Eriophyton wallichii, an alpine perennial endemic to the Himalaya-Hengduan Mountains. In previous study, it has showed that these extrafloral leaves can protect interior flowers from temperature fluctuations caused by drastic solar radiation fluctuations, but these leaves may also protect interior flowers from rain wash and UVB damage, and we do not know which one is the main function. In this study, we investigated whether rain and UVB protection are the main functions of overlapping leaves covering flowers and their potential impact on pollination. We first measured the intensities of UVB radiation in open air, beneath leaves and corollas, and then examined pollen susceptibility to different intensities of UVB and rain in the laboratory to estimate whether corollas per se protect interior pollen from UVB and rain damage. We also carried out pollination treatments and observed pollinator visitation of flowers with and without leaves in the field to assess whether the overlapping leaves covering flowers impair pollinator attraction. Our results showed that (1) water and strong UVB significantly decreased pollen germinability, but corollas per se could protect pollen from UVB and rain damage; (2) no autonomous self-pollination and apomixis occurred, and pollinators were essential for the reproduction of E. wallichii; however, flower coverage by overlapping leaves did not limit pollination. We suggested that rain and UVB protection was not the main function of overlapping leaves covered flowers, given that this protection can be provided by corollas per se. Alternatively, this extrafloral structure in E. wallichii may have evolved in response to extreme high temperatures associated with the strong solar radiation fluctuations. This indicates that, even in alpine plants, extreme high temperature may affect the evolution of plant extrafloral structures.Extrafloral structures are supposed to have evolved to protect flowers from harsh physical environments but might have effects on pollination. Overlapping leaves cover flowers in  Among tper se , 7 or exper se , 8, 9. Iper se . For exaithin it . In contithin it –15.Rheum nobile, protecting pollen from damaging rain, UVB and low temperature [R. nobile flowers release specific volatiles rather than visual cues [R. nobile far exceed the costs, such as increased herbivore attraction or resource consumption [The evolution of floral/extrafloral structures is shaped by simultaneous selection exerted by both biotic forces, including pollinators and herbivores , and theperature . Howeverual cues . Overallsumption , 17.Although abiotic factors have played important roles in the evolution of protective floral and extrafloral structures in alpine plants , the keyEriophyton wallichii (Lamiaceae) is a perennial herb endemic to the Himalaya-Hengduan Mountains (HHM). Notable features of this plant include highly pubescent leaves, corollas and calyxes. In particular, woolly and overlapping leaves  cover its flowers  Which environmental factor best explains overlapping leaves covering flowers? (2) Does E. wallichii rely on pollinators to reproduce? If so, do the overlapping leaves reduce pollinators’ visitation rates?In the present study, we investigated the relative importance of different abiotic factors in driving the evolution of the overlapping leaves covering flowers, and also assessed whether overlapping leaves make the flowers less attractive for pollinators in No specific permits were required for the described field studies. The field studies were conducted on public land and did not involve endangered or protected species.Eriophyton wallichii is a perennial herb endemic to the HHM subnival belt, and this species is only found in alpine extreme gravelly slopes or screes at altitudes ranging from 4,200 to 5,200 m . It usually has a large rhizome system but does not seem to propagate vegetatively via rhizomes, i.e. no such reproduction has been observed . The woolly leaves  are circular or sub-circular and overlap flowers (or fruits). One to three subsessile flowers are concealed within each leaf, and each plant typically has 15.55 ± 0.96 flowers . Most  of the corollas are covered by leaves (n = 10 flowers) pollen grains, yielding a P/O-ratio of 15754 [n = 14 flowers) when they open, with a sugar concentration of 21.91 ± 0.7% (n = 34 flowers) [y leaves . Floweriy leaves  and compy leaves . Floral E. wallichii, Phyllophyton complanatum, Rhodiola crenulata, Fritillaria delavayi and Rheum nobile . The fields featured a monsoon climate, with 80% of precipitation occurring in the growing season from June to September. The mean annual precipitation (1982–1984) at the nearest meteorological station , 95 km from the study sites, was 680–790 mm, with 30 mm in May, 130 mm in June and 500 mm between July and September [-2 s-1 at 14:00 h (strongest solar radiation on sunny days) on 17 and 18 July, 2013 at the Puyong Pass site.Field experiments were conducted during the summers of 2012 and 2013 at two sites located in the subnival belt of Daxue Shan Mountain in Shangri-la County, Yunnan Province, SW China: one beside Huluhai Lake  and the other at Puyong Pass . The distance between the two sites was about 70 km . Both sites had an open, stony habitat and the vegetation of the two sites was dominated by eptember . The anneptember . The inset al. [t-tests to determine differences in rates of both germination and bursting between pollen exposed to water and the sucrose solution. PASW Statistics 18 was used for all statistical analyses reported in this paper [To test the effect of rain on pollen viability, we followed the germination methods of Huang et al. . We randet al. . Three ris paper .-2 s-1), provided by a UV analyzer . Anthers of another group were exposed to 4.5 ± 0.2 μW cm-2 s-1 UVB, the mean intensity recorded beneath corollas without covering leaves. The third (control) group was not exposed to UVB. After 4 h, pollen grains from the 10 flowers subjected to each treatment were placed in 10% sucrose solution (at room temperature), and the number of grains from each flower that had germinated after a further 4 h was counted under a light microscope. Three replicate sets of pollen from each flower were used (to determine the average), and each treatment was replicated ten times (n = 10 flowers). One-way ANOVA was used to compare pollen germination rates following exposure to the three UVB intensities.Using UV radiometers supplied by the Photoelectric Instrument Factory of Beijing Normal University, we measured the ground-level UVB intensity and UVB intensities beneath two corollas with and without covering leaves in the same individuals (seven randomly selected plants) at 14:00 h (strongest solar radiation on sunny days) on the 17 and 18 July, 2013 at Puyong Pass population. To test responses of the plant’s pollen grains to UVB, 30 flowers from 30 different individuals  with newly dehisced anthers were selected randomly and separated into three groups of 10 in the laboratory (at room temperature). The anthers of one group were exposed to UVB (302 nm) at the mean intensity recorded in the open air  in 2012. To assess apomixis rates, anthers of five randomly selected plants were removed after their flowers had opened but before anther dehiscence, then each selected plant was netted with a nylon mesh bag (10 × 10 threads cm-2) to exclude floral visitors. To test if pollinators were required for seed set, intact floral buds of another 10 plants were netted. To test if pollinators were a limiting factor, flowers of a further set of five plants were supplementally pollinated by hand after their flowers had fully opened, using in each case pollen grains from three individuals located at least 10 m away. Thirty naturally pollinated individuals were used as openly pollinated controls. Each manipulated individual was marked with a sign at the base of the stem. All bags used in the treatments were left in place until the end of the experiments. Fruits from all of the individuals used in the tests were collected on the 25th of September, 2012 and then transported to the laboratory to evaluate the fruit set and seed set under each treatment. Three individuals with nylon bags were damaged by yaks from both the Huluhai Lake and Puyong Pass populations, 22 individuals without nylon bags from the Huluhai Lake population were infected by larvae and 24 individuals from the Puyong Pass population were infected by larvae. We removed these damaged individuals. Fruit set was calculated as the number of fruits with seeds divided by the total number of flowers, and seed set as the number of fully developed seeds divided by four (the number of ovules per flower). To test for pollen limitation, we applied an independent-sample t-test to determine whether supplemental hand pollination significantly increased frequencies of seeds relative to open pollination.To examine possible contributions of self-pollination to the reproduction of t-tests.Pollinator behavior was observed on sunny days when pollinators were abundant between 10:00 and 17:00 h from 2–6 August, 2012 at the Puyong Pass population. To investigate if the overlapping leaves influenced their visitation rates, we carefully removed all the leaves covering flowers of 55 randomly selected individuals (with 322 flowers). Sixty-five intact individual plants (with 350 flowers) of similar sizes were used as a control group. During 40 (30 min) observation periods , we recorded insects that entered into the corollas of both sets of plants. Visitation rates were standardized as both visits per flower per hour (VFH) and visits per individual per hour (VIH). The difference in pollinator visitation rates (VFH and VIH) between individuals with and without leaves was assessed using independent-sample t = 16.484, P < 0.001; t = 0.873, P = 0.394; The pollen germination rate was significantly higher in 10% sucrose solution (36.74 ± 1.57%) than in 0% sucrose solution  did not decrease pollen germinability compared to the control treatment (0 μW cm-2 s-1). However, pollen exposed to strong UVB  germinated significantly less frequently  species, which pushed their bodies into the corollas to forage for nectar at the base of the corolla tubes. As they crept into the flowers, pollen grains became stuck to their backs and were then available to be transferred to stigmas of the same flower, other flowers of the same individual or other individuals during a continuous visit. The mean visitation rates to the control group (0.84 ± 0.13 VFH and 1.49 ± 0.19 VIH) did not differ significantly from those of the treated group (0.91 ± 0.14 VFH and 1.82 ± 0.29 VIH) at either the individual , alpine plants are subject to wide and often rapid temperature variations. Pubescence of leaves and inflorescences is known to be a common plant adaptation to low temperatures , 14, 24.et al.  have alr calyxes  effectivE. wallichii are directly exposed to peaks of solar radiation, the flowers and fruits are exposed to high temperatures and wide temperature fluctuations, which reduce pollen germinability and seed production [E. wallichii.Further, solar radiation can be very intense for short periods in alpine habitats, especially at midday , 15, 26.oduction . This pooduction . Thus, cE. wallichii , which could have advantages to attract pollinators. Indeed, the mean visitation rates of E. wallichii . Such foraging behavior can result in considerable self-pollination, enhancing pollination success. Thus, coverage of the flowers by overlapping leaves does not seem to cause a fitness cost.Seed production in the presence of pollinators and lack of seed production in their absence indicate that pollinators are essential for the reproduction of allichii . Howeverallichii ; pollen allichii . As only species . When BoE. wallichii. Our results indicate that rain and UVB protection are not the major function of overlapping leaves covered flowers. Potentially damaging high temperatures during strong solar radiation fluctuations might be responsible for the evolution of the overlapping leaves covering the plant’s flowers as they provide important protection for the reproductive structures. Moreover, these overlapping leaves clearly do not have a negative effect for attracting pollinators. Among the abiotic agents, in addition to UVB, rain and low temperatures, extreme high temperatures caused by large solar radiation fluctuations may have affected the evolution of plant structures in alpine plants.In summary, the objectives of this study were to test whether UVB and rain protection are responsible for the evolution of the overlapping leaves covering flowers and their potential impact on pollination in


Introduction. Failure of the vascular pulmonary remodeling at birth often manifests as pulmonary hypertension (PHT) and is associated with a variety of neonatal lung disorders including a uniformly fatal developmental disorder known as alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV). Serum serotonin regulation has been linked to pulmonary vascular function and disease, and serotonin transporter (SERT) is thought to be one of the key regulators in these processes. We sought to find evidence of a role that SERT plays in the neonatal respiratory adaptation process and in the pathomechanism of ACD/MPV. Methods. We used histology and immunohistochemistry to determine the timetable of SERT protein expression in normal human fetal and postnatal lungs and in cases of newborn and childhood PHT of varied etiology. In addition, we tested for a SERT gene promoter defect in ACD/MPV patients. Results. We found that SERT protein expression begins at 30 weeks of gestation, increases to term, and stays high postnatally. ACD/MPV patients had diminished SERT expression without SERT promoter alteration. Conclusion. We concluded that SERT/serotonin pathway is crucial in the process of pulmonary vascular remodeling/adaptation at birth and plays a key role in the pathobiology of ACD/MPV. FOXF1 abnormalities were detected in up to 70% of ACD/MPV cases; however, the exact mechanism through which these genetic abnormalities lead to ACD/MPV is yet to be elucidated [There is a remarkable change in the structure and function of the pulmonary vasculature during the transition from fetal to extrauterine life. In the fetus, the pulmonary vasculature is a high resistance low flow system, and thus right ventricular output is shunted to the placenta for gas exchange. At birth, even with the first breath, pulmonary vascular resistance falls dramatically, and pulmonary blood flow increases rapidly. Failure of this transition can lead to serious neonatal lung disorders characterized by the clinical syndrome of Persistent Pulmonary Hypertension of the Newborn (PPHN) , 2. A leucidated .The role of vasoactive mediators in achieving successful pulmonary vascular remodeling at birth has begun to emerge . BioavaiCurrent data suggests that disorders associated with PPHN have an overall mortality rate of 10–20%, although ACD/MPV is uniformly lethal despite the use of inhaled nitric oxide therapy often combined with extracorporeal membrane oxygenation , 26. As Thus, we hypothesize that human pulmonary SERT may play a role in the adaptation of fetal pulmonary vasculature to extrauterine life and that abnormalities of SERT activity lead to neonatal pulmonary hypertension. Here we present a study of SERT expression in pre- and postnatal human lungs and in cases of newborn and childhood PHT of varied etiology including ACD/MPV.Approval for this study was obtained through review by the University of Pittsburgh and Baylor College of Medicine Institutional Review Boards.Microarray of pulmonary tissue obtained from multiple autopsy cases was employed to establish the specificity and sensitivity of SERT immunostaining of lung endothelium. Additionally, SERT immunostaining of the vascular endothelial of different organs was similarly assessed.Physiologic temporal-spatial expression of SERT was studied using lung sections from 30 autopsy cases encompassing a range of ages, including 27 cases of 12, 13, 16, 18, 34, 39 and 40 weeks of gestational age (WGA) and 3 postnatal lungs of ages 1, 5, and 10 years. Cases with significant pulmonary pathology were excluded from this group.n = 14), congenital alveolar dysplasia (n = 1), congenital diaphragmatic hernia (n = 5), bronchopulmonary dysplasia (n = 3), secondary PHT in congenital heart disease (n = 1), and idiopathic primary PHT (n = 5). For these 29 cases, patient histories and histologic sections of the lung were reviewed to confirm the initial diagnosis. All ACD/MPV patients were tested for mutation in FOXF1 and 4 were tested for rearrangements at 16q24 locus. Among the 14 ACD/MPV patients, three had mutations in FOXF1. Of the 11 without identified mutation, 4 were further identified with rearrangements at 16q24 locus with deletions identified in three  and CD31  antibodies were used as follows: after the routine steps of deparaffinization and dehydration SERT antigen was retrieved by steam-heat treatment in 1 mM EDTA + 10 mM Tris buffer at pH 9.0. After incubation of primary SERT antibody for 30 minutes in room temperature at a dilution of 1/4000, diaminobenzidine (DAB) staining was carried out with utilization of the avidin-biotin-peroxidase system. All sections were counterstained with hematoxylin. Diffuse brown membranous endothelial staining was interpreted as positive while absence of brown staining was deemed negative. For positive controls brain sections including the Raphe nucleus (rich in serotoninergic neurons and recommended by manufacturer) were used; adrenal tissue was used as negative control . The ACDSERT immunohistochemistry was assessed and recorded as positive and negative. In addition, the terms diffuse and patchy were added for the distribution pattern of the stain.The PCR was carried out in a TC-512 thermocycler  utilizing 100 ng template DNA using the following protocol: 5 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 60°C, 2 min at 72°C, and final extension for 10 min at 72°C. The PCR products were purified using a Sephadex G-50 column and sequenced on a ABI 3130XL DNA Genetic Analyzer  following the manufacturer's protocol. The primers used were as follows: forward-GCCAGCACCTAACCCCTAATG and reverse- GAATACTGGTAGGGTGCAAGGAG.The autopsy tissue microarray showed that in normal lungs SERT is expressed specifically in pulmonary endothelial cells with a diffuse crisp brown staining after birth . We alson = 3, n = 13, n = 5, n = 6, Lungs from the antenatal period included all stages of lung development including pseudoglandular n = 3, , canalicSERT expression was absent or only very minimal in the pulmonary microvasculature in every ACD/MPV case Figures . The ACD FOXF1 abnormalities  while the morphologic changes of ACD/MPV begin earlier, likely before the canalicular stage (starting at ~20 WGA) . TherefoDue to the rarity of ACD/MPV and because it has largely been defined histologically with most diagnoses made at autopsy examination, it has been very challenging to identify cases in which tissue appropriate for complex molecular studies can be obtained –43. Ther FOXF1 in ACD/MPV patients, the identification of genetic abnormalities in the SERT gene (either in the coding or in the regulatory regions) could provide a potential explanation for the diminished SERT protein expression.The genetics of SERT promoter regulation is complex and several abnormalities have been described that can lead to a decrease in SERT protein expression –36. We aMost pediatric pathologists, radiologists, and neonatologists consider ACD/MPV to be a difficult to diagnose neonatal lung disease. Our study suggests that absence of SERT immunostaining in the pulmonary vasculature in infants and beyond is an excellent surrogate marker for this disorder, currently diagnosed only by microscopic examination of the lung with hematoxylin and eosin staining . The uniBecause diminished SERT expression was unique to ACD/MPV patients with all other patients with neonatal or childhood PH diseases having normal SERT expression, our study provides evidence of a role for SERT-serotonin regulation in the pathogenesis of ACD/MPV. Since the precise mechanism leading to ACD/MPV is unknown, our findings could lead to focused studies to test SERT-relevant pathways  in ACD/MWe conclude that abnormalities in pulmonary SERT protein expression contribute to the pathomechanism of ACD/MPV. We speculate that the absence of SERT activity leading to high levels of serotonin results in the maintenance of high pulmonary vascular resistance and low pulmonary blood flow seen during fetal life and contributes to the characteristic vascular abnormalities and pulmonary hypertension of ACD/MPV.


Inapparent avian exposure was suspected for the sporadic infection of avian influenza A(H7N9) occurring in China. This type of exposure is usually unnoticed and difficult to model and measure. Infected poultry with avian influenza H7N9 virus typically remains asymptomatic, which may facilitate infection through inapparent poultry/bird exposure, especially in a country with widespread practice of backyard poultry. The present study proposed a novel approach that integrated ecological and case-control methods to quantify the risk of inapparent avian exposure on human H7N9 infection. Significant associations of the infection with chicken and goose densities, but not with duck density, were identified after adjusting for spatial clustering effects of the H7N9 cases across multiple geographic scales of neighborhood, community, district and city levels. These exposure risks varied geographically in association with proximity to rivers and lakes that were also proxies for inapparent exposure to avian-related environment. Males, elderly people, and farmers were high-risk subgroups for the virus infection. These findings enable health officials to target educational programs and awareness training in specific locations to reduce the risks of inapparent exposure. Avian influenza AH7N9) virus, detected strictly in birds in the past, finally spread to humans and animals in China in February/March of 20135N9 virus,Direct poultry contact and exposures to poultry in live-poultry markets (LPMs) are potential sources of H7N9 infection89101213161718212 pixel) without H7N9 cases were randomly selected to generate pseudo-absence data on infection. However, the risk of poultry-related exposure might be underestimated as some of the selected areas of pseudo-absence might have locally high poultry densities relative to their human population.Inapparent poultry exposures are very common in people’s daily lives in China, although this type of exposure is difficult to be noticed, identified, and measured. The usual way to record such exposure was through recall interviews and questionnaire surveys. However, the ‘inapparent’ nature also means that an interviewee might not even be aware of the presence of poultry or wild birds in his/her surroundings to provide an accurate account. Measurement irregularities also occurred because of perceptual differences in ordinal frequency  or intensity . Poultry and LPM densities have been used as proxies for estimating the risks of outdoor environmental exposure to poultryThe objective of this study was to examine the effects of inapparent avian exposure on human H7N9 infection. Spatial/ecological and case-control studies were integrated to estimate the exposure risks at the individual level after adjusting for spatial and personal demographic factors across multiple spatial scales. The novelty of this case-control study was the use of two different diseases of comparable but unrelated infectious etiology as cases and controls. Due to limited availability of data for noncases, this study employed H7N9 as cases and tuberculosis (TB) as controls. Specifically, the study made an attempt to answer the following questions: 1) Are poultry densities, species, and distances/proximity to inland waters  associated with human H7N9 infection and what are the relative contributions of each variable to the occurrence of human H7N9 infection? 2) Do these variables vary geographically and exert interactive impacts to increase the risks of infection? and 3) Who are the target high-risk populations: male vs. female, adult vs. elderly, or farmer vs. worker vs. other occupation subgroups?p = 0.831), gender (p = 0.986), and occupation (p = 0.106). Over 97% of cases occurred in adults aged 15 years or older, with 51% belonging to the working-age population (aged 15–60). The number of male cases was 93 which almost doubled the number of females. The proportion of farmer infection was around 41% and four times higher than that of the worker subgroup in non-agricultural occupations. The ‘other’ subgroup included agricultural workers in forestry, animal husbandry, fishery, agricultural machinery, and hunting, which accounted for 47.9% of all infected cases.km) for rivers and 100.9 km for lakes for the case group and 39.4 km and 183.8 km respectively for the control group. Areas surrounding the cases had consistently higher poultry densities than those near the controls. Statistically significant case-control differences (p < 0.05) were found in the average chicken and goose densities across multiple spatial scales: neighborhood (<1 km), community (1–3 km), district (3–5 km), and city (5–8 km). Duck density did not show such significant case-control differences at the first two spatial scales. The above variables were further categorized into ordinal groups to examine their association with risks of human H7N9 infection  between the occurrences of H7N9 infection and the shortest distances to inland waters. For the poultry densities, the q statistics were 0.032 to 0.058 for chicken, 0.031 to 0.059 for goose, and 0.005 to 0.030 for duck. Chicken and goose showed significant association with H7N9 infection at the four spatial scales while duck did not. The infection risks were further elevated by interaction effects between inland waters and poultry densities.The stratified/conditional logistic regression analysesThis is a novel study that integrates epidemiology and health geography to quantify the effects of inapparent exposure to poultry and proximity to inland waters on human H7N9 infection through a set of individual-level cases (H7N9) and their matched controls (TB). Spatial stratified heterogeneity analyses were applied to examine the connections between poultry densities and the shortest distances to inland waters, both were proxies for inapparent avian exposure and their associations with the virus infection. Spatial clusters, which could be a confounding factor in the analyses, were estimated to adjust for potential, albeit very limitedkm), compared to higher risks at the community, district or city levels  within a distance of 8 km . Goose ekm . These rkm . FurtherIndividual characteristics also affect susceptibility to infection833This study identified the male population, accounting for 65% of all infection cases, as a high-risk subgroup although a previous study in Zhejiang did not find a substantial difference in poultry exposure between the two gendersThe farmer subgroup reported significant and higher risks of infection with the H7N9 virus through chicken and goose across a large distance, as indicated by the mORs . FarmersThis study had a few limitations. Firstly, serum specimens were not collected from the controls to determine seropositivity to H7N9 virus. Although no known cases of simultaneous infection of H7N9 and TB had been reported in the province of Zhejiang, the statistical power of estimating the effects of inapparent exposure would be reduced when some of the controls were confirmed seropositive to avian influenza virus. Secondly, although disease clusters were estimated to correct for the potentials of spatial transmission among people , our datIn conclusion, this study operationalized a means to account for inapparent avian exposure and demonstrated the risk trends of H7N9 virus in human infection associated with poultry density, species, and avian-related environment such as proximity to inland waters. The risks vary in population by age, gender, and occupation. Thus, health education to bring awareness of this risk is needed to protect the public from inapparent exposure to avian influenza viruses. The research findings can also gauge the development of protective measures and strategies in accordance with population characteristics and the spatial separation of poultry farms.Zhejiang, located south of the Yangtze River Delta, is a province on the east coast of China . It had 19The study employed two types of disease data with spatial locations: H7N9 (cases) and TB (control). Other data included the spatial distribution of three poultry species  and their numbers, as well as locations of inland waters .Human H7N9 infection was defined according to the diagnostic and treatment program issued by the National Health and Family Planning Commission of the People’s Republic of ChinaTB data were obtained as controls from the Zhejiang CDC for the same time period covered by H7N9 cases. The controls were randomly selected but matched in proportions by age , gender , and occupation . The number of TB controls was four-times the size of H7N9 cases. The controls were also geocoded by their nominal addresses and the spatial distribution (as green solid triangles) was displayed in km2 were obtained from a previous studyPoultry densities  were used as proxies for risks of inapparent avian exposure. Digital maps of poultry density in high resolution format of 1×1 This is a spatial case-control study that employed spatial point and spatial stratified heterogeneity8www.arcgis.com) and the Matlab v.2010b mapping toolbox.Inapparent avian exposure was modeled using the spatial distribution of poultry species, their respective densities, and the shortest distances to inland waters. Multiscale spatial analyses  were applied to estimate inapparent avian exposure across four spatial scales. Since 52% of the Chinese population commuted between 2 to 8 kilometers every dayP-values were generated to determine statistical significance.The Wilcoxon rank sum statistics was used to examine the distribution of disease between groups of individuals with different demographic characteristics  for the case and control groups. The geographical detector methodK functionDiseases can cluster spatially, temporally, or spatiotemporally to potentially cause spatial confounding. Stratified/conditional logistic regression analyses were applied to model the risks of human H7N9 infection associated with average poultry densities and proximity to inland waters after correcting for effects of disease clustering. The co-effects of inapparent exposure to different poultry species and proximity to inland waters were also modeled using multivariate logistic regression and adjusting for disease clustering. The local Subgroup analyses were also conducted to model exposure risks by gender, age, and occupation groups. Children under 15 years of age were excluded from the study due to low counts. The modified effects of inapparent exposure to poultry on the risks of H7N9 infection were examined in conjunction with the shortest distances to inland waters using multivariate logistic regression after adjusting for disease clustering.All of the above analyses were conducted across four spatial scales. The study employed the Epi package in R v.2.10  to carry out the case-control analyses using stratified/conditional and multivariable logistic regression models. The results were reported as mORs and their corresponding 95% confidence intervals.How to cite this article: Ge, E. et al. Estimating Risks of Inapparent Avian Exposure for Human Infection: Avian Influenza Virus A (H7N9) in Zhejiang Province, China. Sci. Rep.7, 40016; doi: 10.1038/srep40016 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Mythimna separata, reared at the densities of 1, 2, 5, 10, and 30 larvae per 650-mL jar. Concomitant with the increased phenoloxidase (PO) activity and total haemocyte count in the larvae at high densities , the octopamine level was also increased. In contrast, the dopamine level was decreased, and the 5-hydroxytryptamine level was not significantly affected. Injection of octopamine induced significant increases in the total haemocyte count and PO activity. Conversely, epinastine, a specific antagonist of octopamine, decreased the total haemocyte count and PO activity. Another octopamine antagonist, phentolamine, inhibited the activity of PO and lysozymes. In addition, injection of dopamine induced a significant increase in PO activity and decreased the total haemocyte count and lysozyme activity. These results suggested that both octopamine and dopamine mediate the increases in total haemocyte count and PO activity in the crowded larvae.Similar to pathogenic infection, high population density alters insects’ prophylactic immunity. Density-dependent prophylaxis has been reported in many polyphenic insects, but the regulatory mechanism underlying this phenomenon remains unclear. The biogenic monoamines are known to play critical roles in mediating insect immune responses. In the current study, the immune capacity and the levels of three biogenic monoamines were investigated in the polyphenic larvae of  Individuals experiencing crowded conditions are predicted to be more resistant to pathogens than those experiencing low-density conditions, through facultative allocation of physiological resources to defence mechanisms. This phenomenon, termed “density-dependent prophylaxis”1, has been observed in many species that display phase polyphenism, including Spodoptera exempta2, Mythimna separata3, Spodoptera littoralis4, Locusta migratoria5 and Loxostege sticticalis6. Insects can mount an immune response when challenged with an infection7. In fact, an increased immune response is also observed in insects under high-density conditions. Increasing phenoloxidase (PO) activity, total haemocyte counts and lysozyme activity had been observed in some species from high-density conditions6. The immune system plays an important role in insects’ prophylactic response to pathogens. However, the mechanism regulating the immune response underlying high-density prophylaxis is not very clear.Insects infected with pathogens are ubiquitous in nature. It has been found that insects living in high-density populations have evolved adaptive prophylactic mechanisms to cope with pathogens8. The immune system requires information about the environment in order to respond adaptively8, and the nervous system integrates sensory information and sends that information to other systems, including the immune system9. Linking the nervous system and immune organs are the biogenic monoamines10. Three biogenic amines have been implicated in the regulation of animal immune function: dopamine (DA), octopamine (OA) and serotonin . In response to bacterial infection, OA and 5-HT enhanced both haemocytic phagocytosis and nodule formation in the beet armyworm Spodoptera exigua11. OA also increased the number of circulating haemocytes in S. exigua12. In addition, the OA level increased in the haemolymph during immune challenge13, and OA enhanced phagocytosis in cockroach haemocytes14. 5-HT mediates haemocyte phagocytosis through the receptors 5-HT1B and 5-HT2B15. The dopamine-dependent signalling system operates in haemocytes to mediate phagocytic functions16. N-β-alanyldopamine synthase was induced in the mealworm, Tenebrio molitor, and in the medfly, Ceratitis capitata, by infection with Escherichia coli17. Genes for biogenic amine receptors were also expressed in the immune system of the Texas field cricket, Gryllus texensis18. All these data seem to indicate that the action of biogenic amines is probably linked with the immune function of insects.Connections between the immune and nervous systems are ubiquitous in animalsMythimna separata (Lepidoptera: Noctuidae), a major long-distance migratory insect pest of grain crops in China and other Asian countries, shows typical density-dependent phase polymorphism21. Larval colour polymorphism, survival rates, development rate, feeding behaviour of larvae, and flight capacity, fecundity and energetic reserves in adults are affected by larval density23. Mitsui and Kunimi24 (1988) found that larvae from gregarious (high density) phases were more resistant than solitary (low density) phase larvae when perorally inoculated with nuclear polyhedrosis virus (PsNPV). Our preliminary results also showed that the resistance to the entomopathogenic fungus Beauveria bassiana and the bacterium Bacillus thuringiensis of larvae raised at high density was higher than that of larvae raised at low density. Therefore, M. separata larvae showed density-dependent prophylaxis.The oriental armyworm, S. exempta, S. littoralis, L. migratoria, L. sticticalis, under crowded conditions6. The levels of octopamine, dopamine and 5-hydroxytryptamine were significantly higher in crowded Gryllus bimaculatus than in isolated individuals25. It is assumed that under larval crowding stress, M. separata may produce increased levels of biogenic amines, subsequently leading to increased immunity. Accordingly, the purpose of the present study is to examine (1) the effect of larval density on the immune response and levels of biogenic amines in M. separata and (2) the immune responses of M. separata injected with biogenic amines and their antagonists.PO activity, total haemocyte counts and lysozyme activity have been reported to cause increased resistance to disease in phase species, such as Larval density significantly affected the PO activity  than with low population density (solitary phase). Our results showed that PO activity and total haemocyte count of M. separata larvae from conditions of 5, 10, or 30 larvae/jar were significantly higher than those of larvae reared at 1 larva/jar. This suggests that the changes in immune parameters imposed by the larval density, such as increases in PO activity and total haemocyte counts, caused increases in the resistance of M. separata to disease.An insect mounts an immune response when challenged with an infectionIt was noteworthy that although the PO activity of larvae reared at 10 larvae/jar was significantly higher than that of larvae reared at 2 larvae/jar, their weights were not significantly different. This situation is similar to that observed for total haemocyte counts. The weight of larvae reared in isolation was significantly greater than that of larvae reared at a density of 2 larvae/jar, but their total haemocyte counts were not significantly different. We therefore suggest that the variation in immune competence of larvae at different larval densities is not related to the size of the individual.26. It has been shown that the levels of biogenic monoamines are altered in various insect species in response to unfavourable conditions. Our results showed that octopamine levels in the haemolymph of M. separata larvae reared at 5, 10 and 30 larvae/jar were significantly higher than those in larvae reared at 1 and 2 larvae/jar. Iba et al.25 found that the contents of octopamine in the corpora cardiaca were significantly higher in the crowded crickets than isolated ones. Octopamine was also elevated in the larvae of Galleria mellonella after bacterial infection27. The octopamine levels in the haemolymph of both the locust Schistocerca americana gregaria and the cockroach Periplaneta americana were significantly higher when exposed to mechanical, thermal and chemical stress than under normal conditions28. When worker honey bees (Apis mellifera) were fed pollen and exposed to CO2, brain dopamine levels were significantly lower than in untreated controls that were also fed pollen29. In contrast, we found that the dopamine levels after rearing at 5, 10 and 30 larvae/jar were significantly lower than after rearing at 1 or 2 larvae/jar. No significant difference in the dopamine level in the corpora cardiaca was observed between crowded and isolated crickets25. This inconsistent result may be due to different unfavourable conditions, different degrees of density stress, and different species (or regions of the body measured). On the other hand, the 5-HT level was not significantly affected by larval density. This is consistent with the results that the 5-HT level in the corpora cardiaca was not significantly different between crowded and isolated crickets25. Therefore, M. separata larvae may increase the octopamine level and decrease the dopamine level of haemolymph in response to high population density without changing the level of 5-HT.Most animals show a coordinated series of physiological changes in response to stress stimuli31.The close correlation between the increasing octopamine level and the decreasing dopamine level may be related to the biosynthesis of biogenic amines. That is, in the process of octopamine biosynthesis, dopamine can be converted to tyramine by dopamine dehydroxylase, followed by β-hydroxylation of tyramine to form octopamine32. Octopamine induced haemocyte phagocytosis and nodule formation in the cockroach Periplaneta americana33. Octopamine also increased the number of circulating haemocytes in S. exigua12. Similarly, octopamine accelerated the removal of bacteria from haemolymph in the greater wax moth, Galleria mellonella27. In response to bacterial infection, octopamine markedly enhanced both haemocytic phagocytosis and nodule formation in Spodoptera exigua larvae11. Our results showed that PO activity and total haemocyte counts were significantly stimulated by injection of various concentrations of octopamine solution. Epinastine is known as a highly specific antagonist of insect neuronal octopamine receptors34. When various concentrations of epinastine were injected into the haemolymph of M. separata larvae, significant decreases in the PO activity and total haemocyte counts in the larvae were observed. Treatments with phentolamine, another octopamine antagonist, at various concentrations  and with a high concentration (1 µg/mL) of phentolamine significantly decreased total haemocyte counts and lysozyme activity, respectively. However, a low concentration (0.01 µg/mL) of phentolamine induced significant increases in PO activity and lysozyme activity. This may be because the phentolamine was recognized as foreign matter, leading to failure of its inhibitory effect. These results imply that octopamine may enhance haemocyte immune response and that octopamine also plays an important role in the larval resistance to infection. However, injection of octopamine can also result in a decline in resistance to bacteria. For example, the survival of crickets (Gryllus texensis) challenged with Serratia marcescens was decreased upon injection of octopamine35. Huang et al.36 also found that at a low concentration, octopamine stimulated haemocyte spreading and phagocytosis in the larvae of the striped stem borer, Chilo suppressalis, whereas at high concentrations, octopamine inhibited spreading and phagocytosis. Adamo37 also found that octopamine treatment decreased disease resistance in insects, despite the fact that it enhanced individual metrics of immune function. Therefore, the effects of octopamine on immune function may be related to the concentration of octopamine and to the insect species. Moreover, the effect of octopamine on immunity may be complex, and it requires further investigation.Octopamine has been reported to mediate the cellular immune response in some insects38. Wu et al.16 reported that dopamine enhanced haemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis. Chang et al. (2006)39 found that dopamine depressed total haemocyte counts and PO activity in the tiger shrimp Penaeus monodon. Our findings showed that dopamine induced PO activity but decreased total haemocyte counts and lysozyme activity. These inconsistent results for immune parameters may be related to the tested time after injection and to the animal species.Dopamine can also modulate immune functionsAlthough the concentration of dopamine in larvae reared at higher densities  is lower than that in larvae reared at lower densities (1 and 2 larvae/jar), the concentration of octopamine shows the opposite pattern. Moreover, among larvae reared at high densities, the content of octopamine exceeded that of dopamine. Therefore, compared with dopamine, octopamine plays a more dominant role in the modulation of the PO activity. Further, total haemocyte counts were increased by octopamine but decreased by dopamine. Due to the high content of octopamine and the low content of dopamine in larvae reared at higher densities, the total haemocyte counts were increased, and the inhibitory effect was blocked. However, the lysozyme activity of larvae reared at higher densities was not markedly affected by the lower dopamine contents.M. separata demonstrates density-dependent prophylaxis and that the octopamine and dopamine signalling pathways play important roles in immune modulation by adjusting PO activity and the total haemocyte count.In conclusion, the present study suggests that Mythimna separata was established with eggs supplied by the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences, Beijing, China. Larvae were maintained at 23 ± 1 °C and under a 16 h light:8 h dark photoperiod until pupation and adult emergence. Adults emerging on the same day were collected and held together in a plastic cage with a 5-L capacity to allow mating and oviposition. Adults were provided daily with 10% (wt/vol) glucose solution as supplemental food. Eggs were laid on plastic cage with gauze until hatching. Larvae were maintained in plastic cages with a volume of 5 L and were fed daily with fresh leaves of maize (Zea mays L.). When larvae stopped feeding, sterilized soil containing 10% water was added to the cage to a depth of 5 cm to provide a substrate for cocoon formation, pupation, and adult emergence.A laboratory colony of M. separata colony had been maintained for two generations when the experiments began. Newly hatched larvae were grouped and reared at five density treatments of 1, 2, 5, 10, or 30 larvae/jar . The number of larvae for each density treatment was maintained throughout the feeding period. Excess food was provided by adding fresh leaves every morning. All insects were maintained at a constant temperature of 23 ± 1 °C with 70–75% relative humidity, and a 16 h light:8 h dark regime.The On the second day of the fifth larval instar, larvae were cooled to torpor on ice and then sterilized by swabbing with 75% ethanol. An abdominal proleg was cut with a fine scalpel, and haemolymph was collected with a microcapillary pipette and transferred into a 1.5-mL centrifuge tube on ice to prevent melanization. Haemolymph samples were collected from groups of 30 larvae from each rearing density , and haemolymph from each rearing density were pooled as one biological replicate for the assays of PO and lysozyme activity and for haemocyte counts. Each biological replicate included three technical replicates. Three biological replicates were established for each density treatment. All the samples were then frozen at −80 °C until evaluation.et al. (2001)5. Briefly, 50 μL of haemolymph was added to 150 μL of phosphate buffer (pH = 7) in a plastic tube. Haemolymph PO activity was assayed spectrophotometrically by adding 1500 μL of 0.1 M L-dopa and 1000 μL of phosphate buffer (pH = 7) to 200 μL of haemolymph phosphate solution. Changes in the absorbance of the mixture within 3 min at 492 nm were measured using a time-dependent program and a lambda 25 UV/VIS spectrometer . Protein content was measured in the samples according to the method described by Bradford40. PO activity is expressed as PO units per milligram of protein, where 1 U was the amount of enzyme required to increase the absorbance by 0.001 min−1.PO activity was analysed as described by Wilson Total haemocyte count was determined by adding 5 μl of haemolymph that had been stained with Giemsa for 5 min to the counting chambers of a haemocytometer. The haemocytes in the central square and four corners were counted under phase-contrast illumination and averaged to give an estimate of the number of haemocytes.Lysozyme activity was determined using a lysozyme kit . First, 0.2 mL of haemolymph obtained as described above (see “Haemolymph sampling”) was mixed in a test tube with 2 mL of bacterial culture medium, and the two absorbance values at 530 nm were determined in a UV-2000 spectrophotometer  after 20 and 140 seconds, respectively. The lysozyme activity was determined using the two values according to the manufacturer’s instructions.On the second day of the fifth instar, the larvae from the density treatments of 1, 2, 5, 10 and 30 larvae/jar were weighed on an electronic balance , and the length and width of larvae from five density treatments were also assessed with callipers. For each density treatment, data from seven larvae were obtained.The octopamine, dopamine and 5-HT were determined based on the Insect Biogenic Amine ELISA Kit . Standard curves were established based on standards of octopamine, dopamine and 5-HT, respectively. The haemolymph obtained from the procedure described above (10 μL), sample diluents (40 µL) and HRP-Conjugate reagent were combined and incubated for 60 min at 37 °C in microELISA wells. Each well was aspirated and washed with washing solution, followed by the addition of chromogen solution A (50 μL) and chromogen solution B (50 μL) and incubation for 15 min at 37 °C, after which 50 μL of stop solution was added. The absorbency at 450 nm was measured using a microplate reader . The biological and technical replicates for the octopamine, dopamine and 5-HT assays were the same as described for the immunity assays.Octopamine, octopamine antagonists (epinastine and phentolamine) and dopamine were purchased from Sigma-Aldrich. Octopamine, epinastine, phentolamine and dopamine were dissolved in phosphate-buffered saline (PBS) and sterilized with 0.2-μm membrane filters. PBS was prepared at pH 7.4 with 50 mM sodium phosphate buffer and 0.7% NaCl. Various concentrations of octopamine , epinastine , phentolamine , and dopamine  solutions were injected into haemocoel through the abdominal proleg of fifth-instar larvae in a volume of 2 μL, using a 10-μL micro-syringe after surface sterilization with 70% ethanol. Injected larvae were placed in Petri dishes with leaves of corn under normal rearing conditions for 24 h.Haemolymph samples were collected from groups of 30 larvae injected with octopamine, an octopamine antagonist (epinastine or phentolamine) or dopamine, as indicated. Haemolymph from larvae from each treatment were pooled as one biological replicate for the assays of PO and lysozyme activity and for haemocyte counts. Three biological replicates were established and used for three technical replicates. The haemolymph was collected and then frozen at −80 °C for the assays of phenoloxidase activity, total haemocyte count and lysozyme activity.All numeric values obtained from this study are presented as the mean ± SE. All data were assessed and showed a normal distribution using the Kolmogorov-Smirnov test. Differences among the different treatments were tested using one-way ANOVA followed by Duncan’s multiple range test. All statistical procedures were performed with SPSS 10.0.


Arabidopsis ecotype Col-0 and one of its most affected mutants called 2+]i increase 1reduced hyperosmolality-induced [Ca. Kinetic chlorophyll fluorescence curves were useful for understanding the drought tolerance mechanism of Arabidopsis. Conventional fluorescence parameters provided qualitative information related to drought stress responses in different genotypes, and the corresponding images showed spatial heterogeneities of drought stress responses within the leaf and the canopy levels. Fluorescence parameters selected by sequential forward selection presented high correlations with physiological traits but not morphological traits. The optimal fluorescence traits combined with the support vector machine resulted in good classification accuracies of 93.3 and 99.1% for classifying the control plants from the drought-stressed ones with 3 and 7 days treatments, respectively. The results demonstrated that the combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging with the machine learning technique was capable of providing comprehensive information of drought stress effects on the photosynthesis and the secondary metabolisms. It is a promising phenotyping technique that allows early detection of plant drought stress.Plant responses to drought stress are complex due to various mechanisms of drought avoidance and tolerance to maintain growth. Traditional plant phenotyping methods are labor-intensive, time-consuming, and subjective. Plant phenotyping by integrating kinetic chlorophyll fluorescence with multicolor fluorescence imaging can acquire plant morphological, physiological, and pathological traits related to photosynthesis as well as its secondary metabolites, which will provide a new means to promote the progress of breeding for drought tolerant accessions and gain economic benefit for global agriculture production. Combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging proved to be efficient for the early detection of drought stress responses in the  Agricultural drought has become one of the major challenges in global agricultural production and food security. Plant responses to drought stress are complex, as plants varieties with different stress sensitivity might have different strategies for eliciting various mechanisms of drought avoidance and tolerance to maintain growth. Plant phenotypic traits related to morphological, physiological, and pathological traits are usually measured to evaluate the plant performance under different stresses. They are influenced by both environmental and genetic factors, and are essential in breeding research. There is an urgent need for developing advanced phenotyping techniques in drought-tolerant breeding programs.Arabidopsis is a model system for dicot plant species with characteristics of small size, short life cycle, high viability, fertility, and small genome. Also, the whole genome sequencing of Arabidopsis was completed in 2000. Arabidopsis is used widely to study functional genomics and phenomics of plants. With phenotyping the model plant Arabidopsis, it is possible to benefit the progress of breeding and provide economic benefits for global crop production. However, traditional plant phenotyping methods are labor-intensive, time-consuming, and subjective, which becomes one of the major bottlenecks to exploit genetic information for genomic analysis  and far-red (740 nm) regions from chlorophyll a, and blue (440 nm) and green (520 nm) regions from plant phenolic compounds. Previous studies have successfully applied multicolor fluorescence for evaluating fruit qualities  and one of its most affected mutants called 2+]i increase 1reduced hyperosmolality-induced [Ca (osca1) as reported by Figure 1. Cultivation method of Arabidopsis followed -2 s-1 cool-white fluorescent illumination. Plants were watered with 6 ml 1% nutrient solution every day. At ten-leaf stage (day 28 after sowing), 59 pots of each genotype were withheld from watering for 8 days as the drought stress treatment based on the preliminary experiment, and the other 59 pots of each genotype continued to be watered daily during the whole experiment as the control. A total of 236 pots were used in this experiment with 208 pots for non-destructive fluorescence imaging, and 28 pots for destructive measurement of the leaf stomatal conductance. Meanwhile, 96 pots randomly selected from 208 pots were used for the measurements of morphological parameters and malondialdehyde (MDA).The -2 s-1) and AL 1 , and two cool-white LED panels for SF (0–3000 μmol m-2 s-1) and AL 2 , with the angle of 45°. The distance between the plant canopy and the lens was 22 cm. After dark adaptation for 20 min until the PSII reaction centers opened, the kinetic chlorophyll fluorescence curves and images of plants were acquired with an optimized quenching kinetic protocol as follows. Plants were first exposed to the dark for 17 s. In the dark environment, Fo was measured with a 4040 ms flash of light. Fm was then measured at 5.56 s with a 320 ms saturating flash at ∼2300 μmol m-2 s-1, followed by exposing plants to actinic light at a constant of 100 μmol m-2 s-1 for 70 s. Fp was measured at 23.12 s. Ft_Ln was measured before any of the saturating flash, and later, the signal declined to Ft_Lss. Fm_Ln and Fm_Lss were measured with the SF at 32.24, 42.24, 52.24, 72.24, and 92.24 s. By following the actinic light period, fluorescence signals during dark relaxation were recorded by measuring Ft_Dn, and acquiring Fm_Dn with the SF at 122.24, 152.24, and 182.24 s. Based on the basic chlorophyll fluorescence signals, the maximum quantum yield of PSII photochemistry (Fv/Fm), instantaneous non-photochemical quenching during light adaptation (NPQ_Ln), steady-state non-photochemical quenching (NPQ_Lss), instantaneous fluorescence decline ratio in light (Rfd_Ln), steady-state PSII quantum yield (ΦPSII_Lss), PSII quantum yield during light adaptation (ΦPSII_Ln), coefficient of non-photochemical quenching during dark relaxation (qN_Dn), coefficient of non-photochemical quenching during light adaptation (qN_Ln), coefficient of photochemical quenching during light adaptation (qL_Ln), coefficient of photochemical quenching in steady-state (qL_Lss), variable fluorescence in steady-state (Fv_Lss), and variable fluorescence during light adaptation (Fv_Ln) can be derived as equations (1–10)  was used to obtain the kinetic chlorophyll fluorescence curves and images of plants every day upon the onset of drought stress. The system consisted of a high-speed CCD camera with the spatial resolution of 1392 pixels × 1040 pixels, a prime lens , four LED panels, and a sample handling unit with a manually adjustable vertical stage. The light source included two orange-red LED panels for MF  :n is related to the moments of irradiance of suturing flashes. Finally, a total of 89 chlorophyll fluorescence images were obtained based on this protocol.where BF, GF, RF, and IrF for each measurement, and a total of 16 multicolor fluorescence images were obtained.For multicolor fluorescence image acquisition, a UV (320–400 nm) LED panel was used as the excitation source. Multicolor fluorescence images in blue (440 nm), green (520 nm), red (680 nm), and far-red (740 nm) wavelength regions excited by UV light were acquired sequentially. In order to improve the signal-to-noise ratio, five images with the exposure time of 4 s were averaged to calculate In order to evaluate the effect of drought stress on the plant morphological and physiological characteristics, and to determine the most useful phenotypes to understand the early response to drought stress, parameters including projected leaf area, rosette leaf number, plant height, flowering plant number, leaf stomatal conductance, and MDA were determined after performing fluorescence imaging.The projected leaf area was calculated from the total pixels of the chlorophyll fluorescence image. The total rosette leaf number was counted every day after drought stress. The plant height was measured from the base of the stem to the tip of the main inflorescence of the plant from day 3 to day 8 after drought stress, and at the same time, the flowering plant number was also recorded by counting the new flowering plant.Leaf stomatal conductance was measured using a leaf porometer  after performing the calibration with a filter paper of 100% humidity as a reference (according to the user menu). The rosette leaves of the plants cannot be measured directly due to the size of leaves. Therefore, nine rosette leaves were randomly cut from one pot of each genotype, and then leaves of each treatment were examined immediately. Since leaves were still small at day 1 and also drought-stressed leaves wilted at day 7, the stomatal conductance was only recorded from day 2 to 6 after drought stress.osca1 plants were selected for MDA measurement at the last day of the experiment (day 8 after drought stress). 0.1 g leaf tissue of each repetition was homogenized in 5 mL of 0.1% trichloroacetic acid (TCA), and the homogenate was centrifuged  at 10,000 × g for 5 min at 4°C. 20% TCA containing 0.5% TBA was added to 1 mL supernatant. The mixture was then boiled for 30 min at 95°C, followed by the centrifugation at 10,000 × g for 10 min. The absorbance of the supernatant was measured at 532 nm and 600 nm with a microplate spectrophotometer . MDA content was then calculated using an extinction coefficient of 155 Mm-1 cm-1  method . Leaves m-1 cm-1 .osca1, and Pearson’s correlations among fluorescence, morphological and physiological traits were visualized in a network to gain a better understanding of the phenotypic relationship.A total of 105 fluorescence images including 89 chlorophyll fluorescence images and 16 multicolor fluorescence images were acquired from each pot. The image preprocessing was performed including the selection of ROIs, background segmentation, and computation of mean fluorescence parameters from the corresponding ROIs. After preprocessing, ANOVA was implemented first to identify the differences between the control and the drought stress treatments among 5 morphological and physiological related parameters, 4 commonly used chlorophyll fluorescence parameters, and 4 basic multicolor fluorescence parameters. To further explore fluorescence parameters, feature selection by using SFS algorithms with Fisher criterion was then performed to extract the most important features that could differentiate the drought-stressed plants from the control. SFS algorithm is a bottom-up procedure that starts with an empty subset of features and repeatedly adds one new feature with the best function criterion value in the subset each time . This pr1.In order to differentiate the drought-stressed plants from the control based on the selected features, SVM was used for the two-class classification. The SVM classifier can separate a given set of labeled training data with a hyper-plane that is maximally distant from the training data set. It employs a linear kernel function to solve a convex quadratic programming problem . The twoosca1 under control and drought stress treatments at day 1, 3, 5, 7, and 8 after exposure to the drought stress are presented in Figure 2. The parameters included projected leaf area, rosette leaf number, plant height, and flowering plant number. It was visually observed that both wild-type and osca1 presented wilting leaves under the drought-stressed condition. In general, the mutant osca1 wilted more seriously than wild-type. The investigation of morphological traits including the projected leaf area and the rosette leaf number indicated that low water availability had significant effects on the plant growth . The projected leaf area of wild-type and osca1 decreased evidently after 3–8 days and 5–8 days post-drought treatment, respectively. While the significant decrease in the rosette leaf number with drought stress was observed at day 7 in wild-type and at day 5 in osca1. Drought stress had no significant effect on the plant height except for osca1 at day 8 . The average plant height of osca1 was higher than wild-type in the control condition. The increased flowering plants of wild-type and osca1 under drought treatment compared to the control treatment indicated earlier flowering time with the drought stress .The RGB images and the morphological parameters of wild-type and Figure 3A presents stomatal conductance of wild-type and osca1 plants under the drought stress condition as well as the control condition. There is a clear decrease in the stomatal conductance values in both wild-type and osca1 plants with the drought stress treatment over time. MDA contents of the control and the drought-stressed plants at day 8 are shown in Figure 3B, which is considered as a reliable indicator of lipid membrane peroxidation . The slopes of the curves from Fp to Fm_L2 for both genotypes reduced significantly after 7 days exposure to drought stress , indicating the slow fluorescence quenching process.To understand the photosynthetic performance of the control and the drought-stressed plants, chlorophyll fluorescence curves were measured from day 1 to day 8 under drought stress by using the chlorophyll fluorescence quenching protocol . TypicalFv/Fm, NPQ_L2, Rfd_L3, and ΦPSII_Lss . Overall, wild-type and osca1 had similar patterns under the control and the drought conditions. The mean values of Fv/Fm in control plants were relatively consistent with the values at 0.85 ± 0.002 over time, while significant decreases in drought-stressed plants were observed at day 7 (P < 0.05) and day 8 (P < 0.001) with the values at 0.84 ± 0.01 and 0.82 ± 0.04 for wild-type and osca1, respectively. NPQ_L2 of both genotypes showed significant decreases in the drought stress treatment starting from day 3 compared with the control set, indicating that this parameter was sensitive to drought stress. Similar patterns were also found in Rfd_L3 except wild-type at day 7. The values of ΦPSII_Lss in all the treatment groups increased from day 1 to 5 and then decreased. The significant difference between the control and the drought stress treatment was detected at day 5 for both genotypes.Commonly used chlorophyll fluorescence parameters averaged from ROIs were also investigated to further explore the plant response to drought stress on photosynthetic performance, including Figure 6 presents representative chlorophyll fluorescence images, including NPQ_L2 and ΦPSII_Lss, according to the parameters that have the potential to explore the spatial heterogeneity. The intensity of NPQ_L2 in the drought stress treatment decreased compared with the control as early as day 3, while the spatial variation was not obvious. Images of ΦPSII_Lss decreased compared with the control as early as day 3, and this trend spread from the leaf tips and edges to the entire canopies indicating the spatial heterogeneity.The parameters of chlorophyll fluorescence only showed the average trends of the canopy without reflecting the spatial variation. BF, GF, RF, and IrF were selected from the full data set . RF and IrF are emitted by chlorophyll a, and BF and GF are emitted by phenolics, which could provide information related to the primary and secondary metabolism, respectively , qN_D2, Fv_Lss, qL_L4, qN_L2, ΦPSII_L4, qL_L1, NPQ_L1, and Fm_D1 for wild-type, and fluorescence blue/far-red ratio (BF/IrF), NPQ_L2, NPQ_Lss, fluorescence green/blue ratio (GF/BF), Fv_L3, qN_D2, qL_Lss, ΦPSII_L3, and ΦPSII_L4 for osca1. Trait–trait correlations among the total 16 selected fluorescence, 4 morphological and 2 physiological traits were shown in a correlation network , which presented phenotypic similarity of traits related to the drought stress response. Traits of the same categories showed good correlations  with each other. Physiological traits correlated well with fluorescence traits. MDA and stomatal conductance displayed high correlations with fluorescence traits, and MDA had the highest correlation with the fluorescence trait of NPQ_L2  among the selected 16 fluorescence traits while stomatal conductance had the highest correlation with IrF/BF . However, the correlation between morphological traits and fluorescence traits were not significant. Flowering plant number only correlated with qN_D2 . Projected leaf area, rosette leaf number, and plant height showed poor correlation with fluorescence traits .Although commonly used fluorescence parameters and images can provide a basic evaluation of plant phenotypes of the drought stress response, it is difficult to perform a quantitative analysis to clearly differentiate the drought-stressed plants from the normal ones. Therefore, machine learning techniques were employed to further explore the fluorescence images. The optimum feature subsets of two genotypes selected by SFS were fluorescence far-red/blue ratio  revealed that leaf rolling followed by wilting might be an initial morphological survival mechanism against drought stress that could reduce the transpiration rate, thereby improving water use efficiency. The decline in the projected leaf area and the leaf number demonstrated the progress of plant senescence or damages under drought stress. Both genotypes of Arabidopsis conferred a drought escape strategy of early flowering during the reproductive stage , which could ensure their survival under drought stress. Earlier flowering of osca1 in drought stress treatment implied its sensitivity to drought stress and shorter survival compared to wild-type under drought stress , which was consistent with the study reported by osca1 with impaired OSCA1 might not induce the accumulation of abscisic acid (ABA) trigger stomatal closure at time. Previous studies have demonstrated that the activity of antioxidant enzymes was correlated with plant tolerance to abiotic stresses, such as drought stress, and MDA was the final product of plant cell membrane lipid peroxidation and one of the important signs of membrane system injury  after 8 days post-drought stress indicating a higher sensitivity to drought stress, which agreed with the findings of Plants manifest multiple defense mechanisms involving various morphological and physiological alterations in response to drought stress . Investit stress . These cnt level . Stomatant level . The stom injury . In thisosca1 exhibited different photosynthetic strategies to utilize the absorbed irradiance under drought stress. Wild-type can efficiently keep homeostasis of photosynthetic apparatus and the plants under the drought-stressed treatment yield almost similar chlorophyll fluorescence transients to the control at the early stage of drought stress . At the later stage when plant leaves turned wilted, the results of decreased patterns of drought stress treatment were similar to the findings of Drought stress is one of the most severe limitations to photosynthesis, which causes the diffusion limitations through the stomata and the photosynthetic metabolism. Kinetic chlorophyll fluorescence imaging is a useful tool to monitor subtle changes in the photochemical and non-photochemical process. Different patterns of chlorophyll fluorescence quenching curves implied that wild-type and NPQ_L2, Rfd_L3, and ΦPSII_Lss were considered as straightforward and practical parameters to monitor the response to drought stress . Fv/Fm of both genotypes were not significantly different between the control and drought stress treatments until 7 days’ drought stress, indicating that Fv/Fm might not be a good indicator of short-term plant drought stress. Rfd_L3 presented the potential photosynthetic capacity of plants. The Rfd_L3 of drought-stressed groups of two genotypes showed a declining pattern after drought stress induction, suggesting that Rfd_L3 was more sensitive to drought stress than Fv/Fm , which was in agreement with a reported study of tobacco  might be a result of the gradual loss of photosynthetic quantum conversion which could increase the red chlorophyll fluorescence yield or the breakdown of chlorophylls that the plants suffered a period of lack of water , due to the lack of re-absorption of the emitted chlorophyll fluorescence band in vivo chlorophyll protein complexes , indicating that SFS feature selection methods could provide valid information related to the drought stress response with only a few number of traits. Additionally, it was noted that the classification accuracies of both genotypes was 87.5% at least for the plants as early as day 3 after drought stress treatment, which revealed that the drought stress could be detected by the fluorescence imaging at the early stage before the wilted symptoms appeared. Advanced machine learning methods showed superior ability to detect drought stress response. The classification accuracy of osca1 was higher than that of wild-type at day 3, which indicated a higher sensitivity of osca1 to drought stress.The SVM classifier combined with the SFS feature selection method has shown a good classification performance. Although fluorescence parameters were not well correlated with the morphological parameters based on Pearson’s correlation analysis, the correlation network showed trait similarity of the same categories and high correlations between fluorescence parameters and physiological parameters (Arabidopsis. The kinetic chlorophyll fluorescence curves have the potential to detect the drought tolerance mechanism of Arabidopsis. Even though some conventional fluorescence parameters provided qualitative information related to the drought stress response in different genotypes including the spatial heterogeneities of drought stress response within the leaf and the canopy level, the selected fluorescence parameters by SFS presented a higher correlation with physiological traits, and they have shown a better result to classify the drought-stressed and the control plants by using SVM classifier. The results demonstrated that the combination of kinetic chlorophyll fluorescence and multicolor fluorescence imaging with the machine learning technique was capable of providing comprehensive information of drought stress effect on the photosynthesis and the secondary metabolisms. The technique proposed in this study can be applied not only to the evaluation of drought stress in Arabidopsis but also to other crops.This study has shown the potential of kinetic chlorophyll fluorescence and multicolor fluorescence imaging for the early detection of drought stress responses in two different genotypes of HC and JY designed the experiment. JY, DS, HX, and HW performed the experiment. HC, JY, and DS contributed to the data analysis and wrote the manuscript. FY and YH provided suggestions on the experiment design and discussion sections. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oxidative stress and inflammation are underlying pathogenic mechanisms associated with the progression of several pathological conditions and immunological responses. Elucidating the role of signalling lipid classes, which include, among others, the isoprostanes, nitro fatty acids, prostanoids, sphingoid bases and lysophosphatidic acids, will create a snapshot of the cause and effect of inflammation and oxidative stress at the metabolic level. Here we describe a fast, sensitive, and targeted ultra-high-performance liquid chromatography–tandem mass spectrometry metabolomics method that allows the quantitative measurement and biological elucidation of 17 isoprostanes as well as their respective isomeric prostanoid mediators, three nitro fatty acids, four sphingoid mediators, and 24 lysophosphatidic acid species from serum as well as organ tissues, including liver, lung, heart, spleen, kidney and brain. Application of this method to paired mouse serum and tissue samples revealed tissue- and serum-specific stress and inflammatory readouts. Little correlation was found between localized (tissue) metabolite levels compared with the systemic (serum) circulation in a homeostatic model. The application of this method in future studies will enable us to explore the role of signalling lipids in the metabolic pathogenicity of stress and inflammation during health and disease.The online version of this article (10.1007/s00216-018-0912-2) contains supplementary material, which is available to authorized users. Oxidative stress is characterized as a condition where the levels of reactive oxygen species (ROS) necessary for cellular redox biology –5 increa2-FAs, prostaglandins, sphingoids and LPAs as well as the cLPA species presents an opportunity to study the cause and effect of oxidative stress, inflammation and cell proliferation at the metabolic level. Secondly, studying the combined effect of these signalling mediators will be particularly helpful as these signalling molecules could have distinct and sometimes even opposing functions.Several lipid classes, including the prostaglandins, sphingoid bases and lysophosphatidic acids (LPAs) have been implicated in the activation of signalling pathways regulating inflammation, oxidative stress and cell proliferation among other physiological responses via dedicated cellular receptors. Prostaglandins Fig. b are enz2-FAs, four sphingoid mediators, 16 LPAs and 6 cLPA species. Application of the method developed to patient-derived serum and tissue samples will help in determining the underlying links between inflammation and oxidative stress in disease. We applied the method developed to a homeostatic mouse model with paired tissue and serum samples, providing a stress and inflammation readout for serum as well as brain, lung, liver, heart, kidney and spleen tissues.Several different methods have been developed for the analyses of these metabolites, each focusing on specific compound classes and biological matrixes. The analyses of prostanoids and isoprostanes is commonly accomplished with large volumes of plasma/serum (more than 200 μL), accompanied by solid-phase extraction coupled with liquid chromatography (LC)–tandem mass spectrometry (MS/MS) –30. Comp2-FAs were purchased from Cayman Chemicals  (see Table Ultra-performance LC grade acetonitrile (ACN), isopropyl alcohol (IPA), methanol (MeOH), ethyl acetate (EtOAc) and water were purchased from Biosolve . 1-Butanol (BuOH) was obtained from Boom . Acetic acid, ammonium hydroxide, butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA) and ammonium acetate were from Sigma-Aldrich . Sodium dihydrogen phosphate dehydrate and citric acid were obtained from Merck . Standards and deuterated standards for the isoprostanes, prostanoids and NO2-FAs and 4000 nM for the LPA and sphingoid base standards and was labelled “C8”. This C8 solution was diluted to levels C7 to C1, with C1 being the lowest calibration level at 0.75 and 2.3 nM, respectively. From these calibration stock mixes (C8 to C1), 20 μL was added to 150 μL sample to construct the calibration curves. Table Standard and deuterated standard stock solutions were prepared in MeOH containing BHT (0.2 mg/mL). A calibration stock solution was made, with a concentration of 1304 nM for the isoprostanes, prostanoids and NOControl human serum used in the method validation was purchased from Harlan Sera-Lab .g at 4 °C for 10 min, after which the serum was transferred to a clean Eppendorf vial, snap frozen and stored at –80 °C. All procedures were approved by the Institutional Review Board for Animal Experiments at Leiden University Medical Center .C57BL/6 mice were housed at 21 ± 1 °C, 40–50% humidity, with a 12-h light-dark cycle, with ad libitum access to water and a standard rodent diet. Eight male adult mice were anesthetized with isoflurane and euthanized by cervical dislocation. After the chest cavity had been opened, blood was collected with a 22-gauge needle from the heart and left to coagulate on ice. The heart, lungs, liver, spleen, kidneys and brain were harvested in that sequence, cleaned of excess visceral fat, hair, tissues and blood, and snap frozen in liquid nitrogen. After the blood had coagulated on ice for approximately 30 min, the samples were centrifuged at 2000g, after which 900 μL of the upper organic phase was collected and transferred to a clean 2-mL tube. The LLE was repeated by addition of 400 μL BuOH saturated with water and 400 μL EtOAc to the remaining aqueous phase. After mixing and centrifugation, 800 μL of the organic phase was collected, and the total organic fraction was dried under a vacuum. Then 40 μL of ice-cold 90% MeOH injection solution was added to the dried residue as a reconstitution solution. Reconstituted samples were vortexed before centrifugation for 10 min at 4 °C and 25,300g, and were subsequently transferred to inserts in injection vials before LC–mass spectrometry (MS) analyses and stored in the autosampler at 5 °C.Serum aliquots (150 μL) were thawed on ice, after which 5 μL antioxidant (0.2mg BHT and 0.2 mg EDTA) solution and 10 μL of ISTD solution were added and the serum was briefly vortexed. Samples were then acidified through the addition of 350 uL of 0.2 M citric acid and 0.1 M disodium hydrogen phosphate buffer at pH 4.5. LLE was accomplished by the addition of 1 mL of a 1-butanol–ethyl acetate (1:1 v/v) solution. Samples were mixed for 2 min, then centrifuged for 10 min at 4 °C and 25,300g, followed by the addition of 150 μL of pH 4.5 buffer as well as 1 mL of a BuOH–EtOAc (1:1 v/v) solution. The collection of the organic phase and the repeated extraction steps were as detailed in the previous subsection for serum extraction.Snap-frozen tissue samples were transferred to a freeze dryer, where tissues were dried overnight, mechanically homogenized, aliquoted and stored at –80 °C before extraction. Dried tissue amounts of 5 mg were suspended in 200 μL of a 0.2 M citric acid and 0.1 M disodium hydrogen phosphate buffer at pH 4.5, to which approximately 500 mg of 0.5-mm stainless steel beads  was added. Then 5 μL antioxidant (0.2mg BHT and 0.2 mg EDTA) and 10 μL ISTD solution were added, and tissue samples were homogenized in a bullet blender at maximum speed for 10 min. Afterwards, samples were centrifuged for 30 s at 232518 column  maintained at 40 °C. The mobile phases consisted of water and 0.1% acetic acid (mobile phase A), ACN–MeOH (7.5:2.5 v/v) and 0.1% acetic acid (mobile phase B), and IPA (mobile phase C) with a flow rate of 0.7 mL/min. The pH of the mobile phases ranged between 3.2 and 3.5 during the chromatographic gradient. To increase the column load ability, a stacked injection was used which consisted of stacking 10-μL sample volume with 20 μL of mobile phase A in the needle before injection of this “solution stack” onto the column. The analytes were eluted with a gradient starting at 5% mobile phase B and 0% mobile phase C an progressing to 75% mobile phase B and 25% mobile phase C between 0 and 9 min; the final conditions were held for 1 min, after which the column was reequilibrated to the starting conditions from 10.15 to 13 min.LC was performed with an LCMS-8050 system  and an Acquity BEH C18 column  maintained at 40 °C. The mobile phases consisted of water, 5 mM ammonium acetate and 0.0625% ammonium hydroxide (mobile phase A) and ACN–IPA (8:2 v/v) and 0.1% ammonium hydroxide (mobile phase B) with a flow rate of 0.6 mL/min. The pH of the mobile phases ranged between 10.3 and 8.5 during the chromatographic gradient. The injection volume was 5 μL. The metabolites were eluted with a linear gradient starting at 10% mobile phase B and progressing to 100% mobile phase B in 5 min; the conditions were kept at 100% mobile phase B for 0.75 min, after which the column was reequilibrated to the starting conditions from 5.75 to 8 min.LC was performed with an LCMS-8050 system  and a Kromasil EternityXT-1.8 CThe Shimadzu LCMS-8050 system consists of a UHPLC system connected to a triple-quadrupole mass spectrometer with an electrospray ionization source. The electrospray ionization source parameters were as follows: interface temperature 300 °C, desolvation line temperature 250 °C, heat block temperature 400 °C, nebulizing gas flow rate 3 L/min, heating gas flow rate 10 L/min and drying gas flow rate 10 L/min. The analytes and ISTDs were measured by multiple reaction monitoring (MRM) in either positive or negative ion mode with the complete optimized target list (collision energy and dwell time) in Table The target list Table  includedThe retention times of putatively identified metabolites were compared with those standards and had to have similar retention time frames.Elution sequences were compared with those of the available standards. For example, the most unsaturated acyl species of any given length was eluted first, followed by progressively more saturated species until the saturated one .The putatively identified metabolites had to have the characteristic fragmentation patterns associated with the LPA and cLPA species. For the LPA species the deprotonated parent ion [M − H]− had to fragment into a dehydrated glycerol phosphate 152.90 m/z fragment as well as a phosphate 78.90 m/z fragment. For the cLPA species the deprotonated parent ion [M – H]− had to fragment into a unique free fatty acid fragment as well as a phosphate 78.90 m/z fragment.Because of the shortage of LPA standards, an MS screening approach was used to putatively identify other LPA species. Precursor ion scans together with single reaction monitoring (SRM) was used to search for and identify putative LPA species with use of known fragmentation patterns. Metabolites were identified on the basis of the following criteria:The targeted LPA species are listed in Table Method performance was investigated, and included linearity, limits of detection, retention time stability, interday and intraday precision, extraction recovery and the matrix effect.Calibrations curves were prepared in four replicates with seven concentrations ranging over four orders of magnitude to assess the limit of detection (with use of a blank matrix consisting of pure water) and linear range (with use of control serum). For each calibration curve, the ratio of the analyte area and its corresponding ISTD area was plotted against its nominal concentration, with no weighting factor being applied. The limit of detection was determined as the lowest concentration that resulted in a peak with a signal-to-noise ratio greater than 3 according to the ASTM method. Furthermore, the lower limit of quantification used to determine the linear range was determined as the lowest concentration that resulted in a peak with a signal-to-noise ratio greater than 10 according to the ASTM method.Retention time stability reflects the stability of the chromatography over progressive runs, to ensure correct peak identification based on retention time during data processing. The retention time stability was investigated through our determining the relative standard deviations (RSD) of the metabolite retention time across a sequence of 100 injections. Retention times are considered stabile if the RSD is less than 1%. The autosampler performance was investigated through our determining the RSD of the peak areas of 12-[(cyclohexylcarbamoyl)amino]dodecanoic acid (CUDA). CUDA is an exogenous compound added to the injection solution at a level of 100 nM. Autosampler performance was deemed to be adequate when the CUDA peak areas had an RSD of less than 5% across a batch.Precision is defined as the closeness of the measurements of individual samples when the procedure is applied to multiple aliquots of a homogenous matrix; hence it is reported as the RSD of the measurements . The metRecovery reflects the extraction efficiency of the LLE procedure for the metabolites in a specific biological matrix and should be reproducible (low RSD). Both the serum and different tissue recoveries were determined by our comparing the response of an ISTD added to the sample before LLE with the response obtained from the ISTD added after LLE. Serum recoveries were determined in quadruplicate with control serum aliquots at three different levels . The relative ratios of metabolite peak areas to the peak areas of their corresponding ISTDs were used for statistical analyses. Principal component analyses (PCA) and heatmaps based on Euclidean distance measure and the Ward clustering algorithm were performed with the R script-based online tool MetaboAnalyst (version 3.0), a comprehensive tool suitable for analysing metabolomics data . Spearma2-FAs  had reasonable extraction efficiencies (70–100%) as did the LPA, cLPA and sphingoid metabolites.The different recoveries of the ISTDs from the six tissues reflected the diverse nature of these tissues Fig. . It is i2-FAs experienced high levels of the matrix effect during the LC–MS chromatographic run, and together with the reduced extraction efficiency of this class of metabolites, this highlights the challenges in measuring them. The use of ISTDs for the analysis of especially those metabolites showing higher matrix effects is therefore critical; these non-endogenous ISTDs should be added at the same concentration as the corresponding metabolite in different tissues and allow a better comparison across different tissues in biological studies, and this is true for semiquantitative (as in our case) as well as quantitative metabolomics measurements.From investigation of the matrix effect for different tissues Fig. , brain tn = 4) at the C4 (medium) level  and inflammatory metabolic profile. Of the seven biological matrices investigated, spleen had the highest number of metabolites (53 of 60 metabolites) detected, and serum had the lowest number (35 of 60 metabolites). This result is expected as most of these metabolites are signalling mediators which are produced locally. We also compared the variation of each metabolite due to the analytical variation  with the biological variation of that metabolite  at the endogenous metabolite levels in the different tissues Table . In almoNext we investigated the natural projection of the metabolite levels in the various tissues and serum samples using unsupervised multivariate PCA. Inspection of the PCA results revealed that the samples of different tissue types and the serum samples were completely separated, as shown in Fig. 2-FAs  and prostaglandin F2α (PGF2α) being the dominant species. The heart and liver samples both have the lowest levels of prostaglandins detected. The kidney samples have high levels of especially PGE2 and PGA2, and the lung has high levels of prostaglandin F1α. The reasons for these unique prostaglandin profiles could be attributed to their signalling functioning and its relation to the specific tissue function. The spleen is an interface between the circulating blood and lymph systems critical for innate and adaptive immune responses, especially against bacterial and fungal infections. It is also essential in regulating erythrocyte level. The presence of high levels of prostaglandins in the spleen highlights the immunological importance of these lipid mediator species in orchestrating the differentiation, migration and response of leucocytes [2 in the kidney relates to its homeostatic role in fluid metabolism and hemodynamic effects. PGE2 regulates blood flow in the kidney and sodium excretion, influencing blood pressure [2 has been identified as the main central nervous system prostaglandin in rats, functioning in a protective manner [1 (and to a lesser extent PGE2), and other samples having much lower levels. Even though these mice present baseline levels, some physiological state cannot be excluded, and the differences found might be subtle, or important.To compare the different metabolite levels in the different tissue samples, heatmaps were used to visualize the individual measurements. The signalling lipid profile was split into the prostaglandins Fig. , panel aFAs Fig. , panel bFAs Fig. , panel cFAs Fig. , panel aucocytes , 49. Higpressure –52. PGD2e manner , 54. The2-FAs  was detected in all six tissues and serum, and might be a more sensitive readout for oxidative stress. The downstream metabolite of 8-iso-PGF2α, 2,3-dinor-8-iso-PGF2α, was strongly detected in serum and kidney tissue, revealing the metabolizing and excretion of oxidative stress markers via urine. Nitro-oleic acid was detected in all tissues and serum except for brain, heart and kidney. Fu et al. [2-FAs. Overall, the spleen presented the highest levels of oxidative stress markers. It is interesting to note that the different tissues all had different types of isoprostanes present. As lipid oxidation and nitration via ROS and RNS is a non-enzymatic, non-specific process, the homeostatic levels are possibly reflective of low-level stress and redox biology processes and that this is to some extent probably tissue function related.Next we zoomed in on the homeostatic stress-related lipid profile consisting of the isoprostanes and NOFAs Fig. , panel bu et al.  reportedLastly, we focused on the LPAs and sphingoids Fig. , panel cAs shown in the preceding sections, each tissue clearly has a distinct homeostatic metabolic stress and inflammatory profile primarily related to tissue function; therefore, it is interesting to determine how reflective serum is as a systemic readout of these different tissue profiles. Because of the relative ease and non-invasive nature of the collection protocols for most biofluids , they are frequently the sample material of choice in studies investigating health and disease, although a possible drawback of biofluids is that they represent a systemic readout of a highly dynamic system rather than allowing tissue-specific readouts. For the metabolites studied in this method, it is still rather unclear whether the serum profile reflects the physiology of the various tissues, as it is difficult to determine the origins of most metabolites in serum. It is generally believed that localized disease perturbations in the body are severe enough to spill over into the circulation, where they can be measured in a less invasive manner. This is also the fundamental principle in disease biomarker studies, where systemic circulating metabolites are sensitive and specific for a particular disease, acting as a biomarker (biosignature or disease fingerprint) , 62. How2 derived from a glutathione-dependent PGD2 synthase is produced by antigen-presenting cells, of which the spleen is a rich source under homeostasis, supporting this finding. The isoprostane 8,12-iPF2α VI correlated strongly between serum and kidney tissue samples, suggesting the excretion of this metabolite via urine. Both positive and negative LPA correlations were observed between the different tissues, the interpretation of which remains unclear. Especially spleen and brain tissue indicated a strong correlation, with four unsaturated LPA species identified as significantly negatively correlated.Three metabolites correlated significantly between serum and spleen tissue, and one correlation was found for kidney and heart tissues; these were all different metabolites. Urade et al.  reportedFrom the data taken collectively, few correlations were found between circulating serum and tissue metabolism when we investigated the baseline stress and inflammatory profiles of healthy mice. Furthermore, the subtle correlations could allude to organ regulation of signalling metabolites as well as intraorgan metabolic compartmentalization. It is quite well known that different organs are composed of different tissue subtypes. For example, the spleen consists of white and red pulp, with each having unique properties, functions and quite possibly also metabolism. Thus, when metabolic questions related to health and disease are being studied the choice of the proper sample material is of critical importance to accurately reflect the pathological condition investigated..Oxidative stress can be assessed with different techniques depending on the experimental design and sample material available. Currently the use of in vitro assays measuring hydrogen peroxide levels and glutathione ratios  is viewed as the most reliable approach in assessing the oxidative stress status. Newer technology such as Seahorse XF provides the opportunity to gain unique insights into mitochondrial functioning under different non-endogenous stressor conditions ideally suited for in vitro studies. However, the in vivo suitability of this live-cell analyses has some limitations when inadequate sample collection procedures have been followed. The application of the method presented in this article is applicable to both in vitro (cell lysate and medium) and in vivo (biofluids and tissues) studies. The non-specificity of free-radical attack makes the lipid peroxidation markers (isoprostanes) a generic readout for oxidative stress, which can be broadly applied. Furthermore, the inclusion of other signalling lipids adds to the understanding of inflammatory processes, and these mediators could potentially provide different readouts depending on the biological questions investigated. However, many questions remain about their function, in part because only a few tools can accurately measure their location specifically in vivo2-FAs reflective of lipid peroxidation and nitration, as well as an inflammatory readout based on the prostaglandins, LPAs, cLPAs and sphingoids. Application of this method to biological questions related to health and disease will broaden our understanding of oxidative stress and inflammation at the metabolic level. The application of this metabolomics profiling method to healthy mice found that the systemic (serum profile) readout for stress and inflammation markers had little correlation to the localized readout of the six tissues tested under baseline conditions. However, we did identify specific serum metabolites correlating with levels in the spleen, heart and kidney as well as significant tissue–tissue metabolic correlations. In addition, each tissue type presented a unique homeostatic stress and inflammation profile. This might be due to the tightly controlled nature of these potent biological signalling lipids during homeostasis. In the event of a severe health perturbation, the reflective nature of this panel of metabolites in serum needs to be evaluated as the localized perturbation can spill over into a system readout, while also affecting other tissues and organs. Localized metabolomics studies have the potential to aid in biomarker discovery, elucidation of pathogenic mechanisms, prediction of disease severity, risk stratification and measurement of therapeutic intervention.As oxidative stress and inflammation are central to many known diseases, the development of a method able to provide systemic and localized readouts is of the utmost importance. In addition, in some tissues and some diseases (cardiovascular), nitrosative stress is an important process. The method developed and validated in this work provides a stress readout based on the isoprostanes and NOESM 1(PDF 1.40 mb)