emerge from chemical screens that target aging.We hypothesize that compounds that enable an animal to maintain protein homeostasis during aging could be active against multiple states of diverse conformational diseases. These compounds can be identified by using


Pancreatic cancer is a complex, aggressive, and heterogeneous malignancy driven by the multifaceted interactions within the tumor microenvironment. While it is known that the tumor microenvironment accommodates many cell types, each playing a key role in tumorigenesis, the major source of these stromal cells is not well-understood. This review examines the contribution of bone marrow-derived cells (BMDC) to pancreatic carcinogenesis, with respect to their role in constituting the tumor microenvironment. In particular, their role in supporting fibrosis, immunosuppression, and neovascularization will be discussed. It is long known that adult stem cells have remarkable flexibility in their differentiation repertoires. This plasticity allows adult stem cells, particularly those of bone marrow origin, to engraft alternative non-haematopoietic locations and differentiate into cell types appropriate to their new niche. This is particularly evident when the recipient organ is damaged  comprise approximately 4% of pancreatic cells, and exist in a periacinar dissemination. There is increasing evidence to suggest that PSC are key participants in the pathogenesis of pancreatic exocrine diseases, particularly in the production of the abundant fibrous stroma, which is a feature of pancreatic cancer  originate from BMDC and possess the capacity to differentiate into mature endothelial cells, contributing to the complex process of tumor neovascularization. There is evidence that tumor angiogenesis can be stimulated by tumor cell secreted CXC chemokine ligands CXCL5 and CXCL8, via their common receptor CXCR2  in pancreatic tumors, and contribute to neovascularization by homing to fast growing tumors and incorporating into blood vessels as atypical VEGF-secreting endothelial cells (Beckermann et al., There is mounting evidence that BMDC contribute to the neovascularization in pancreatic cancer, however, pancreatic cancer remains a hypovascular tumor with significant perfusion impairment. With a distinct lack of efficacy of anti-angiogenic therapies for pancreatic cancer [reviewed in (Feig et al., hGFAP promotor to develop targeted gene/drug therapy (Ding et al., In conclusion, pancreatic carcinogenesis is an extremely aggressive, and complex malignancy with very few effective therapeutic modalities. Pancreatic cancer is characterized by an extensive desmoplastic stroma that interacts with the tumor cells, where together they drive tumor progression, invasion and metastasis. In addition, the stroma provides a physical barrier, denying the tumor cells access to chemotherapeutic agents. Future research priorities should be focused on further understanding the complex tumor-stroma relationships, which is key for facilitating the development of potential novel therapeutic strategies. Such strategies may include reducing tumor neovascularization by targeting the ablation of BM derived EPC (Li et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Xtreme Everest 2 (XE2) was part of an ongoing programme of field, laboratory and clinical research focused on human responses to hypoxaemia that was conducted by the Caudwell Xtreme Everest Hypoxia Research Consortium. The aim of XE2 was to characterise acclimatisation to environmental hypoxia during a standardised ascent to high altitude in order to identify biomarkers of adaptation and maladaptation. Ultimately, this may lead to novel diagnostic and treatment strategies for the pathophysiological hypoxaemia and cellular hypoxia observed in critically ill patients. XE2 was unique in comparing participants drawn from two distinct populations: native ancestral high-altitude dwellers (Sherpas) and native lowlanders. Experiments to study the microcirculation, mitochondrial function and the effect that nitric oxide metabolism may exert upon them were focal to the scientific profile. In addition, the genetic and epigenetic (methylation and histone modification) basis of observed differences in phenotype was explored. The biological samples and phenotypic metadata already collected during XE2 will be analysed as an independent study. Data generated will also contribute to (and be compared with) the bioresource obtained from our previous observational high-altitude study, Caudwell Xtreme Everest (2007). The study of human adaptation to high altitude has a long history. We haveIn 2007, the University College London Centre for Altitude Space and Extreme Environment (CASE) Medicine conducted the largest comprehensive prospective observational study to be performed at altitude: Caudwell Xtreme Everest (CXE)-5. This Data from CXE suggest that in lowlanders, there is significant inter-individual variability in changes in nitric oxide biology during ascent to high altitude. SubgrouIt is possible that Sherpas have additional phenotypic advantages that are not evident even in high-performing lowland individuals at altitude. Thus, studying them in comparison to a cohort of lowland volunteers may identify traits that are difficult to tease out when looking only at lowland participants. As direct descendants of highland Tibetans who may have dwelt at an altitude of over 4,000 m for the past 20,000 years, the Sherpas of Nepal display exceptional phenotypic adaptation to this environment. The remote location of the Tibetan plateau also means that gene flow from other populations is likely to be minimal. Famed for scaling the Himalayan Giants whilst supporting lowland mountaineers, Sherpas demonstrate a high degree of physical resilience to hypoxia carrying heavy loads with apparently minimal effort. Whilst genetic inheritance and natural selection are likely to have led to traits that favour survival at high altitude, the physiological basis underlying their performance remains elusive. Of note, they have not been shown to demonstrate augmented systemic oxygen delivery, in contrast to what is commonly seen in lowland elite athletes at sea level. Strikingly, when compared to other resident high-altitude populations who have inhabited such heights for shorter periods of time (Andeans and Ethiopians), Tibetans demonstrate the lowest haemoglobin concentration,13. The The XE2 study design shared a core dataset and ascent profile with CXE but addeDetailed phenotypic characterisation included measurements of microcirculatory blood flow and tissue oxygenation by forearm plethysmography, near-infrared spectroscopy, tissue laser Doppler and side-stream darkfield imaging of the sublingual microcirculation. Mitochondrial function was studied with real-time cellular respiration measurements in fresh skeletal muscle samples, as well as metabolomic, proteomic and lipidomic screening of plasma, skeletal muscle and urine samples. Nitrogen oxide metabolism was studied in multiple compartments including plasma, urine, exhaled breath and saliva. These data will be combined with a core physiological dataset including daily physiological dairy incorporating simple cardiorespiratory variables and symptom scores (e.g. Lake Louise Score) and measHypoxaemia is commonplace amongst critically ill patients yet optimal management strategies remain uncertain. Mechanisms that lead to beneficial adaptation, as opposed to maladaptation, need to be identified in order to develop individualised treatment strategies. The current standard response to hypoxaemia in critically ill patients is to aim for at least normal arterial oxygenation ; however, this may not be beneficial and in certain circumstances, may be injurious. Whilst Identifying cellular and metabolic differences between lowland individuals and those adapted over centuries to the hypoxia of high altitude may identify beneficial mechanisms of adaptation for subsequent evaluation in the clinical setting. Sherpa physiology may reveal novel target pathways that are amenable to pharmacological manipulation in the critically ill, such as the nitrate-nitrite-nitric oxide axis. Attempting to mimic the most effective human hypoxia-tolerant phenotype could provide new directions in critical care medicine.The model of studying healthy volunteers ascending to altitude for the purpose of gaining insight into hypoxaemic patients is not without limitations. It is possible that mechanisms elicited at altitude may differ from those seen in pathology, environment factors other than hypoxia may confound data obtained in extreme conditions and differing levels of fitness may influence responses. However, comprehensive phenotype-genotype analysis in large groups of healthy volunteers exposed to an identical, graded hypoxic stimulus may unveil otherwise concealed mechanisms of importance,20.In keeping with our core mission, the aim of XE2 is to create a large-scale biobank of samples linked to a phenotype database. This will be used to identify adaptive mechanisms and drive a translational research agenda. The consistency of data collection across the research expeditions run by the Caudwell Xtreme Everest Hypoxia Research Consortium has resulted in a unique bioresource for the study of human adaptation to hypoxia. Biomarkers and metabolic pathways can be exploited for patient benefit in an ongoing manner, and the resource can be interrogated repeatedly as new techniques evolve.The unique physiology of the Sherpa people may hold the key that unlocks the secret of successful hypoxic adaptation. The bioresource developed from XE2 (and CXE) provides a unique opportunity to explore human hypoxic adaptation and thereby drive improvements in the management of hypoxaemic critically ill patients in the years to come.CASE: Centre for Altitude Space and Extreme Environment; CXE: Caudwell Xtreme Everest; CASE: Centre for Altitude Space and Extreme Environment; XE2: Xtreme Everest 2.The authors declare that they have no competing interests.All authors  conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.


Many cancers are associated with aging . MetformWhat is the mechanism by which aging may increase cancer incidence? Although many molecular changes correlate with aging, the presence of senescent cells capable of secreting inflammatory cytokines may be involved. This senescence associated secretory phenotype (SASP) consists of multiple cytokines, chemokines, growth factors and extracellular matrix degrading enzymes that can potentially affect normal tissue structure . The SASAt the molecular level, we found that metformin interfered with the activation of protein kinases IKK a and b, which are responsible for activating NF-kB, an essential transcription factor for SASP activation. Intriguingly, metformin did not reduce the expression of anticancer cytokines such as interferon and interferon target genes in senescent cells, suggesting that it modulates SASP to reduce its inflammatory potential but retaining its antitumor activity. In addition, metformin did not affect the senescent cell cycle arrest caused by oncogenic ras in primary human cells, suggesting again that it can modulate the SASP without allowing proliferation of potentially malignant cells. The primary site of action of metformin is considered to be the complex I of the electron transport chain . HoweverMany questions remain to be addressed in order to fully characterize metformin actions. Our results were obtained using cultured senescent fibroblasts and macrophages; other cell types should be studied as well. In addition, it remains to be determined if metformin can achieve this anti-SASP activity in vivo or whether it can influence the clearance of senescent cells by modulating the SASP. Anisimov and colleagues reported that metformin extends life span in female mice but not males  and it w