Wheat and maize are among the world’s most important crops, covering nearly 400 million ha of Earth’s land surface (an area roughly twice the size of Mexico) and playing a major role in the diets and livelihoods of millions of people. Leaf characteristics (or leaf traits) of these crops—including photosynthetic rates—are widely used in models that predict how wheat and maize will respond to environmental change. Using a large global database, we evaluate how leaf traits of wheat and maize differ across cultivars, and link this variability to climate conditions. In doing so, we assess how leaf traits can be expected to vary under climate change, and in turn, influence predictions of future food security. Amax) and leaf nitrogen (N) concentrations, for wheat and maize. We evaluated intraspecific LES trait variation, and intraspecific trait–environment relationships. While wheat and maize occupy the upper 90th percentile of LES trait values observed across a global species pool, ITV ranged widely across the LES in wheat and maize. Fertilization treatments had strong impacts on leaf N, while plant developmental stage (here standardized as the number of days since planting) had strong impacts on Amax; days since planting, N fertilization and irrigation all influenced SLA. When controlling for these factors, intraspecific responses to temperature and precipitation explained 39.4 and 43.7 % of the variation in Amax and SLA, respectively, but only 5.4 % of the variation in leaf N. Despite a long history of domestication in these species, ITV in wheat and maize among and within cultivars remains large. Intraspecific trait variation is a critical consideration to refine regional to global models of agroecosystem structure, function and food security. Considerable opportunities and benefits exist for consolidating a crop trait database for a wider range of domesticated plant species.Leaf Economics Spectrum (LES) trait variation underpins multiple agroecological processes and many prominent crop yield models. While there are numerous independent studies assessing trait variation in crops, to date there have been no comprehensive assessments of intraspecific trait variation (ITV) in LES traits for wheat and maize: the world’s most widespread crops. Using trait databases and peer-reviewed literature, we compiled over 700 records of specific leaf area (SLA), maximum photosynthetic rates ( Functional traits refer to the structural, chemical, physiological or phenological properties of plants and plant parts, which mechanistically influence plant performance  across environmental gradients (Amax). At the opposite end of the LES are ‘resource acquisitive species’ that express high SLA, high leaf N and high Amax  has been hypothesized and tested, as a suite of covarying leaf traits that can be used to describe plant functional biology . Although crop growth models are parameterized with functional trait data, crops are commonly represented by species-level mean trait values. Accounting for intraspecific variation in LES traits for crops has been identified as a key avenue for refining predictions of agricultural yield (Triticum spp.) and maize (Zea mays)—could aid in refining simulation models of food, nutrient, water and energy fluxes across nearly 400 million ha of crop land  in influencing structure and function in natural or experimental ecosystems , T. durum—and Z. mays (Poaceae). Focusing on T. aestivum, T. durum and Z. mays is consistent with species-level taxonomy for the ‘Maize’ and ‘Wheat’ commodity groups recognized by the Food and Agricultural Organization of the United Nations (FAO) (see www.fao.org/economic/ess/ess-standards/commodity). , and less than three peer-reviewed publications with trait data for each species).) We initially sought to assess ITV at the within-cultivar level. However, trait observations for any particular cultivar across multiple studies were prohibitively low to allow comprehensive assessments of ITV within any cultivar, beyond the results reported in a given source publication .Our analysis focused on two species of wheat—T. aestivum, T. durum and Z. mays, we focused our data compilation on key LES traits that are important inputs into many agricultural vulnerability models, particularly those employed by the IPCC (Supporting Information—Table S1). Data compilation was done following two approaches. First, we submitted structured data requests to the TRY database (Amax) and leaf N on both a mass and area basis , leaf area, leaf phosphorus concentrations, leaf lifespan and leaf dry mass, but there were few data available on these traits, so they were not included in our analysis here.For Zea mays’). Our search targeted (but was not limited to) the period of 2000–15 in order to focus our analysis on the most recent wheat and maize genotypes, the majority of which have been introduced since 2000 , as well as climate data including mean annual temperature (MAT) and total annual precipitation (TAP). We also sought to include information on solar irradiance, vapour pressure deficit (VPD) and potential evapotranspiration (PET), but these variables were not consistently reported among studies and were therefore not incorporated into our formal analysis. Where climate information was not included in these studies, historical climate data were obtained via the closest weather station, and using a 0.6 °C change in growth temperature (GT), per 100 m in elevation  fixing plants present, crop planting density, soil pH, N fertilizer application rate, irrigation rates, plant age and day of sampling. Although explicit efforts were made to standardize these data for analysis, since they were not consistently reported among studies , and were challenging/impossible to standardize in certain instances (such as fertilization treatments which were reported in multiple inconsistent ways), they were generally omitted from analyses here (but see below).All trait measurements were additionally categorized by study area type, which was listed as one of ‘field’, ‘greenhouse’ or ‘growth chamber’. Any pot experiments within the compilation were categorized as either field or greenhouse, depending on the conditions of the experiment. If both field and greenhouse locations were used within one study, the data were separated when possible. Other ancillary information included the plant developmental stage at time of sampling. Due to inconsistent methods of reporting across studies, we sought to re-categorize this information for analysis. For example, any one of the following was commonly given: date of sowing; date of sampling; number of days after a given development stage; or the plant developmental stage at time of sampling. This reporting also included varying developmental stage models . Trait observations were more readily available in the primary literature (n = 663 observations) as compared to the TRY database (n = 122 observations), and the large majority of data were based on field-grown crops , as compared to growth chambers (n = 168 observations from 16 studies) or greenhouse-based studies (n = 45 observations from nine studies); one study entailed n = 2 observations, one from both field- and greenhouse conditions, while n = 8 observations from five studies did not provide growth condition information.Our data compilation resulted in a total of 721 leaf level observations for n = 292 observations across 31 studies) and Asia (n = 136 observations across 21 studies), followed by Australia (n = 95 observations across four studies), South America (n = 32 observations across four studies) and North America (n = 136 observations across four studies); Africa was represented by n = 4 observations, and n = 1 observation from one study was not associated with any geographical information (see Supporting Information—Table S2).Geographic representation of crop trait data varied with the largest number of sources derived from studies based in Europe  and the variance explained by both the fixed and random factors   .3 and C4 plants) used to define the LES .To understand where wheat and maize fell within the LES, we compared mean crop trait values to the trait variation observed in the GLOPNET database, which is the initial global data set  might confound relationships between traits and GT and TAP. To do so, we fit a model of the form:i represents the value of the ith trait measurement in our data set, which is predicted as a function of five fixed effects including (i) β0, which represents an overall model intercept; (ii) β1, which is the parameter estimate associated with nitrogen fertilization ; (iii) β2, which is the parameter estimate for irrigation ; (iv) β3, which is the parameter estimate for the type of study ; and (v) β4, which is the parameter estimate that represents the influence of days since planting (D). In this model, the influence of crop genotype (Gi) on a predicted trait value was included as a random effect, and εi represents the error associated with each individual trait observation.where traitsee , in our second step we fit and compared a number of linear mixed-effects models to evaluate relationships between traits and both GT and TAP. These full models included species identity, GT, TAP and all species-by-environment interactions as fixed effects, and variables that had a significant influence on traits (as per Supporting Information—Table S3) as random effects. These full models were of the form:Based on the results of these analyses ij represents the predicted trait value measured on the ith leaf of the jth species, and β0 represents an overall intercept. In these models, (i) β1 represents the parameter estimate associated with the jth species ; (ii) β2 is the parameter estimate for GT; (iii) β3 represents the parameter estimate for TAP; (iv) β4 represents an interaction term between S and GT (a parameter applicable only to Z. mays); and (v) β5 is the interaction term between S and TAP . Random effects in these models  were irrigation (Iij), the type of study (Fij), nitrogen fertilization (Nij) and the number of days since planting (Dij) on predicted trait values; εij represents the error associated with each individual trait observation. Sample sizes of these models . In general, the full models presented were associated with either the lowest AIC score, or had AIC scores that were ≤2 greater than the AIC scores for the next most parsimonious model fit see . Therefore, in our next step we assessed the statistical significance of all fixed effects in see , leaving us with final predictive models of each individual trait  scores to compare the full models in al trait . For eacT. aestivum had the most extensive LES trait coverage with n = 496 observations, while Z. mays traits were represented by n = 207 observations; T. durum was represented by only n = 18 observations. For T. aestivum, leaf N concentration was the most well-represented trait (n = 218), followed by Amax (n = 173) and SLA (n = 105) , followed by Amax (n = 80) and SLA (n = 39). Sample sizes for T. durum were considerably lower for Amax (n = 9), leaf N (n = 8) and SLA (n = 1)  . Similar (n = 1) .Amax was highest in Z. mays (27.4 ± 1.7 (SE) µmol CO2 m−2 s−1), followed by T. durum (24.3 ± 3.4 µmol CO2 m−2 s−1) and T. aestivum (16.4 ± 1.2 µmol CO2 m−2 s−1), with wheat species differing significantly from maize  = 16.0–28.8 mm2 mg−1) and 6-fold in T. aestivum , while leaf N varied over 2-fold in T. durum , 5-fold in Z. mays  and nearly 10-fold in T. aestivum , 19-fold in T. aestivum  and 2-fold in T. durum , and leaf N values fell within the 90th and 99th percentile (which ranged in GLOPNET from 31.9 to 50.5 mg g−1). Mean SLA values for T. aestivum and maize fell within the 85th percentile (which ranged in GLOPNET from 20.5 to 24.1 mm2 mg−1), although mean SLA for T. durum fell only within the 40th percentile of GLOPNET values , while for every 1 mm increase in precipitation SLA declined by 0.02 ± 0.003 mm2 mg−1 .From a modelling perspective, studies have suggested that accounting for intraspecific variation in SLA and/or op yield . Our resAmax, SLA and leaf N, which highlights systematic differences among these traits in the world’s two most widespread crops , an understanding of the linkages between ITV in LES traits and environmental conditions should be taken into account when parameterizing process-based models with trait data. Our compiled data set (available in the Dryad data repository: doi:10.5061/dryad.4r55n) could be used as a direct source of Amax and SLA values for crop model parameterization, since these data are linked to specific environmental conditions (GT and TAP) and plant ontogenetic stages (i.e. time since sowing). Similarly, our regression models on trait–environment relationships  suggest that wheat and maize occupies ~215 and 120 million ha of cropland, respectively, distributed widely across the globe . Navigating the landscape of publically funded or proprietary crop data from agronomic institutions may be an avenue for expanding crop trait databases.Crop trait compilations could be expanded through data provided by agronomic institutions . AlthougSmall-scale, regional- or site-specific vulnerability assessments of crop growth and yield are gaining importance in the assessment of agroecosystem structure and function, with a growing recognition that large-scale models can be broadly informative but limited in terms of supporting management decisions at a farm scale. Advances in techniques for downscaling climate models now allow for high-resolution climate change projections at fine spatial scales. But even so, there remain systematic deficiencies in even the most computationally intensive models  to reproduce observed yields  in Jena, Germany. TRY is or has been supported by DIVERSITAS, IGBP, the Global Land Project, the UK Natural Environment Research Council (NERC) through its programme QUEST (Quantifying and Understanding the Earth System), the French Foundation for Biodiversity Research (FRB) and GIS ‘Climat, Environnement et Société’ France.A.R.M. conceived the project, compiled data, lead data analyses and prepared the manuscript. C.E.H. lead data compilation, contributed to data analysis and helped prepare the manuscript. B.E.L.C. contributed trait data. J.H.C.C. contributed trait data and advised on data analysis. J.C. contributed trait data and advised on data presentation. W.A.G. contributed to climate data analysis. J.K. contributed trait data and facilitated trait data compilation through the TRY Functional Trait Database. C.K.F.T. compiled data and assisted with data analysis. All authors discussed the results, commented on the manuscript and contributed to revisions.None declared.Supporting InformationClick here for additional data file.


The current study sought to perform a meta-analysis to compare the preoperative staging of endoscopic ultrasonography (EUS) and multidetector computed tomography (MDCT) in gastric carcinoma.Articles published between January 1, 2000, and April 1, 2016, that compared EUS with MDCT were included, and data were presented as 2 × 2 tables. The sensitivities, specificities and summary receiver operating characteristic (ROC) curves for T and N staging were calculated using a bivariate mixed effects model. Data were weighted by generic variance and then pooled by random-effects modeling.P = 0.030). For lymph node involvement, the sensitivity value for EUS (91%) was also significantly higher than that for MDCT (77%) . However, the specificity values of both EUS and MDCT were quite low, at 49 and 63%, respectively. No significant differences in T2–4 staging between EUS and MDCT were noted.Eight studies comprising 1736 patients were included in this meta-analysis. For T1 staging, the sensitivity value for EUS (82%) was significantly higher than that for MDCT (41%) (relative risk (RR): 2.06, 95% confidence interval (CI) 1.07–3.94; This meta-analysis indicates that EUS may be superior to MDCT in preoperative T1 and N staging. Additionally, the low specificity values of EUS and MDCT for N staging merits attention. On a worldwide scale, gastric cancer is the fourth most common malignancy and the third leading cause of cancer-related death . With eaRegarding the imaging for preoperative staging of gastric cancer, endoscopic ultrasonography (EUS) and multidetector computed tomography (MDCT) are the most commonly used techniques , especiaThe PubMed and Web of Science databases were searched systematically for all relevant articles published between January 1, 2000, and April 1, 2016, that compared EUS with MDCT. The following key words were used in these literature searches: “EUS” OR “endoscopic ultrasound” OR “endosonography” OR “endoscopic ultrasonography” OR “computed tomography” OR “contrast-enhanced computed tomography” OR “multidetector computed tomography” and “stomach cancer” OR “gastric cancer” OR “gastric adenocarcinoma” and “sensitivity” OR “specificity” OR “accuracy” OR “diagnostic”. The reference lists of the articles retrieved were manually reviewed to identify additional relevant references.Studies were included if they met the following criteria: (1) histologically proven gastric adenocarcinoma in more than 30 patients without any preoperative chemotherapy and/or radiation therapy; (2) histopathologic findings after gastrectomy used as the reference standard; and (3) data sufficient to construct a 2 × 2 contingency table. Studies were excluded based on the following exclusion criteria: (1) animals or ex vivo studies; (2) review articles, meta-analyses, abstracts, case reports, letters, and conference proceedings; and (3) studies published in a language other than English. The most recent or complete article was used if multiple articles described the same population.Data were extracted and summarized independently by two authors . Two adjudicating senior authors  resolved any disagreement. The following data from each included study were extracted: first author, study design, year of publication, and sample size (number of patients).Considering the lack of consensus to assess the quality of non-randomized clinical trials, studies used in this meta-analysis were selected based on data completeness and inclusion criteria.χ2 test to assess heterogeneity with the level of significance set at 10%, and the I2 statistic was used to quantify heterogeneity.A bivariate mixed effects model was performed to acquire summary estimates of sensitivity and specificity and to fit summary receiver operating characteristic (ROC) curves. We used the relative risk (RR) for the comparison of the sensitivity and specificity of the two modalities. Our studies reported results with 95% confidence intervals (CIs). We used the The fixed-effects model was used if there was no significant heterogeneity between studies; otherwise, the random-effects model was performed . We alsoAnalyses were performed using STATA SE 12.0 for Windows .A total of 5675 articles were identified using our search strategy. After screening their titles and abstracts, 5576 identical studies were excluded. The remaining 99 studies were fully reviewed. Ultimately, eight studies including 1736 cases were included for the final analysis –16, 19 , with significant between-study heterogeneity . No significant publication bias was observed (P = 0.106). The specificity values for EUS and MDCT were 89 and 97%, respectively, and this difference was not significant   was significantly higher than that for MDCT (41%) , with significant between-study heterogeneity . The specificity values for EUS (84%) and MDCT (86%) were similar   was marginally higher than that for MDCT (48%)  Fig. .Fig. 3FoP = 0.749). EUS and MDCT imaging exhibited similar specificity estimates of 87%  and MDCT 64%) was noted , with moderate between-study heterogeneity . No significant publication bias was observed (P = 0.073). Specificity was comparable between modalities, with values of 49% for EUS and 63% for MDCT   was significantly higher than that for MDCT (77%)  are the most common and valuable tools for the preoperative evaluation of gastric cancer. EUS was first introduced in clinical practice in the 1980s and exhibits high accuracy in detecting the depth of gastric cancer , 21. MorWith the increase in global aging, the proportion of gastric cancer patients older than 70 years is increasing, resulting in an increase in morbidity and mortality after curative gastrectomy. This trend has led to the need for less invasive treatment options, such as EMR and ESD, for early gastric cancer (EGC). Isomoto et al. demonstrated that 5-year overall and disease-specific survival rates after ESD reached 97.1 and 100%, respectively, indicating the excellent prognosis of ESD for EGC . HoweverFeng et al. revealed that MDCT showed higher sensitivity than EUS with regard to lymph node metastasis , whereasNotably, the pooled specificity values of EUS and MDCT were both quite low , indicating that the pooled value might not be reliable, even if EUS or MDCT suggested negative lymph node involvement. It is possible that in some cases, pathological positive lymph nodes may appear to be small without obvious enhancement. Zhao et al. also demonstrated that the rate of lymph node metastasis of EGC is quite high 25.27%), and EMR or ESD should be cautiously used in high-risk EGC patients [%, and EMThere are also some limitations in the present meta-analysis. First, the main limitation is that most of the included studies were retrospective, with the exception of three prospective studies. Second, significant between-study heterogeneity was noted in this meta-analysis. Finally, the incorporation of various classification systems may have influenced the results of this meta-analysis. However, we applied strict criteria to include high-quality studies, and the use of random-effects modeling could adequately address the heterogeneity. In addition, T staging of all editions is similar; therefore, the comparison of pooled data was not influenced. Given that N staging varies in different editions, the present meta-analysis compared only the preoperative identification of N0 versus N+ disease of EUS and MDCT to make the included studies comparable.This meta-analysis indicates that EUS may be superior to MDCT in preoperative T1 and N staging. Additionally, the low specificity of both EUS and MDCT for N staging merits further attention.


Aedes aegypti and Aedes albopictus as well as the YFV-enzootic mosquitoes Haemagogus leucocelaenus and Sabethes albiprivus from the YFV-free region of the Atlantic coast were highly susceptible to American and African YFV strains. Therefore, the risk of reemergence of urban YFV epidemics in South America is major with a virus introduced either from a forest cycle or by a traveler returning from the YFV-endemic region of Africa.Yellow fever virus (YFV) causing a deadly viral disease is transmitted by the bite of infected mosquitoes. In Brazil, YFV is restricted to a forest cycle maintained between non-human primates and forest-canopy mosquitoes, where humans can be tangentially infected. Since late 2016, a growing number of human cases have been reported in Southeastern Brazil at the gates of the most populated areas of South America, the Atlantic coast, with Rio de Janeiro state hosting nearly 16 million people. We showed that the anthropophilic mosquitoes  Flavivirus (family Flaviviridae). It is a single-stranded, positive sense RNA virus with a genome of approximately 11 kb. Until now, seven lineages have been identified: five in Africa, with two in West Africa (West Africa I and II) and three in East and Central Africa , and two in the Americas (South America I and II)3. The YFV strains circulating in the Americas derived from a Western African lineage ancestor, and most isolates from Brazil belong to the South American genotype I5. These findings are in line with the hypothesis that YFV emerged in Africa and was imported into the American East coast from West Africa during the slave trade which has undoubtedly favored the introduction of the African YF mosquito Aedes (Stegomyia) aegypti (Linnaeus)4.Yellow fever (YF) is an arboviral disease endemic to tropical regions of Africa and South America that may cause hemorrhagic fever in humans. The etiologic agent is the yellow fever virus (YFV), the prototype member of the genus 6. The 2015–2016 YFV epidemic in Angola exemplifies the threat posed by the reemergence of this virus; a six-month urban outbreak in Angola reported 4,000 suspected cases and ~600 deaths. The epidemic reached the Democratic Republic of Congo, and viremic people dispersed to densely populated and Aedes-infected zones, in and outside Africa (e.g. China)7.Despite the availability of effective vaccines, YF remains an important public health issue in Africa and South America, with an annual incidence of around 200,000 cases and 30,000 deathsAe. aegypti, it has remained an enzootic virus restricted to rainforests and savannah-like forests in the Americas since the 1940s8.Yellow fever is a zoonotic disease with two main epidemiological transmission cycles: the forest and the urban cycles. Traditionally, the YFV is transmitted within a forest cycle between non-human primates (NHPs) and canopy-breeding mosquitoes. During epizootics of YF, humans can be contaminated if they live close to the forest fringe or when entering into the wild. While YFV in Africa circulates within a forest cycle before emergence in an urban cycle involving humans and th to the early 20th century with devastating urban epidemics. However, the development of two attenuated vaccines in the 1930s as well as a continental eradication program of Ae. aegypti led to a clearance of urban YF3. Brazil was certified free of Ae. aegypti in 19579. Over the ensuing decades, human YFV infections have been acquired only in the forest cycle, despite the reinvasion of Ae. aegypti in Brazil from the late 1960s onwards10. Today, the forest YFV cycle is still very active in Brazil, and generates outbreaks every 6–10 years in the Southern, Southeast and Central-West regions, and every 14 years, in the Amazon11. Humans are contaminated by the bite of forest canopy-dwelling mosquitoes of the genera Haemagogus (primary vectors) and Sabethes (secondary vectors)4.In the Americas, the disease was a major human health issue from the 18Aedes-infested cities having low vaccination coverage14. Thus, the YFV lineage belonging to the South America genotype I (named 1D) responsible for epizootics from 1998 to 20014 has been replaced by a new viral lineage (1E) causing massive deaths of howler monkeys (Alouatta caraya) and human cases . If the re-establishment of Ae. aegypti posed a threat by itself16, the invasion and spread of Ae. albopictus since the late 1980s have stepped up the risk of urban YFV outbreaks in Brazil  and one from Senegal (S-79)). When examining AE-GOI, mosquitoes were infected and disseminate YFV from 7 days post-infection (dpi), and viral particles were only detected in mosquito saliva at 14 dpi . Therefore, all further analyses were conducted considering 14 and 21 dpi together.Interestingly, out of 447 female mosquitoes studied at 3 dpi, no mosquitoes showed infection. Therefore, specimens examined at 3 dpi were not considered in further analysis. Moreover, considering all Ae. aegypti populations from Brazil were competent vectors of YFV, three populations   Fig. , Table 2Ae. aegypti populations regardless of the YFV strain , Goiânia (AL-GOI) and Rio (AL-RIO) were experimentally infected with three YFV strains . When comparing viral infection, AL-MAN and AL-RIO presented low  for the three YFV, although for AL-MAN, dissemination tended to be higher with viral strain 1E  with 74018-1D and 50% (4) with 4408-1E) and surprisingly, was not able to transmit the S-79 YFV from Africa. The population AL-GOI collected in the epizootic/epidemic region were able to excrete similarly all three YFV (p > 0.05) but was less efficient than AL-RIO  and 38.6% (when infected with 4408-1E). Whatever the viral strain, the infection rate was not different between AE-CON and AL-CON  and DIR higher than 68.75% (S-79). TR did not differ significantly (p > 0.05), varying from 36.36% (S-79) to 50% (74018-1D) strains. Thus Ae. aegypti and Ae. albopictus from Congo, city of Brazzaville presented similar vector competence indices when infected with YFV strains belonging to both the South America I and West Africa genotypes.Viral transmission measured by TR was slightly lower except when infected with the YFV 4408-1E strain (73.33%). On the other hand, AL-CON presented roughly similar patterns than AEHg. leucocelaenus and Sa. albiprivus were as susceptible as Ae. aegypti and Ae. albopictus, mosquitoes were infected with the three YFV strains. Both enzootic mosquito species showed pattern of infection similar to Ae. aegypti while Ae. albopictus was significantly less often infected (as previously shown in this study). Overall, dissemination occurred in 64.3% of mosquitoes, this rate of dissemination was not different between the four species (p = 0.34), between the three viruses (p = 0.14). Overall, transmission was observed in 36.6% and was not different between the four species (p = 0.85) nor between the three viruses (p = 0.95). IRs were higher than 48%, DIRs higher than 44% and TRs higher than 40% suggesting a limited role of both barriers, midgut and salivary glands, in the virus migratory route inside the mosquito. When mosquitoes with infectious saliva were considered according to the initial number of females tested, transmission efficiencies (TE) varied from 10.81% to 20% for Hg. leucocelaenus and 23.33% to 31.66% for Sa. albiprivus . Thus enzootic YFV vectors from Brazil were highly competent to transmit YFV from Brazil as well as from West Africa.To examine if enzootic mosquitoes from Brazil, Ae. aegypti and Ae. albopictus from YFV-free regions in Brazil were as susceptible as their counterparts in the endemic Amazon region to transmit YFV. Mosquitoes from the epizootic/epidemic region were broadly less competent suggesting that Ae. albopictus and to a lesser extent, Ae. aegypti, were unlikely to play the role of active bridge vector in the emergence zone even if it cannot definitively be excluded.Our results showed that Ae. aegypti has been eradicated from Brazil in 19579, YFV had been maintained within a forest cycle in endemic/enzootic regions where waves of epizootics coincided with the renewal of non-human primate populations, taking an interval of 6–10 years22. Consequently to the reintroduction of Ae. aegypti and the new establishment of Ae. albopictus in the 1970–80 s, YFV distribution has extended to the Central-West of Brazil (state of Mato Grosso), the Northeast (state of Bahia), Southeast , and the Southern23. In 2008–2009, an YFV outbreak hit Rio Grande do Sul24 and São Paulo15 caused by a new lineage 1E replacing the former 1D4. We showed that Brazilian Ae. aegypti and Ae. albopictus as well as wild vectors  were equally susceptible to the two lineages ruling out differences of vector competence as a cause of viral replacement.Since the main YF-vector Hg. leucocelaenus was the most dominant species in forests close to Rio de Janeiro beside Ae. albopictus25. We showed that Hg. leucocelaneus as well as Sa. albiprivus were highly susceptible to all three YFV strains corroborating their role as YFV-enzootic vectors26. They were able to experimentally deliver infectious viral particles in saliva after infection with the two YFV strains, the former 1D and the new viral lineage 1E, as well as the West African YFV strain isolated from Senegal in 1979. Their pattern of YFV infection, dissemination and transmission are roughly similar to those of domestic Ae. aegypti and Ae. albopictus from Brazzaville in Congo acting as potential urban vectors in Central Africa27. YFV is at the gates of the most densely populated zone in South America, the Brazilian Atlantic coast, where are located large cities like Rio de Janeiro . Ae. albopictus colonizes regions surrounding the urban environment where Ae. aegypti remains the main vector of arboviruses pathogen to humans . As expected, we showed that both mosquito species were able to excrete YFV from day 1428. Populations of Ae. aegypti and Ae. albopictus from -endemic, -epizootic/epidemic, and YFV-free regions were compared for their performance to be infected, disseminate and transmit YFV. We brought out that mosquitoes from the YFV-endemic (i.e. Manaus) and YFV-free regions (i.e. Rio de Janeiro) were more capable after oral infections to expectorate the two American (74018-1D and 4408-1E) and one African (S-79) YFV strains than mosquitoes from the epizootic/epidemic area (i.e. Goiânia). These two geographically distant regions are linked by an emergence region where mosquitoes are susceptible to YFV infection. Ae. albopictus as well as Ae. aegypti from Goiânia are able to deliver the three YFV strains in their saliva after experimental infections but with much less efficiency. However, a poorly competent vector may play an important role in transmission if other conditions are met, e.g. high vector densities, high human-biting rate and high daily survival rates29. Moreover, it has been shown that human population has extensively contributed to YFV dispersal2. Some environmental/ecological barriers by inhibiting movements of vectors and hosts can prevent YFV spillovers from a forest cycle. However, these barriers became more and more anecdotal in Brazil as with population growth, cities are increasingly close to YFV-enzootic forests. Besides, because of their original habitat degradation and their high propensity to explore new environments, NHPs such as capuchins and marmosets have densely invaded parks and urban areas in Rio de Janeiro and other cities in the Atlantic coast. These new urban invaders colonize a large area around cities including patches of forests31 where sylvatic YFV vectors can be abundant32. Considering all these conditions, it is difficult to understand why YF is not already in the urban areas of Brazil. However, it seems that this is changing with the recent detection of human cases at less than that 100 km apart from city of Rio de Janeiro .Enzootic mosquitoes 32. Most cases were found in cities suggesting that transmission implicates urban vectors, mainly Ae. aegypti. Imported cases from Angola were later confirmed in China33 stressing the risk of spread outside Africa in Aedes-infested countries through non-immunized travelers. All four mosquito species, Ae. aegypti, Ae. albopictus, Hg. leucocelaenus and Sa. albiprivus from Rio de Janeiro were highly susceptible to Brazilian as well as West African YFV lineages. The city of Rio de Janeiro is an important touristic and trade center hosting nearly 6.5 million inhabitants, and where annually converge 26 millions of Brazilians/tourists in airports and 19 million via roads (http://cidades.ibge.gov.br/xtras/perfil.php?codmun=330455). If the virus is introduced from an infected traveler into Rio de Janeiro, opportunities to initiate a vectorial transmission of YFV are multiple: (i) by anthropophilic mosquitoes such as Ae. aegypti and Ae. albopictus which are highly susceptible to YFV and (ii) by YFV-enzootic mosquitoes Hg. leucocelaenus and Sa. albiprivus colonizing the forest near Rio de Janeiro. Therefore, vaccination of travelers visiting Rio de Janeiro should be highly advised to limit the risk of introducing the virus from YFV-endemic areas.The emergence in Angola in January 2016 exemplifies the threat of YFV spreading outside its historical cradle. Starting from Luanda where YFV caused nearly 600 deaths, it reached neighboring countries, the Democratic Republic of Congo (DRC) in March 2016 and Uganda in April 2016An effective YFV vaccine has been available since the 1930s. Unfortunately, incomplete coverage in regions at risk of infection is responsible for several thousands of deaths every year. In the Americas, to prevent enzootic spillovers with introduction of YFV into the urban cycle, people in contact with the jungle should be rapidly vaccinated in priority to prevent a potential urbanization of YFV.The Institut Pasteur animal facility has received accreditation from the French Ministry of Agriculture to perform experiments on live animals in compliance with the French and European regulations on care and protection of laboratory animals. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the Institut Pasteur and by the Institutional Ethics Committee on Animal Use (CEUA-IOC license LW-34/14) at the Instituto Oswaldo Cruz. Mosquito collections in the Atlantic forest in Rio de Janeiro were approved by local environmental authorities . No specific permits were required for performing mosquito collection in the urban and suburban areas in Brazil and Congo. This study did not involve endangered or protected species.Ae. albopictus, four Ae. aegypti, one Haemagogus leucocelaenus (Dyar & Shannon) and one Sabethes albiprivus Theobald . Similarly, four species  collected in the Rio de Janeiro area were tested  of Ae. aegypti and Ae. albopictus was used for experimental infections. For Hg. leucocelaenus which cannot be maintained in laboratory conditions35, the F0 generation derived from eggs collected fortnightly in 2015 was directly used for infections. In the case of Sa. albiprivus, mosquitoes from a colony established in the laboratory since 2013 were used.Ten American and African mosquito populations originated from three contrasting regions  were challenged with YFV: four ald Fig. , Table 4ed Table . PopulatHg. leucocelaenus, we added senesced leaves besides yeast powder in water. We reared ~100 larvae/pan for Ae. albopictus and Ae. aegypti, and ~50 larvae/pan for Hg. leucocelaenus and Sa. albiprivus. The emerged F0 adults were morphologically identified36 maintained in insectaries  and supplied with both 30% sucrose and/or honey solutions. Ae. albopictus, Ae. aegypti, and Sa. albiprivus females were fed three times a week on anesthetized mice to produce eggs.Ovitraps were provided with 1 to 3 wooden paddles. Eggs were hatched by submerging paddles in dechlorinated tap water for two consecutive days. Larvae were reared in pans (25 × 25 × 10 cm) containing one liter of dechlorinated tap water, supplemented with yeast powder renewed every 2–3 days. In the case of 37, and IEC-4408 (YFV-4408), isolated from a howler-monkey in 2008, from the lineage 1E (GenBank KY861728), and one strain [S79-P4 (YFV-S79)] from the West African lineage, isolated from a human case in Senegal in 197938. YFV-74018 and YFV-4408 were isolated from serum in Ae. albopictus C6/36 cell culture, and passaged four times on the same cell line, while YFV-S79 was passaged twice on newborn mice and two times on C6/36 Ae. albopictus cells. Viral stocks of all strains were produced on C6/36 Ae. albopictus cells and stored at −80 °C until used for the mosquito experimental infection assays.Mosquitoes were challenged with three YFV isolates: two belonging to the South America genotype I, isolated from Brazil, corresponding to two distinct lineages FIOCRUZ 74018/MG/01 (YFV-74018), isolated from a human fatal case in 2001, belonging to the lineage 1DSa. albiprivus which were starved for 48 h. Females were fed on an infectious blood meal containing two parts of washed rabbit erythrocytes, one part of the viral suspension supplemented with a phagostimulant (ATP) at a final concentration of 5 mM maintained at 37 °C. The infectious blood-meals contained a final viral titer of 106 PFU/mL. Mosquito feeding period was limited to 1 hour. Only fully engorged females were incubated at 28 °C constant temperature, 80% RH and 16 h:8 h light:dark cycle, with daily access to 10% sucrose or honey solution. For each combination mosquito population-YFV strain, samples of 20 mosquitoes were examined at 3, 7, 14 and 21 days after virus exposure for determining vector competence indices. Infection rate (IR) refers to the proportion of mosquitoes with infected body among the engorged ones. Disseminated infection rate (DIR) corresponds to the proportion of mosquitoes with infected head among the previously detected infected mosquitoes . Transmission rate (TR) represents the proportion of mosquitoes with infectious saliva among mosquitoes with disseminated infection39. Even if this is an indication, TR should not be regarded as a measure of transmission from mosquitoes to a vertebrate host.Six- to 8-day-old females were grouped in feeding boxes  and starved for 24 h, except for Ae. albopictus C6/36 cell culture in 96-well plates. After 1 h incubation at 28 °C, 150 μL of 2.4% CMC (carboxymethyl cellulose) in Leibovitz L15 medium supplemented with 10% FBS was added per well. After 5 days incubation at 28 °C, cells were fixed with 10% formaldehyde, washed, and revealed using hyperimmune ascetic fluid specific to YFV as the primary antibody and Alexa Fluor 488 goat anti-mouse IgG as the second antibody (Life Technologies)28. To estimate viral transmission, mosquito saliva was collected in individual pipette tips containing 5 μL FBS for 30 min as previously described40. FBS containing mosquito saliva was expelled into 45 μL of Leibovitz L15 medium, inoculated on Ae. albopictus C6/36 cell culture in 96-well plates and stained as described above.Mosquitoes were processed as follows: abdomen and thorax (herein after referred to as body) were tested for determining infection, head for dissemination and saliva for transmission. To determine viral infection and dissemination rates, each mosquito body and head were respectively ground in 500 μL and 300 μL of Leibovitz L15 medium (Invitrogen) supplemented with 2% fetal bovine serum (FBS), centrifuged at 10,000 × g for 5 min at +4 °C and inoculated onto monolayers of Rates  were described using median and inter-quartile range (IQR). The effect of species, population, YFV strain and duration on the rates was investigated using logistic linear regression models. Two-by-two interaction between species, population, YFV strain and duration was systematically investigated. Statistical analyses were conducted using the Stata software . P-values < 0.05 were considered significant.Supplementary information


Piriformospora indica facilitating growth/development and stress tolerance has been demonstrated in various host plants. However, global metabolomic studies are rare. By using high-throughput gas-chromatography-based mass spectrometry, 549 metabolites of 1,126 total compounds observed were identified in colonized and uncolonized Chinese cabbage roots, and hyphae of P. indica. The analyses demonstrate that the host metabolomic compounds and metabolite pathways are globally reprogrammed after symbiosis with P. indica. Especially, γ-amino butyrate (GABA), oxylipin-family compounds, poly-saturated fatty acids, and auxin and its intermediates were highly induced and de novo synthesized in colonized roots. Conversely, nicotinic acid (niacin) and dimethylallylpyrophosphate were strongly decreased. In vivo assays with exogenously applied compounds confirmed that GABA primes plant immunity toward pathogen attack and enhances high salinity and temperature tolerance. Moreover, generation of reactive oxygen/nitrogen species stimulated by nicotinic acid is repressed by P. indica, and causes the feasibility of symbiotic interaction. This global metabolomic analysis and the identification of symbiosis-specific metabolites may help to understand how P. indica confers benefits to the host plant.Root colonization by endophytic fungus  Piriformospora indica of the Sebacinales, Basidiomycota, can colonize a broad spectrum of plants1. The mutualistic symbiosis further promotes plant growth/biomass, and confer resistance/tolerance against biotic and abiotic stress3. Due to its axenically cultivability, P. indica has been extensively used for studying the mechanisms and evolution of mutualistic symbiosis4. Finally, being able to promote growth significantly and deliver biotic/abiotic stress acclimation ability to its host, P. indica has great potential for biotechnological applications and future research for bio-agricultural engineering5.P. indica represents a missing link between a saprophytic fungus and an obligate biotrophic mutualist6. It holds genes for a biotrophic lifestyle, and lacks genes for nitrogen metabolism, but also matches characteristics of symbiotic fungi and obligate biotrophic pathogens6. The symbiotic interaction ultimately leads to growth promotion, increased biomass production, stimulation of the uptake of phosphate and other limiting ions, and enhanced tolerance to biotic and abiotic stresses, as known from mycorrhizal fungi8. Genetic and biochemical analyses revealed possible signal transduction processes induced by P. indica in the root cells of host plants11. Moreover, Stein et al.12 has shown that P. indica-induced resistance against powdery mildew in Arabidopsis host involves jasmonic acid (JA), but not salicylic acid (SA). Changes in plant abscisic acid (ABA), gibberellic acid (GA), JA, and SA levels affected root colonization in barley, and changes in the GA biosynthetic pathway suppressed P. indica-mediated defense responses13. Furthermore, sustained exposure to ABA enhances the colonization potential of P. indica on Arabidopsis roots14. Once inside the roots, the fungus benefits from the plant’s photoassimilates, which further promotes colonization and proliferation in particular environmental stress conditions16. As in mycorrhiza, the fungus reprograms the plant transcriptome and proteome17. The P. indica-responsive genes provide a powerful toolbox to elucidate signaling and metabolic pathways involved in establishing the symbiosis and promoting plant performance.The decoding of the fungal genome revealed that P. indica with Chinese cabbage roots7. Besides an overall stimulation of root and shoot growth by the endophyte, the substantial increment of lateral root development results in a bushy root phenotype. Such a strong stimulation of lateral root development has not yet been described for another plant species colonized by P. indica. This has prompted us to investigate the symbiotic interaction of P. indica with Chinese cabbage roots in more details, since the strong effect of the fungus on root growth and proliferation might be important for agricultural applications. We have demonstrated that the observed phenotype is associated with a strong, transient increase of the auxin level in the roots, which results in upregulation of Chinese cabbage genes correlated with growth- and auxin signaling-related functions, such as genes for P-type/V-type H+-ATPases and nutrient/ion transporters, root hair-forming phosphoinositide phosphatase 4 (RHD4), cell wall loosening/synthesizing proteins involved in cell wall growth, and auxin transportation/responses2. Some of the genes induced during the biotrophic phase of P. indica colonization harmonize the auxin level with the degree of root colonization. In-depth microscopic analyses of the root structure after successful symbiosis showed that the fungus stimulates primarily growth and development of the root maturation zone7.In the past decade, we studied the beneficial interaction of A. thaliana with P. indica19. Recently, Strehmel et al.20 investigated the metabolic response of A. thaliana to P. indica under hydroponic condition. Results showed that the profiles of primary and secondary metabolites were obviously changed in colonized roots, while far less significant change in leaves. The increased concentrations were found in organic acids, carbohydrate, ascorbate, glucosinates and hydroxycinnamic acids, and a decreased concentration found in nitrogen-rich amino acids. This important finding has contributed to validate the symbiotic mechanism between plant host and P. indica.While relatively much is known about the transcriptomic and proteomic changes in roots in response to colonization by beneficial fungi, systematic analyses of metabolomic profiles are still rare. Thus far, the induction of salicylic acid catabolites and jasmonate as well as glucosinate metabolism have been demonstrated correlating with the colonization of P. indica, we performed a comprehensive metabolomic analysis, in which the profiles of P. indica-colonized and uncolonized Chinese cabbage roots, and the profile of P. indica hyphae were comparatively surveyed. The data allow the identification of symbiosis-specific metabolites in P. indica-colonized Chinese cabbage roots, such as GABA and IAA derivatives. The results are helpful for validating the beneficial function triggered by P. indica-colonization. Most importantly, the mechanisms that growth promotion and abiotic/biotic stress tolerance are simultaneously enhanced by P. indica could be demonstrated based on the metabolomic data.Following our previous study, in order to further understand the mechanisms of stress tolerance and growth promotion in Chinese cabbage caused by colonization of P. indica colonization, three metabolomic libraries were generated: two from Chinese cabbage root extracts with and without P. indica inoculation, and one from a hyphal extract of P. indica. In all, 549 of a grand total 1,126 metabolites were identified from the GC-MS analysis. Of the 549 metabolites annotation uncovered, 298 compounds in preparations from P. indica-colonized roots, 229 compounds from uncolonized roots and 169 compounds from P. indica hyphae  were three to five times higher (mass per gram of dry weight) for s7 Table , such asose Fig.  through 21. It has been proposed to function as an osmo- and redox regulator and a plant-signaling molecule23. From the quantitative analysis, the GABA level was much higher in P. indica-colonized roots compared to the uncolonized controls, suggesting an important role in symbiosis  was present in P. indica-colonized roots, but below detectability in uncolonized roots and P. indica hyphae  to promote stress tolerance.It has been well known that plants produce various types of oxygenated fatty acids, termed “oxylipins”, in responses to microbial infection or biotic/abiotic stresses. Usually, α-linolenic acid and arachidonic acid (C18:2 and C18:3) were the major precursors oxidized to 2-hydroperoxy by lipoxygenaseP. indica reduced the niacin level in Chinese cabbage roots to half the amount of uncolonized roots  to the leaves, the seedlings displayed more tolerance to 150 mM NaCl and 72 h after spraying, their performance under high temperature stress (35 °C) was clearly better compared to the water-treated controls , the black rot disease that affects Brassicacae worldwide, they performed better than the control seedlings  on Chinese cabbage seedlings for 0.5 or 1 hour caused higher Hion Fig. . These rP. indica to optimize root colonization and plant performance. As shown in Fig. P. indica belong to the primary and secondary metabolisms, defense compounds, and signaling compounds. Their regulation by P. indica is consistent with the beneficial effect of the fungus on the performance of Chinese cabbage. In a previous study with root tissue of A. thaliana, Strehmel et al.20 demonstrated that nearly all metabolite levels increased upon co-cultivation in root extracts, except those in leaves. These included carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. Our current findings with Chinese cabbage resemble those from A. thaliana. Generally, the primary metabolism is activated, and the levels of metabolites in primary metabolism are increased. This indicates that P. indica can perform largely similar symbiotic function with Chinese cabbage and A. thaliana. Although some cases of different level, such as for pyruvate and niacin, were observed in Chinese cabbage when compared to A. thaliana. This might be caused by species-specific differences, but also by the fact that the response of Chinese cabbage to P. indica colonization seems to be stronger than the responses described for A. thaliana. Overall, the metabolomic profiles demonstrate that P. indica stimulates the energy status of the roots, trigger the accumulation of metabolites, which promote growth, and they are consistent with developmental processes and phenotypic changes.In this paper, we identified plant host metabolites, which are stimulated or repressed by 30, the higher the pyruvic and malic acid levels it could likely activate the host’s TCA cycle for energy generation required for root growth and consumption by the fungus. Furthermore, the fungus could provide additional pyruvate and malic acid to the roots via lactic and acetic acids, which accumulate during previously described fungal-growth in the maturation zone7. Pyruvaldehyde, which accumulates in P. indica-colonized roots, is a cytotoxic side-product of glycolysis and is synthesized from the pyruvate metabolism. It is detoxified to S-lactoylglutathione by lactoylglutathione lyase, which is induced for oxidative stress tolerance in plants32. Moreover, lactoylglutathione lyase is identified in our previous EST library to be induced by P. indica after colonization of Chinese cabbage roots. Therefore, P. indica may stimulate the accumulation of these metabolites in this symbiosis to support the enormous growth requirement and stress adaptation.We have demonstrated an elevation of metabolites involved in the pyruvate metabolism and detected higher levels of pyruvate, pyruvaldehyde and malate of the TCA cycle in colonized roots Figs  and 6. SOryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa33. The P. indica - derived IAM  has led to the biosynthesis of auxin and the activation of auxin-responsive genes in IAM Fig.  that we 36. We observed different regulation of aromatic acids and defense-related precursors: while some of them are up-regulated , others are down-regulated (in particular tryptophan). Within the plant kingdom, the phenylalanine metabolism along amino acid metabolisms are important for the synthesis of phenol derivatives such as coumarin, monolignal, lignin, spermidin, flavonoid and tannin. Genes involved in plant phenolic secondary metabolism such as phenylammonia-lyase37, which is frequently used as case studies of plant defense evolution38, has been identified in our EST library7  to alanine and succinic semialdehyde (SSA)43. SSA is toxic for plants and there are two known pathways for SSA metabolisms: oxidization to succinate via SSA dehydrogenase (SSADH) or reduction to γ-hydroxybutyrate by glyoxylate reductase44. SSADH is located in mitochondria46, and appears to be the main enzyme for SSA removal since mutants for this enzyme suggest its significant role in heat stress adaptation and development22.GABA has been reported as a non-proteinous amino acid functioning as an important neurotransmitter in animal systemse40 Fig. . The pro48. The perception mechanism of BABA has been identified in Arabidopsis. Recently, BABA was detected in Arabidopsis and several plants including B. rapa, Z. mays, etc.,. It is very clear that BABA was naturally present in planta, and having identical function with its isomers: AABA (α-aminobutyrate) and GABA (γ-aminobutyrate)49. In agreement with previous studies, our treatment of BABA confirmed that the homologous priming component, GABA induced by P. indica, could activate a PAMP-triggered immunity (PTI) response against pathogen infections , a GABA homolog, was shown to induce resistance against root- and soil-borne pathogens in tomato and cotton, as GAGAons Fig. .A. thaliana mutants fin4-1 and fin4-3 which lack de novo niacin biosynthesis, has lower ROS levels after fungal infection and does not triggered PTI or other immune responses50. Our results show that niacin-treated Chinese cabbage produces higher levels of nitric oxide and H2O2  attack  were induced while others were not affected or decreased54. The reduced dimethylallyl-PP level detected in Chinese cabbage could also lead to lower cytokinin levels which ultimately establish a cytokinin-to-auxin ratio favorable for root growth.Niacin or nicotinic acid is involved in the ROS burst in response to fungal infection. 2O2 Fig. . The lowica Fig. . Moreoveica Fig. . This meack Fig. . Previou57. Consequently, mutants deficient in lipid unsaturation show enhanced thermal tolerance59. Oleic acid and arachidonic acid were shown to act as signaling molecules that modulate plant stress responses61. We have observed that P. indica colonization increase the production of saturated fatty acids and decrease the production of unsaturated fatty acids in B. campestris. In our study, the increased level of polysaturated fatty acids and the decreased polyunsaturated fatty acids observed in P. indica-colonized roots suggested that the cell membrane fluidity is higher and innate immunity is lower. When plants are challenged by pathogens, lipase are activated to release unsaturated fatty acids which are subsequently transformed to oxylipins with diverse functions against microbial and insect attacks25. JA and its precursors, oxo-phytodienoic acids are regulating defense mechanism and balance growth and defense responses26. Although the oxylipin levels were not separately analysed in our work, the JA level is higher in P. indica-colonized roots  were donated by Ming-Hong Seed Company, Taichung City, Taiwan. Seeds were sterilized with 75% alcohol for 10 min, then placed on a petri dish containing ½ MS nutrient medium65. Plates were incubated at 22 °C under continuous illumination (100 μmol m−2 s−1) for seed germination.Seeds of Chinese cabbage (−2 s−1). P. indica was cultured as described previously67 on Kaefer medium. Seedlings were removed from the plates 7 days after co-cultivation and the roots were used for the extraction of secondary metabolites and GC-MS analyses. The results are presented on the basis of root fresh weights. Alternatively, the seedlings were transferred to pots and grown in a walk-in growth chamber, as described previously11.Seven days after seed plating on ½ MS medium, the growing seedlings were transferred to fresh plates containing ½ MS medium. One to six seedlings were used per petri dish and one fungal plague or one agar plague without fungus of 5 mm in diameter per seedling was placed at a distance of 1 cm from the roots. The colonized seedlings along with uncolonized ones were incubated at 22 °C under continuous illumination (50–55 μmol mt-butyldimethylsilyl) trifluoroacetamide] and the extraction reagents were ethylacetate, acetone and distilled water.All chemical reagents were of analytical grade. The silylation reagents were MTBSTFA  were immediately frozen in liquid nitrogen after removed from the whole plant. The frozen samples were then incubated overnight (16 h) in a Labconco FreeZone® Freeze Dry System which was pre-chilled with liquid nitrogen.Roots from seven-day-old Chinese cabbage  and water. 500 ml EA and 50 ml of milli-Q water were added to each sample in the dark. The suspensions were sonicated for 5 minutes before churning in order to dissolve the secondary metabolites in the heterogeneous mixture of EA and water. The two-phase suspensions of the three preparations were incubated overnight in the dark phases to obtain optimal phase separation, and the phases were then separated with separation funnels in the dark. The EA phases were used for further analyses and the solvent was removed in the dark with a rotary evaporator. The partitioning was carried out three times and the water fractions  were discarded. Finally, the weights of the dried EA extracts were determined.69. Dry materials of the three samples Hyphae, Uncolonized root, and Colonized root were re-suspended in GC-MS grade methanol (200 ng/μl). Silylation was performed using MTBSTFA under 80 °C for 2 h. The separation conditions on an Rtx-5MS column (30 m × 0.25 mm × 0.25 μm) for GC-MS analysis was set as follows: column flow, 1 ml/min helium; injector temperature, 250 °C; GC temperature program: 40 °C for 1 min, increase to 300 °C in 10 °C/min changes, 300 °C for 8 min; solvent delay, 470 sec; transfer line temperature, 280 °C; pegasus acquisition rate, 10 spectra/sec; mass range saved: m/z 50–600; ion source temperature: 200 °C. GC-MS was performed using the Pegasus® 4D GCxGC-TOFMS from Leco.The analysis was performed as described by Gobel and Feussner® Software from Leco, by setting the threshold for up to 650 hits on m/z similarity and compared with the metabolites present in libraries of the software. Compounds with peak-areas below 700 were undetectable and compounds with less than 650 hits for m/z identification were removed. The identified compounds without silylation were individually analyzed; annotated and synthetic compounds were removed. Their abundance was calculated and quantified relatively to the internal control nonadecanoic acid-trimethylsilyl ester, which was added before the extraction and partitioning procedures and GC-MS analyses. Undetected peaks of any given compound from any given sample preparations: Hyphae, Uncolonized Root, or Colonized Root were calculated using threshold value of 700 for peak area. Moreover, the identified compounds were mapped to metabolomic pathways using the KEGG online software.Three sets of data were collected from three independent biological repetitions; and analyzed using the program ChromaTOFFisher’s Exact test. The MetPA software annotated compounds of P. indica-colonized and uncolonized roots, as well as those of P. indica hyphae were matched to A. thaliana and yeast databanks. Additionally, MetPA software contains a series of programs with matches for the metabolic pathways found in their databases, and assigns the compounds to these pathways with their p-values. Figures and tables were generated from the MetPA analysis.The metabolites identified from the GC-MS were analyzed with the MetPA online software to position them into global metabolic pathways. The analytical settings for MetPA were used for 65. Five days after germination, they were transferred to ½ MS as well as ½ MS containing 100 μM and 200 μM filter-sterilized (45 μM filter) BABA. All six seedlings from one set and each treatment were recorded for stem and root length in centimeters, while two seedlings from each set of treatments were taken for phenotypic analysis by taking photograph of them aligned with each other.One set of Chinese cabbage seeds were sterilized as previously mentioned above, each set containing eighteen, and then were sown unto petri dish containing 1/2 MS nutrient medium65. Five days after germination, they were transferred unto ½ MS as well as ½ MS containing 10 mM and 20 mM filter-sterilized (45 μM filter) niacin ; one set for 30 minutes and the other set for 1 hour respectively. Each set was used for NO quantification by following protocols using diaminofluorescein (DAF-2DA)70 staining and H2O2 by following procedures using titanium oxalate.Two more sets of Chinese cabbage seeds were sterilized, each set containing six, and then were sown unto petri dish containing ½ MS nutrient mediumTwenty-seven Chinese cabbage seeds were sterilized as mentioned above and sown into twenty-seven 6-inch pots containing autoclaved soil/vermiculite mixture. After fourteen days, they were grown into Chinese cabbage plantlets and were used to perform thermal, high-salinity and biotic stress tolerance experiments, as described below in the following procedure.Two sets of Chinese cabbages, each set containing nine 21- DAG plants, were used for the thermal and biotic stress analysis. One set was subjected under a 72 hours thermal stress (35 °C) with three plants treated with 100 μM BABA and another three with 200 μM BABA by spraying each plant with 10 mL of respective concentration unto their leaves. The last three plants were sprayed with 10mL-distilled water, since the solvent vehicle for BABA was distilled water. After 24, 48, and 72 hours, nine of the plants were taken for phenotypic changes observation and a photograph was taken.The second set of Chinese cabbages were used for a 24 hours high-salinity stress analysis. Exogenous application of BABA was identical to what was mentioned above. However, 150 mM of dissolved NaCl was used for the high salinity treatment for all nine plants. After 24 hours, phenotypic changes were recorded by taking photographs of the plants.Xanthomonas campestris pv campestris (Xcc) was used as the pathogen to induce infection in Chinese cabbage. The third leaf of the whole plant from each set was firstly pierced with a hypodermic needle. Next, they were infiltrated with O.D. 0.2 or 108 c.f.u. amount of Xcc through the piercings. After 72 hours (which is the tested time for 0.2 O.D. amount of Xcc to infect Chinese cabbage with symptoms of rotting on leaf), the leaves from each treatment: water, 100 μM and 200 μM BABA were cut and photographed.The final set of Chinese cabbages was used for a 72 hours biotic stress analysis. Exogenous application of BABA was identical to what was mentioned above. Here, Supplementary Information


P=0.06). Univariate analysis showed that therapy failure directly correlated with functional heartburn diagnosis (OR=4.60) and suggested a trend toward a negative correlation with smoking and a positive one with nausea. However, at multivariate analysis, these parameters were not significant. Functional heartburn experienced a lower median percent VAS reduction than acid reflux  even if equal to non-acid reflux (66.6%). In conclusion, a tailored approach to refractory NERD, guided by MII-pH monitoring, demonstrated to be effective and should be promising to cure symptom persistence after conventional therapy failure. Nevertheless, standardized guidelines are advisable.A relevant percentage of non-erosive reflux disease (NERD) is refractory to proton pump inhibitors (PPIs) treatment. Multichannel intraluminal impedance pH (MII-pH) monitoring should give useful pathophysiological information about refractoriness. Therefore, our aim was to assess whether this technique could be useful to guide a 'tailored' therapy in refractory NERD. We retrospectively recruited NERD patients undergoing MII-pH monitoring for unsuccessful treatment. All patients had undergone upper endoscopy, and those with erosive esophagitis were excluded. No patient received PPI during MII-pH monitoring. Subjects were subgrouped into three categories: acid reflux, non-acid reflux and functional heartburn. MII-pH-guided therapy was performed for 4 weeks as follows: patients with acid reflux received PPI at double dose, patients with non-acid reflux PPI at full dose plus alginate four times a day and patients with functional heartburn levosulpiride 75 mg per day. A visual analog scale (VAS) ranging from 0 to 100 mm was administered before and after such tailored therapy to evaluate overall symptoms. Responders were defined by VAS improvement of at least 40%. Sixty-nine patients with refractory NERD were selected . Overall effectiveness of tailored therapy was 84% without statistical difference among subgroups (88.5% acid reflux, 92% non-acid reflux, 66.6% functional heartburn;  Community-based European studies found a NERD prevalence of up to 70%.5About the 40% of the US population is affected by GERD-related symptoms once a month and 20% once a week.6 However, some authors believe that lack of clinical response after a 8-week course of PPI may be sufficient to define refractoriness for NERD.8 Refractory NERD is often a therapeutic challenge. Indeed, in a systematic review, pooled rate of symptomatic response to PPI was 36.7%  in NERD and 55.5%  in erosive esophagitis.9 Several mechanisms may be invoked to explain refractoriness: (i) presence of non-acid/weakly acid reflux, (ii) low tone of lower esophageal sphincter, (iii) poor esophageal clearance, (iv) existence of an acid pocket located close to the gastroesophageal junction, (v) functional heartburn and (vi) poor compliance to the therapy or inadequate optimization of the treatment.6The treatment of NERD consists mainly in modifications of dietary and lifestyle habits and proton pump inhibitor (PPI) assumption. Despite these cautions, there is no improvement of the symptoms in a relevant percentage of patients (refractory NERD). In particular, refractoriness is defined by the persistence of typical symptoms despite receiving a full dose of PPI for at least 12 weeks.11 In the setting of refractory NERD, MII-pH monitoring could be a useful device to understand the reasons of therapy failure, thus addressing a 'tailored' treatment.13 However, few reports have systematically applied MII-pH monitoring to optimize refractory NERD treatment.13Multichannel intraluminal impedance pH (MII-pH) monitoring has been proposed as a tool to improve the management of NERD. Indeed, compared with traditional pH monitoring, it allows the identification of both acid and non-acid reflux episodes, thus leading to differentiate NERD subtypes (i.e. acid and non-acid) and functional heartburn.In the present study, we retrospectively investigated whether a MII-pH-guided 'tailored strategy' could have been beneficial in patients with refractory NERD.P=0.06). This borderline trend for functional heartburn was confirmed in direct comparisons against acid reflux (P=0.13) and non-acid reflux (P=0.06), as graphically shown in Seventy-two patients were eligible according to inclusion criteria; 69 of them accepted to participate to the study. Enrolled patients showed a female–male ratio of 43 : 26 and a mean age of 47.6±15.2 years). Twenty-six had acid reflux, 25 non-acid reflux and 18 functional heartburn. Tailored therapy was effective in 58 out of 69 patients, with a success rate of 84.0% (95% CI: 75.4–92.6). The effectiveness of tailored therapy for acid reflux, non-acid reflux and functional heartburn was, respectively, 88.5% (95% CI: 76.2–100), 92.0% (95% CI: 81.4–100) and 66.6% (95% CI: 44.8–88.4), with a nonsignificant trend (P<0.0001). In patients with acid reflux, VAS score dropped from median 80 mm and interquartile range 20 mm to median 20 mm and interquartile range 30 mm after the therapy (P<0.0001). Similarly, patients with non-acid reflux experienced a decrease in VAS value from median 80 mm and interquartile range 25 mm to median 20 mm and interquartile range 30 mm after the treatment (P<0.0001). Subjects with functional heartburn reported a statistically significant drop (P=0.0002) in VAS scale from median 80 mm and interquartile range 20 mm to median 40 mm and interquartile range 40 mm. These variations in VAS scale before and after the treatment are graphically represented in In our overall cohort, median baseline visual analog scale (VAS) score was 80 mm and interquartile range was 20 mm, whereas, after the treatment, the median was 30 mm and interquartile range was 30 mm, with a significant reduction (P<0.05), even if not different from non-acid reflux , as reported in Therefore, we found a median VAS reduction (delta) of 50 mm and interquartile range of 20 mm. The delta VAS was lower in functional heartburn  compared with acid reflux , but similar to non-acid reflux , as shown in We found an overall median percent reduction of VAS score of 62.5% and an interquartile range of 35.7%. The percent reduction of VAS score of functional heartburn  was lower than that of acid reflux . Smoking habits suggested a trend toward a negative correlation with therapy failure at univariate analysis. .When responders were compared to non-responders . Similar to smoking, at univariate analysis, we found a trend to correlate directly with therapy failure .Nausea was more common among non-responders . Patients with functional heartburn diagnosis showed a significant correlation with the risk of therapy failure , whereas the other MII-pH monitoring categories (acid and non-acid reflux) were not associated with unsuccessful treatment.The diagnosis of functional heartburn was more common in non-responders than in responders . Nausea and diagnosis of functional heartburn were not associated with failure, although a trend was evident both for nausea  and diagnosis of functional heartburn .At multivariate analysis , smoking14 Therefore, a systematic approach to refractory NERD is a therapeutic challenge of great interest. Up to now, MII-pH monitoring has been mainly applied to elucidate some pathophysiological aspects15 even if the investigation-related information may be useful for a tailored therapeutic approach.NERD is a frequent finding in clinical practice; however, refractoriness to PPI treatment is quite common. There is a clear evidence that the response rate to PPI administration is lower in subjects with NERD than in patients with erosive esophagitis.et al.13 performed MII-pH monitoring on 39 subjects with refractory NERD and the test allowed to classify such patients into three main groups . They reported an overall success rate of tailored therapy of 64.1%, with an effectiveness of 83.3% for acid reflux, 50% for non-acid reflux and 66.6% for functional heartburn, thus suggesting the usefulness of the test to guide the treatment.Xiao 13 even if our therapeutic approach was different with regard to functional heartburn (levosulpiride instead of paroxetine) and non-acid reflux .In the present study, we used the same above-mentioned MII-pH-based classification of NERD16 On the other hand, levosulpiride exerts effects on both psychological level and regulation of gastric and esophageal motility. It has been already tested on functional heartburn and has demonstrated an efficacy superior to metoclopramide and domperidone for this symptom relief as well as for postprandial bloating and epigastric pain improvement.18 Additionally, Lozano et al.19 showed that a 15-day course of levosulpiride reduced the symptom score for functional heartburn by 50%, and the prolongation of therapy to 30 days led to symptom disappearance. Similarly, Mearin et al.20 demonstrated that this drug reduced the symptom score of 79.9% in a group of patients with dismotility-like functional heartburn. In the present experience, we found that this strategy was effective in two-thirds of patients, a value that is comparable with similar investigations. Despite the success rate was not inferior to the other NERD categories, we showed that the median improvement of VAS score was statistically lower than that of acid reflux group. This finding could be related to two main aspects: gastrointestinal functional disorders are frequently hard to treat and therapeutic response is subjective being affected by psychosomatic issues.24 This fact was confirmed by the univariate analysis, where diagnosis of functional heartburn was correlated to a high risk of therapy failure. Nevertheless, we believe that a 66.6% rate of response in patients with previous diagnosis of refractoriness is still a relevant gain.In functional heartburn, our choice was because of some concerns. We did not take into account the administration of an antidepressant drug to a group of patients with a benign and non-disabling disorder for possible side effects. Furthermore, similar to many other antidepressant agents, the effect of paroxetine is gradual and generally it takes some weeks to reveal its effectiveness.13 A study investigating MII-pH monitoring for tailored therapy showed that 90.9% of patients with acid reflux improved after PPI dose doubling.25 Our experience confirmed such results, demonstrating a success rate of 88.5% using PPI double dose.In refractory NERD patients with acid overexposure, PPI dose escalation seems to be the most effective strategy.26 For this reason, alginates, which create a mechanical barrier hampering the reflux of gastric content, may be useful for this purpose. In an Italian open-label study on refractory NERD, alginates were effective in controlling heartburn and reducing VAS score, despite that they did not decrease the number of non-acid reflux episodes.27 Similarly, several other studies demonstrated a good profile of alginates in reducing the burden of NERD symptoms.29 Indeed, alginates have been demonstrated to neutralize also non-acid secretions such as pepsin and bile salts.30 Therefore, a simple inhibition of acid secretion by PPI, as proposed by Xiao et al.13 could not be appropriate in patients with non-acid reflux and this may be an explanation for their low success rate (50%). Conversely, herein we demonstrated that alginates plus a full dose of PPI are able to achieve a high response in non-acid reflux (92.0%).The pathophysiological mechanism of non-acid-reflux is an exaggerated relaxation of lower esophageal sphincter, with increased exposure to gastric content.24 Moreover, nausea showed a trend to directly correlate with therapy failure even if not statistically significant. The controversial relationship between smoking habit and refractory NERD stimulates some considerations. Several previous evidences have already stated that smoking is a risk factor for NERD33 and that its cessation leads to clinical improvement.34 However, in our cohort, we found that smokers seemed to have a higher chance of response to tailored therapy, and this observation is supported by some previous studies that have highlighted that non-smokers are at high risk of non-response to PPI therapy.36 Finally, our multivariate analysis failed to find factors associated with tailored therapy response/failure.At univariate analysis, a functional heartburn diagnosis was associated with tailored therapy failure, and this could be an expected finding, as we have already remarked.38 In conclusion, the present report was stimulated by the low number of studies focusing on therapy optimization for refractory NERD according to MII-pH monitoring.25 Additionally, some of these investigations did not quantitatively evaluate symptom improvement by a VAS or Likert numeric scale, as well as an agreement on the treatment according to the type of reflux was not homogeneously ascertained. However, our experience suggests that tailored therapy may be the best therapeutic strategy for refractory NERD even if studies on large populations are needed to reach a final consensus for a standardized strategy in clinical practice.Some limitations of the present study should be emphasized. The retrospective analysis of our results may have affected the uniformity of data retrieval. The sample size could be quite small, although it is more plentiful than previous studies. Owing to the retrospective type of the study, the patients did not assume the same PPI. However, we demonstrated that this type of medication does not influence the response to tailored therapy, similarly to some previous evidence.13 Similarly, further exclusion criteria were: Barrett esophagus, gastric or duodenal peptic ulcer, esophageal diverticula, history of cancer or surgery of upper gastrointestinal tract, severe heart or lung disorders, diabetes, rheumatologic disorders and psychiatric impairment. We further excluded patients assuming non-steroidal anti-inflammatory drugs, aspirin, oral anticoagulants and other drugs associated with esophageal damage .We retrospectively enrolled NERD patients who had undergone MII-pH monitoring in two Gastroenterology Unit in Southern Italy  along the period January 2016–March 2017. We selected patients with reflux symptoms, both atypical and typical, who fulfilled criteria for refractoriness, that is, the absence of symptom improvement after a 8-week course of PPI. All patients performed upper endoscopy before MII-pH monitoring and subjects with endoscopic sings of erosive esophagitis were excluded to avoid potential misleading diagnosis of refractory disorder for the presence of inflammation and/or erosion-related complaint.For each patient, the following data were recorded: sex, age, body mass index, alcohol intake, smoking habits and type of typical and atypical symptoms. All patients gave written informed consent and accepted to participate in the study at the moment of personal interview.All patients undergoing MII-pH monitoring had experienced a 2-week washout from PPI. Therefore, the test was performed 'off therapy'.The test was carried out using the Multichannel Intraluminal Impedance Ambulatory System  with a 24-h recording. Patients had been in fasting conditions for at least 8 h before the investigation. The recorder was calibrated using buffer solutions at pH 4 and 7. The instrument is provided with a data logger equipped with impedance pH amplifiers as well as a catheter containing 1 pH and 6 impedance channels. The catheter was introduced through the nostril into the esophageal body to record pH at 5 cm and impedance at 3, 5, 7, 9, 15 and 17 cm above the lower esophageal sphincter. Successively, subjects were recommended to continue their usual daily activities and assume meals at normal times. They were suggested to keep upright position during daytime and get a clinostatic position only at bedtime. The data logger also recorded events as symptoms, meals and postural variations.et al.39 Reflux episodes were characterized by MII-pH monitoring as acid, non-acid and functional according to Zerbib et al.40The test allowed to identify reflux composition as liquid, gas or mixed according to Vela Therefore, NERD patients were subgrouped into three categories according to the following criteria: (i) acid reflux (exposure to pH<4 for at least 1.1% of record time), (ii) non-acid reflux (symptom association probability to pH>4 reflux episodes >95%) and (iii) functional heartburn .Patients with acid reflux received PPI at double dose (e.g. omeprazole 20 mg twice daily (b.i.d.) or lansoprazole 30 mg b.i.d. or rabeprazole 20 mg b.i.d. or esomeprazole 40 mg b.i.d. or pantoprazole 40 mg b.i.d.).Patients with non-acid reflux assumed PPI at full dose (e.g. omeprazole 20 mg per day or lansoprazole 30 mg per day or rabeprazole 20 mg per day or esomeprazole 40 mg per day or pantoprazole 40 mg per day) plus alginates one stick four times per day.Patients with functional heartburn were given levosulpiride 75 mg per day divided into three doses.MII-pH-guided therapy was performed as follows:All above-mentioned treatments were given for 4 weeks before evaluating their effectiveness.41 ranging from 0 to 100 mm was administered at least 1 week before the MII-pH monitoring and 4 weeks13 after tailored therapy to evaluate overall symptom changing. Responders were defined by VAS improvement of at least 40%, as described by Savarino et al.27A VAS, largely used in literaturepost hoc Dunn test for multiple comparisons were performed. Continuous variables were expressed as median and interquartile range. Fisher’s exact test was applied to categorical variables, which were expressed as percentages. If more than three dichotomous variables were compared, a χ2 test for trend was used. Variables with P<0.10 at univariate analysis were analyzed by binomial multivariate regression analysis42 aimed to investigate factors predictive of failure of tailored therapy. At multivariate analysis, the estimate of risk was expressed as OR and 95% CI, and values of P<0.05 were considered as statistically significant.All continuous variables were tested with the Kolmogorov–Smirnov test, which demonstrated a non-Gaussian distribution. Therefore, nonparametric tests were used. For the comparisons between two paired groups , the Wilcoxon's matched-pair test was used. When the comparison was carried out among three or more independent groups, the Kruskal–Wallis test and Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Vasoinhibins (Vi) are fragments of the growth hormone/prolactin (PRL) family and have antiangiogenic functions in many species. It is considered that Vi derived from PRL are involved in the pathogenesis of peripartum cardiomyopathy (PPCM). However, the pathogenic mechanism of PPCM, as well as heart angiogenesis, is not yet clear. Therefore, the aim of the present study is to clarify whether Vi act directly on angiogenesis inhibition in heart blood vessels. Endothelial cell viability was decreased by Vi treatment in a culture experiment. Furthermore, expression of proangiogenic genes, such as vascular endothelial growth factor, endothelial nitric oxide synthase, and VE-cadherin, were decreased. On the other hand, apoptotic factor gene, caspase 3, and inflammatory factor genes, tumor necrosis factor α and interleukin 6, were increased by Vi treatment. In three-dimensional left ventricular wall angiogenesis assay in mice, Vi treatment also inhibited cell migration, neovessel sprouting, and growth toward collagen gel. These data demonstrate that Vi treatment directly suppresses angiogenesis of the heart and support the hypothesis that Vi induce PPCM. Prolactin (PRL) is a hypophyseal polypeptide hormone in humans that consists of 199 amino acids, with a molecular weight of approximately 23 kDa. PRL has many functions in various species and has been mainly shown to play roles in lactation, maintenance of pregnancy, maternal behavior expression, and angiogenesis promotion. However, an atypical form of PRL, which is cleaved into N-terminal 16 kDa fragment, has been shown to have antiangiogenic functions. Since growth hormone and placental lactogen also exert antiangiogenic effects when cleaved two-thirds from the N-terminal residue , inhibitA recent report showed that the Vi derived from PRL mediate peripartum cardiomyopathy (PPCM) , which iHowever, the direct effect of Vi on heart blood vessels is not clear. The purpose of this study was to investigate the direct effect of Vi on cardiac angiogenesis.Escherichia coli and purified via its His tag  as described previously  and a rat cardiomyoblast cell line (H9c2)  were obt® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA). HUVECs and H9c2 cells were seeded to the wells of 96-well plates at 6 × 104 and 1 × 104 cells/cm2, respectively. After cell adhesion to the wells, the culture media were exchanged for media supplemented with 0.5, 5, and 50 nM Vi or phosphate-buffered saline (PBS) endotoxin-free  as the vehicles. After incubation of the plate for 8 or 24 h, MTS was added to each well. Then, after incubation of the plate for an additional 2 h , 10% SDS was added to each well to stop the reaction. Absorbance at 490 nm was measured using a micro plate reader .Cell viability was evaluated by 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay . The total RNA was reverse transcribed into cDNA using a QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s protocol. The cDNA was then used as the template for real-time PCR with SYBR select master mix (Thermo Fisher Science) and the 7500 real-time PCR system (Thermo Fisher Science). Specific primers for each gene are presented in Table Human umbilical vein endothelial cells were seeded on a 60-mm dish at 1.0 × 10in vitro assay for angiogenesis of the heart was based on previous reports , reconstitution buffer (Nitta Gelatin), and 30 ng/ml mouse vascular endothelial growth factor-164  in DMEM. After gelation, the gel was covered with cover medium consisting of 5% FBS, 2% penicillin/streptomycin, and 30 ng/ml mVEGF164 in DMEM. The collagen gel solution and cover medium included 100 nM Vi or its vehicle in advance. Incubation was performed for 1 week at 37°C in a humidified, 3% O2, 5% CO2 atmosphere. The cover medium was changed every 2 days.Four-week-old female C57/BL6 mice were purchased from CLEA Japan . The hearts were removed immediately after cervical dislocation. The method of  reports , 13. EacAfter incubation, the fragments and gels were fixed in 4% paraformaldehyde and used for whole-mount lectin staining. After permeabilization with Triton X-100, the fragments and gels were immersed with Block Ace  and incubated with endothelium-specific lectin . Cell nuclei were stained with SlowFade Gold antifade reagent with DAPI . Three-dimensional images were obtained using a confocal laser scanning microscope . To evaluate angiogenesis, the number of migrated cells and neovessels and the lengths of neovessels were measured using ImageJ software . The number of migrated cells was calculated by counting cells in the gel. The number of neovessels was calculated by counting lectin-positive vessels that sprouted from the left ventricular fragment toward the gel, and the lengths of neovessels were averaged for all vessel lengths in the gel. The proportion of lectin-positive cells was calculated the positive cell number divided by total cell number.t-test for comparisons between means and defined as a P-value <0.05.Numerical data are expressed as means ± SEM. Significance was evaluated using Student’s The experiment of animal use was carried out in accordance with the recommendations of AVMA Guidelines for the Euthanasia of Animals: 2013 Edition, American Veterinary Medical Association, and the Institutional Animal Care and Use Committee of Meiji University that approved the protocol of this study (IACUC-13-0013).In order to evaluate the effects of Vi on cell viability of endothelial cells and cardiomyocytes, an MTS assay was performed. After 10 h incubation, Vi from 0.5 to 50 nM significantly decreased endothelial cell viability compared with control. Furthermore, cell viability was significantly decreased in 50 nM Vi-treated endothelial cell after 26 h incubation  compared with control. Caspase 3 (CASP3) gene expression, which indicates apoptosis, was significantly increased with Vi treatment compared with control. Although gene expression of tumor necrosis factor α (TNF) was not detected in control after 8- or 24-h incubation, neither Vi treatment induced the TNF expression. Interleukin 6 (IL6) expression was significantly higher with Vi treatment than with control on 8-h incubation.To analyze the effect of Vi on mRNA expression of endothelial cells, real-time PCR was performed after 8- and 24-h incubation  promote angiogenesis and Vi prevents its effects. VEGF increasesd eNOS expression by binding VEGF-R2 . The actlization  and phoslization  through lization  and phoslization . Moreovelization .TNF and IL6, were upregulated by Vi. Vi has a pro-inflammatory function via upregulation of intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin in endothelial cells (It has been suspected that Vi is also involved to myocarditis, which was often observed in PPCM . In thisal cells . Moreoveal cells , there ial cells , and IL6al cells . The bloal cells . Therefoal cells .A previous study reported that left ventricular capillary density is decreased in the PPCM mouse model . It is kIn addition to endothelial cells, migration of non-endothelial lectin-negative cell was also observed in heart culture experiments. Thus, Vi may also inhibit migration of cells other than endothelial cells, such as pericytes and fibroblasts. Moreover, mature vessels that consist of multiple cells were not observed with Vi treatment in culture. Since Vi inhibits pericyte migration toward endothelial cells and vessel maturation , Vi alsoThis study was designed by RN and carried out by RN and EN. RN and TH wrote the manuscript. All the authors have contributed to data collection or interpretation and critically reviewed the manuscript. The final manuscript was approved by all authors.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. The reviewer BMS and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.