Acinonyx jubatus), hunt livestock, we assessed whether livestock constitutes a significant part of the cheetah diet by analysing the stable isotope composition of blood and tissue samples of cheetahs and their potential prey species. According to isotopic similarities, we defined three isotopic categories of potential prey: members of a C4 food web with high δ15N values  and those with low δ15N values , and members of a C3 food web, namely browsers . We quantified the trophic discrimination of heavy isotopes in cheetah muscle in 9 captive individuals and measured an enrichment for 15N (3.2‰) but not for 13C in relation to food. We captured 53 free-ranging cheetahs of which 23 were members of groups. Cheetahs of the same group were isotopically distinct from members of other groups, indicating that group members shared their prey. Solitary males (n = 21) and males in a bachelor groups (n = 11) fed mostly on hartebeest and warthogs, followed by browsers in case of solitary males, and by grazers with high δ15N values in case of bachelor groups. Female cheetahs (n = 9) predominantly fed on browsers and used also hartebeest and warthogs. Mixing models suggested that the isotopic prey category that included cattle was only important, if at all, for males living in bachelor groups. Stable isotope analysis of fur, muscle, red blood cells and blood plasma in 9 free-ranging cheetahs identified most individuals as isotopic specialists, focussing on isotopically distinct prey categories as their food.Large areas of Namibia are covered by farmland, which is also used by game and predator species. Because it can cause conflicts with farmers when predators, such as cheetahs ( Acinonyx jubatus) worldwide Over past years, evidence has accumulated that local extirpation of top predators may have severe and unforeseen consequences for the structure and functioning of whole ecosystems Cheetahs hunt all their prey by themselves and do not kleptoparasitize from other predators or scavenge from carcasses 2 photosynthetic pathway Taurotragus oryx), kudu (Tragelaphus strepsiceros), springbok , steenbok (Raphicerus campestris) and scub hare (Lepus saxatilis) will have lower stable carbon isotope ratios than grazing herbivores such as gemsbok (Oryx gazella), hartebeest (Alcelaphus buselaphus), cattle (Bos taurica), warthog (Phacochoerus africanus) and springhare (Pedetes capensis). We also included guinea fowls (Numida meleagris) in our study, because previous studies suggested that cheetahs feed occasionally on these birds Plants of southern Africa are known to differ in stable carbon isotope ratios because of variations in their COWith respect to the diet of cheetahs, we formulated three hypotheses. Firstly, we expected that members of the same social unit, such as bachelor groups, are more similar in isotopic composition than those of other social units, because members of the same group are likely to share prey. Secondly, we assumed that solitary males, male bachelor groups and females feed on different prey sizes or age classes because males are heavier than females and therefore are likely to subdue larger prey sizes and also calves of large prey species defended by their mothers Field work was conducted on cattle and game farmland in central Namibia between June 2002 and June 2007, and tissue samples for the isotopic discrimination in cheetah tissue were collected between September 2006 and December 2006 at the AfriCat Foundation, a non-profit carnivore conservation facility housing a limited number of captive cheetahs in central Namibia. Our study was approved by the Ministry of Environment and Tourism (MET) in Windhoek, Namibia  and the Animal Care and Ethics Committee of the Leibniz Institute for Zoo and Wildlife Research in Berlin.n = 30), hartebeest (n = 15), cattle (n = 28), warthog (n = 14), guinea fowl (n = 4), springhare (n = 10), eland (n = 2), kudu (n = 10), springbok (n = 7), steenbok (n = 2) and scrub hare (n = 3). They were selected according to their relevance in the human-wildlife conflict concerning livestock (cattle) and trophy species , based on the literature and on availability. The latter aspect precluded the inclusion of common duiker (Silvicapra grimmia), which is also known to be hunted by Namibian cheetahs We obtained muscle tissue of game species from fresh kills of predators, from trophy hunters who hunted legally on farmland and from farmers who killed animals for their own consumption. Cattle samples were collected from individuals at the slaughterhouse of the Meat Cooperation of Namibia in Windhoek. We collected samples from adult individuals of nine prey species which comprise a large proportion of prey species of cheetahs in the study site All samples were dried in a drying oven over 24 hours at 50°C. We put 0.5 mg (±0.1 mg) from each sample in tin capsules  and placed the capsules in an autosampler. Samples were combusted and analyzed using an elemental analyzer  coupled to a Delta Plus isotope ratio mass spectrometer (Thermo Finnigan) in continuous flow. Atmospheric nitrogen was used as the standard for stable nitrogen isotopes and Vienna-PDB for stable carbon isotopes. The methodological approach in analyzing stable carbon and nitrogen isotope ratios has been described in detail in In order to assign prey species to isotopically similar prey categories, we used a combination of statistical analysis and evaluation of pair-wise isotopic differences. The underlying reason for this is that the isotopic composition of two prey species might proof statistically significantly different, yet this outcome may be generated by a relatively large sample size and low variance. In other cases, isotopic differences between two species might not be detectable by statistical means because of large variance and low sample size. Therefore, we used our statistical approach as a guide for assigning potential prey species to isotopic categories.For statistical comparisons, we square root transformed unsigned delta values to meet normal distribution. We then produced a Bray–Curtis similarity matrix of prey species, followed by an ANOSIM to identify potential differences in isotopic values between prey categories Equus asinus asinus). We obtained muscle tissue from seven donkeys for comparative purposes. From each cheetah we collected a small sample of muscle tissue from the hind leg by mechanically restraining the animals and using a biopsy needle. Samples were sun-dried, stored in cryo-vials and shipped to the stable isotope laboratory at the University of Erlangen-Nuremberg, where they were analyzed as outlined above. We tested for isotopic differences in muscle tissue of cheetahs and donkeys with a Mann-Whitney-U-test.To quantify the isotopic discrimination between cheetah and donkey tissue, nine cheetahs were fed at AfriCat for at least two months with only meat of donkey  to determine their diet composition using stable isotopes. Cheetahs were captured at cheetah marking trees, immobilized, sampled and collared as described in n = 21), (ii) as bachelor groups when adult males (n = 23) were captured as a group  or (iii) as females (n = 9) when females were captured either alone (n = 4), with suckling cubs  or with weaned cubs . All cubs were excluded from the analysis. To test whether members of the same group (bachelor groups (n = 10)) are isotopically distinct from individuals in other groups, we performed an analysis of similarity (ANOSIM) using Primer 6  solitary males when males were captured alone (n = 21), males as part of a bachelor group (n = 11) and females (n = 9). We excluded litter mate groups from the analysis because of small sample size for this category.We assessed the relative contribution of the isotopic prey categories to the diet of individual cheetahs by applying a Bayesian isotope mixing model from the package SIAR version 4.1.3 15N by subtracting 3.2‰ from raw δ15N values. We did not correct stable carbon isotope data, because there was no difference in δ13C between donkey and cheetah muscle tissue. Because the trophic discrimination established at AfriCat was based on muscle tissue and the samples from free-ranging cheetahs were RBC, we corrected the result for differences in stable isotope ratios between RBC and muscle tissue. For this we used published data on the closest phylogenetic relative, the red fox (Vulpes vulpes) According to the isotopic information of captive cheetahs fed with donkey meat, we corrected the raw isotope data of free-ranging cheetahs for isotopic discrimination in Vulpes vulpes) 13C data and 2.6‰ from raw δ15N data. During the second analysis, we applied trophic discrimination factors as described by Parng and colleagues for felid species 13C data and 4.1‰ from raw δ15N data.To assess how sensitive the outcome of the Bayesian isotope mixing model is towards deviations in assumed discrimination factors, we repeated the model twice. In the first analysis, we used trophic discrimination factors as described by Roth and Hobson for red foxes  that were reported above, we assessed the dietary specialization of cheetahs by collecting four tissue samples. From each cheetah we collected a fur sample, muscle tissue, RBC and blood plasma. All samples were handled and stable isotope ratios were analyzed at the IZW as described above or in For description of data, we first tested if fur, tissue and blood samples differed in stable isotope ratios using a repeated measure ANOVA for each sample category, using the statistical software SYSTAT 13 . We expected a larger variation in stable carbon isotope ratios in individual tissues than in stable nitrogen isotope ratios because of the higher contrast of potential prey species in stable carbon. We therefore performed the following analysis only with stable carbon isotope data. We calculated a Bray-Curtis similarity matrix and tested whether cheetahs were isotopically distinct according to an ANOSIM using Primer 6  and high δ15N values . Posthoc tests and a similarity analysis differentiated between pairs  and grazing animals with low δ13C values (hartebeest and warthog). We pooled data of some species pairs even when pair-wise comparisons were significantly different, because the statistical separation seem to be driven by low variance and large sample size, yet differences were too small to warrant treatment as a separate isotopic prey category, e.g. in case of cattle and gemsbok.In total, we collected tissue material of 125 potential prey animals, comprising 11 species . For staen pairs . Followien pairs  and an e13C values of cheetah muscle of nine captive cheetahs at AfriCat averaged −15.6±0.9‰. This value did not differ from the mean δ13C of donkey meat  that was fed to cheetahs over 2 months prior to tissue collection. In contrast, δ15N values of cheetah muscle averaged 11.6±0.6‰, which was higher than the mean δ15N of donkey meat . Thus, muscle tissue of cheetahs was enriched in 15N in relation to that of the diet by 3.2‰.δR = 0.814, P<0.001; Members of different groups differed in their isotopic composition , i.e. males of bachelor groups were statistically different in isotopic composition from solitary  and from females , whereas solitary males and females were isotopically more similar . δ13C values averaged −16.2±3.1‰ for solitary males, −13.3±2.8‰ for males in bachelor groups and −17.9±2.0‰ for females . δ15N values averaged 10.9±0.9‰ for solitary males, 11.1±0.9‰ for males in bachelor groups and 11.2±0.9‰ for females. Isotopic data that was corrected for trophic discrimination was within the boundaries of the isospace of the potential prey categories, except for those of males in bachelor groups, which were slightly adjacent to the minimum convex polygon between mean values, but not outside the minimum convex polygon of isotopic data of individual data points of potential prey animals  and to a lesser extent on browsers  and by grazers with high δ15N values , followed by browsers, including eland and kudu as valuable trophy species  and to a lesser extent on grazers with high δ15N values . The model suggested that males in bachelor groups consumed mostly hartebeest and warthog , followed by grazers with high δ15N values  and to a lesser extent on browsers . Finally, female cheetahs were suggested to feed predominantly on browsers , followed by grazers with low δ15N values  and to a low extent on grazers with high δ15N values . We did not calculate a mixing model for raw data corrected according to the discrimination factors suggested for felid species by Parng and colleagues In our sensitivity analysis we investigated the effect of assumed trophic discrimination factors on the output of the Bayesian mixing model. When using trophic discrimination factors as suggested by 13C values corrected for trophic discrimination . δ15N values corrected for trophic discrimination also differed among tissue types  and those of fur and plasma  differed. All other pair-wise comparisons proved not to be significant. Averaged δ13Ccorr values of individual cheetahs ranged from −21.3‰ to −12.2‰ and δ15Ncorr values from 6.5‰ to 11.1‰ . Twenty one out of 36 possible dyadic comparisons of stable isotope ratios, i.e. 58% of all possible comparisons were significantly different . To assess the extent of specialization based on a variance component analysis, we focused on δ13C values since they were most responsible for differences among individuals , guinea fowls that presumably fed on grass seeds and grass-processing insects, and cattle. The second category consisted of grazers with low δ15N, i.e. warthog and hartebeest. The third prey category consisted of members of a C3 food web, namely browsing herbivores such as eland, kudu, springbok, steenbok and scrub hare. Our categorization of species into prey categories was justified by statistical separation and overall small isotopic differences between species pairs that made a separation questionable even in light of a significant outcome of statistical tests. In some cases, statistical detection of relatively small differences was facilitated by relatively large sample sizes, e.g. in the comparison of isotopic data from gemsbok-cattle or gemsbok/cattle-springhare. In some other cases, isotopic data were not distinguishable according to the SIMPER analyses, yet the inability to detect a difference may have originated from low sample sizes and relatively large variation of data, e.g. eland-guinea fowl or scrub hare-guinea fowl pairs. In these cases, we found it justified to pool or separate species pairs accordingly.As a prerequisite of our study, we depended on isotopically distinct prey categories. Our analysis separated three prey categories. We defined one category as grazers with high δStable isotope data of ungulate species of this study were similar to those reported before for free-ranging herbivores in southern Africa 15N in relation to 14N by 3.2‰ when compared to donkey meat, yet we did not measure an enrichment of 13C in relation to 12C in muscle tissue of cheetahs compared to their diet. The discrimination of heavy nitrogen in relation to light nitrogen is similar to what previous studies reported for carnivore species 13C between carnivore tissue and prey tissue. The discrepancy in discrimination factors between their and our findings may be partly explained by the fact that study animals consumed food items that differed in macronutrient composition. The macronutrient composition of food can influence the trophic discrimination of stable carbon and nitrogen isotopes in carnivores 13C in relation to 12C between mammal consumers and their diet of on average 0.5‰. Codron and colleagues 13C in relation to 12C of 2.6‰ between diet and carnivore hair. A larger sample size in our study may have revealed a subtle discrimination of stable carbon isotopes, yet given the presumed small magnitude of this discrimination, we suggest that this has no large effect on the outcome of our mixing models. Indeed, our first sensitivity analysis based on the trophic discrimination measured for Vulpes vulpesIn our feeding experiment with captive cheetahs, we found that muscle tissues were enriched in 13C in relation to 12C of about 5.5‰ for felid species contrasts with our and other studies 15N of 6.9±0.9‰ and an average δ13C of −21.3±3.2‰. The corrected nitrogen isotope values are substantially lower than any of the prey isotope data  and in female cheetahs (1.8%), questioning the relevance of this prey category in the diet of these cheetah categories. Male cheetahs foraging in bachelor groups exhibited a lower 95% CI value of about 10.7%, which suggested that they, indeed, include some grazers with high δ15N values in their diet. However, because we pooled data of four prey species to this prey category, we can not tell to what extent bachelor males included cattle and gemsbok in their diet. Considering all cheetahs of our study, grazers with high δ15N values do not constitute a major part of cheetah diet on Namibian farmland, a notion that is also supported by conventional analyses of cheetah feces In general, solitary and bachelor males prefer hartebeest and warthogs over other prey categories. Furthermore, solitary males and females exhibited relatively low δHyaena brunnea), wolves (Canis lupus) and sea otters (Enhydra lutris) are individual dietary specialists We based our assessment of individual dietary specialization on replicate measurements of stable isotope ratios in four tissues of 9 cheetah individuals. Variation of stable isotope ratios within cheetah individuals was low, yet none of the cheetahs exhibited a unique isotopic signature that differed from all other individuals. This suggests that individual cheetahs overlapped at least partly in their diet. Furthermore, variance/covariance patterns and variance component analysis revealed that most of the individuals were isotopic specialists, i.e. it is very likely that the majority of cheetahs hunted a specific isotopic prey category. Previous conventional and isotopic studies have already pointed out that many mammalian predators, e.g. cheetahs, brown hyenas Click here for additional data file.Table S2Raw isotopic data of cheetahs Click here for additional data file.Table S3Raw isotopic data of 4 tissues in 9 cheetahs , rbc = red blood cells.(DOC)Click here for additional data file.


PPARγ in order to address itsrole in cardiac cell differentiation of mouse embryonic stem cells (mESCs). Peroxisome proliferator-activated receptor γ (PPARγ) is a member of thePPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factorin adipocyte differentiation, it is also associated with a variety of cell functions includingcarbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of  In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chainreaction (qPCR) was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs)formation, was examined by applying the respective agonist and antagonist.PPARγ duringspontaneous formation of CPCs and cardiomyocytes. Our results indicated that duringCPC formation, PPARγ inactivation via treatment with GW9662 (GW) reduced expressionof CPC and cardiac markers. The obtained data revealed an elevation in the expression level of We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis. PPAR transcripts during cardiac differentiation (Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the superfamily of transcription nuclear receptors. They consist of three major isoforms, termed α, β/δ and γ, encoded by distinct single-copy genes. Previous studies have well documented the function of PPARs as regulators of lipid and lipoprotein metabolism, and glucose homeostasis . Moreoventiation . Gata4, Nkx2.5 and Mef2C) start to be fully expressed. Then, the cardio precursor cells emerge regardless of presence of those genes encoding the sarcomeric proteins . However, at the end of the cardiomyocyte differentiation process, both cell type markers are identifiable  unless indicated otherwise. cardiomyocytes mESCs, Royan B1 cell lines derived from the C57BL/6 strain , were keRosiglitazone  was used as a potent specific PPARγ agonist ,19 and Gβ-tubulin V expression level. All measurements were done in triplicate and expression level changes were reported according to the ΔΔCt method. Specific primer pairs . To generate cDNA, Moloney murine leukemia virus (MMLV) reverse transcriptase, 1 µg of each RNA sample and random hexamer primers were used according to the manufacturer’s protocol. Using SYBR Green , qPCR was carried out in a Rotor gene 6000 thermal cycler  as suggested by the protocol. The PCR mixture contained 10 µl RotorGene SYBR Green PCR Master Mix, 3 pmole of each primer, and 25 ng of cDNA for each reaction in a final volume of 20 µl. The relative mRNA concentrations were calculated using the software provided by the manufacturer and normalized to er pairs  were desAn indirect immunofluorescence light microscopy was used to analyze the cells as previously described ,17. The The number of beating EBs was measured in each dish in three different experiments. Furthermore, the area ratio of beating EBs was measured by taking the mean size of 10 separate beating EBs in each group respective to controls . SPSS (version 17) was used to express data as means ± SEM obtained from three independent treatments of the replicated observations. One-way ANOVA was applied to assess globally statistical difference among multiple treatments. Also independent t test analysis was carried out to identify statistical differences between any two treatments. P<0.05 (*) was taken as the level of statistical significance. PPARγ expression level during spontaneous cardiomyocyte differentiation  on day 7 [ were not significantly affected compared with the control, whereas the antagonist, at concentrations of 5 and 10 µM, caused a sharp decrease in the expression of the aforementioned markers (SM22α and SMAα)  . This inAgonist and antagonist treated CPCs were allowed to differentiate to cardiac cells as described above . On day PPARγ (PPARγ activation/inactivation and its potential impact on cardiomyopathic remodeling and cardiophysiology have been intensively reviewed . PPARγ dPPARγ . PPARγ during the spontaneous cardiomyocyte differentiation of mESCs in two stages of cardiac and post-CPC formation. Despite the relatively low expression of PPARγ in the heart, it exerts a prominent role in cardiac function (PPARγ expression increased during CPC formation and remained at a high level even at the terminal stage of cardiac differentiation. This result is in concordance with the data reported by Sharifpanah et al. (PPARγ on days 8 and 18. We then divided the spontaneous cardiomyocyte differentiation in two stages of CPC and cardiomyocyte formation. As depicted on day 7, CPCs were formed as evident by an increase in the expression level of the cardiac transcription factor Nkx2.5. Furthermore, on day 15, the highest level of PPARγ expression level was detected in differentiated cardiomyocytes. To investigate whether this increased level of PPARγ plays a role in cardiyomyogenesis, respective agonist and antagonist treatments were carried out during CPC formation. Our data displayed that the agonist significantly induced the activation of PPARγ which resulted in overexpression of CPC marker Nkx2.5 on day 7 while the respective antagonist caused a reverse effect. Our results are in contradiction with those of Sharifpanah et al. (This study was carried out to address the involvement of PPARγ in different stages of cardiac differentiation. We recently established the stagedependent role of PPARγ modulation on neural differentiation of mESCs by retinoic acid treatment . In herefunction ,27. Our h et al.  whose reh et al.  who showWe conclude that PPARγ influences spontaneous cardiomyocyte differentiation from mESCs at the early stages when CPCs are forming. This conclusion could be an asset along with that of a previous study which indicated that pioglitazoneactivation of PPARγ was followed by improvement of cardiomygenic transdifferentiation of human mesenchymal stem cells. Taken together, PPARγ influences the potentiation of cell commitment at the early stages of cardiomyogenesis. However, further research is required to investigate how PPARγ inactivation inhibits heart cell differentiation.


The purpose of this study was to determine if individuals at greater cardiovascular risk have T1 times were determined at 1.5Tesla before and after gadolinium administration (0.15 mmol/kg) using a modified Look-Locker pulse sequence. The relationship between CMR measures  and 14 established different cardiovascular risk scores were determined using regression analysis. Bootstrapping analysis with analysis of variance was used to compare different CMR measures. CVD risk scores were significantly different for men and women (p < 0.001).CVD risk scores for 1208 subjects  ages 55–94 years old were evaluated in the Multiethnic Study of Atherosclerosis (MESA) at six centers. T1 time showed more statistically significant associations with risk scores  compared to other CMR indices  and partition coefficient  in men. Risk scores, particularly the new 2013 AHA/ASCVD risk score, did not correlate with any CMR fibrosis index.25 minute post gadolinium T1 times at greater delay time (25 minutes) showed better agreement with commonly used risk score indices compared to ECV and native T1 time.Men with greater CVD risk had greater CMR indices of myocardial fibrosis. Thttp://www.mesa-nhlbi.org/, NCT00005487. Overt cardiovascular disease (CVD) encompasses a wide array of late stage outcomes  that are expensive to treat and that may be preventable. In order to better manage cardiovascular health, CVD risk models have been developed to relate an individual’s risk for adverse CVD outcomes with various biomarkers ,2. The F1 relaxivity of myocardial tissue before and/or after administration of a gadolinium based contrast agent using cardiovascular magnetic resonance (CMR) [1 and ECV indices of myocardial fibrosis have been validated in select patient populations [The myocardium responds to physiological and pathological stress by remodeling with increased deposition of interstitial collagen . The prece (CMR) ,14. The ce (CMR) . T1 and ulations ,17.CMR indices of myocardial fibrosis are relatively new biomarkers and their applicability in a general, low to moderate risk population has not yet been determined. The Multi-Ethnic Study of Atherosclerosis (MESA) study provides the opportunity to explore the validity of CMR indices of myocardial fibrosis with respect to risk for CVD in a largely asymptomatic subject pool. The purpose of this study was to determine the relationship between CMR fibrosis indices and CVD risk score models in a large, population-based study. We hypothesize CVD risk scores will be correlated with CMR fibrosis indices.th follow-up examination from 2010–2012 (designated as the “MESA 5” follow-up examination). Institutional review board approval was obtained and all subjects signed informed consent for MESA procedures.Details of the design and organization of the Multi-Ethnic Study of Atherosclerosis (MESA) trial have been reported previously . From 20Smoking was defined as never, former (smoked ≥ 100 cigarettes in lifetime), or current (smoked cigarettes in last 30 days). Hypertension was defined as systolic blood pressure ≥ 140 mm Hg, diastolic blood pressure ≥ 90 mm Hg, self-reported hypertension or use of antihypertensive medication. Hypertrophy of the left ventricle was defined as “no” (absence of LV hypertrophy) or “yes” (presence of LV hypertrophy). Type II diabetes was defined as fasting glucose > 125 mg/dl or use of diabetic medication.Blood was drawn after a 12 hour fast, and samples were stored at −70°C. Lipids, insulin, and glucose were measured at a central laboratory . Lipids were assayed on thawed EDTA plasma within 2 weeks of sample collection, using Centers for Disease Control Prevention/NHLBI standards. High-density lipid cholesterol (HDL-C) was measured using the cholesterol oxidase method  after precipitation of non-HDL-C with magnesium/dextran (coefficient of variation 2.9%). LDL-C was calculated using Friedewald equation . The ser1 mapping protocol in MESA has been described [Study subjects with prior myocardial infarction or focal late gadolinium enhancement were excluded from analysis. The CMR and Tescribed . Pre-con1 maps. Levenberg-Marquardt algorithm allowed a 3 parameter curve fit using MOLLI as a source image to extract T1 time for each pixel. Myocardial T1 time was then determined by careful drawing of the region of interest to encompass the myocardium tissue only, so that the trabeculation, blood pool and epicardial fat were excluded. The partition coefficient (λ) was calculated by using a 3 point linear fit to determine the resulting slope (ΔT1myo/ΔT1blood). Extracellular volume (ECV) as a percentage was evaluated by multiplying the partition coefficient by (1 – hematocrit). Expected associations with greater degree of myocardial fibrosis by CMR are higher ECV and native T1 time, but lower post gadolinium T1 time [MASS research software  was used to create T T1 time -17.Risk scores were based on prior systematic and blinded review by Allan et al. , includi1 times or ECV, the risk scores were treated as continuous variables. Correlations between each risk score and T1 times or ECV were tested using linear regression models adjusted for heart rate. T1 times were studied at both 12 and 25 minutes after gadolinium administration. The Generalized Additive Model (GAM) was employed to test for departures from the linear regression models. If the resulting model suggested non-linearity, the corresponding data points were refitted using a linear spline regression model. Bootstrapping analysis was performed with 10,000 iterations to test for reliability of correlation results [All analyses were stratified by gender to reduce the effects of potential confounders. Risk scores were categorized as a) “low”, “intermediate” and “high”, or b) “low”, “average”, “intermediate” and “high” depending on the conventions of each score . However results .Complete CMR data as well as clinical and serologic data were available Table  for 1231As expected, CVD risk scores were generally greater for men than women Table . Tables 1 time and ECV CMR metrics. Both MESA and Reynolds risk score showed inverse associations with post-contrast T1 time  in men . ECV was positively associated with both MESA and Reynolds risk score .For women, only the MESA and Reynolds risk scores were significantly associated with both T1 time or ECV per unit change in risk percentage. Nonlinear GAM models resulted in improved correlation between the Reynolds risk score and 25 minute T1 time in men (p = 0.011). Similarly, non-linear association was observed for MESA risk score with 25 minute T1 time in men (p = 0.009). Table 1 times or ECV with risk scores below a predefined threshold determined using GAM analysis.Most of the risk scores showed low to moderate changes in T p = 0.00. Table 9Risk scores predicting endpoints for men and women without symptomatic CVD are an increasingly important tool to help identify individuals who may benefit from more intensive or earlier medical intervention. Most risk models use similar risk factors to predict cardiovascular events Table  and thusRisk scores generally have similar endpoint prediction encompassing the likelihood of having coronary artery disease (CAD) that in turn may lead to heart attack and/or heart failure in late stage disease. We hypothesized that this propensity for cardiovascular disease may in turn be related to the development of diffuse myocardial fibrosis. Several previous studies have looked at the agreement amongst various risk scores, including those ones used for the current study. This study sought to indirectly look at agreement between any two risk scores based on their respective effect size and significance of associations with CMR indices. This agreement test assures a consistent comparison between risk scores. As in our study Table , there w1) vs. blood relaxivity before and after gadolinium administration [1 measurements in the ECV calculation, each with its own error/variability in measurement. In contrast, change in T1 time is nonspecific as to etiology and does not entirely reflect myocardial fibrosis. Indeed, increased native T1 time and ECV have been reported in amyloid light chain disease and low T1 times have been reported in Fabry disease [CMR measures of diffuse myocardial fibrosis are relatively new, and long term validation in relationship to cardiovascular outcomes in a general population has not been performed. At present, there is little consensus regarding the ‘optimum’ CMR measure to quantify the degree of fibrosis especially in asymptomatic subjects. The partition coefficient is the slope of the linear relationship between myocardium relaxivity , smoking, diabetes and age are probably those which may be most strongly related to myocardial fibrosis -12. MyocOf several possibilities, it is possible that a greater post gadolinium delay time (i.e. at 25 minutes) discriminates fibrosis and is relatively more reproducible in a multi-center setting, and thus more reliably related to cardiovascular risk factors compared to other T1 times. Nevertheless, it seems reasonable that CMR investigators continue to consider multiple T1 indices and that the optimal T1 index may vary by disease and/or condition being evaluated.1 time in relationship to greater CVD risk. Of interest, ECV appeared to be less sensitive than post gadolinium T1 time in this regard. In the same fashion, native T1 time was also less sensitive than post-gadolinium T1 time. In women, little or no relationship was noted using the CMR indices. One reason for this may be substantially lower levels of both CVD risk and thus lower degrees of myocardial fibrosis in women compared to men.Since CVD risk models have been validated in various cohorts, it is reasonable to question if individuals deemed at higher CVD risk may have greater fibrosis indices by CMR. A critical factor in this analysis is whether CMR is sensitive enough to evaluate early myocardial fibrosis that might be predicted using these CVD risk calculators. Indeed, in men, this does appear to be the case: 10/14 CVD risk scores showed lower post-contrast T1 time and ECV for both men and women. Relative to the older risk scores, this new risk score reclassified substantial number of MESA subjects as “high risk”. This reclassification to high risk levels has been a major criticism of the new ASCVD risk score. Based on our CMR data, the new ASCVD score was not associated with CMR detected myocardial fibrosis.The newly released risk score ASCVD 2013 showed poor correlation with T1 times and ECV. LV mass index characterizes the structure of the heart, also reflective of the degree of diffuse fibrosis present. Potluri et al. previously have shown the inverse relationship between LV mass and T1 changes in more overt diseased cases such as HCM [Although independent of CMR fibrosis markers, the MESA risk score is derived from the same cohort as our study. Thus, it is not surprising that this score offers the greatest correlation between risk score and CMR indices of fibrosis. In addition, the MESA score adds more specific clinical markers to the risk score, including NT-proBNP levels and LV mass index. NT-proBNP is a strong independent predictor of outcomes in outpatients with chronic heart failure. Furthermore, diffuse myocardial fibrosis coincides with the processes leading up to an event of heart failure, indicative of progression of Th as HCM .Like MESA, the Reynolds risk score is a contemporary risk score. The Reynolds risk score includes C reactive protein (CRP) in addition to traditional risk factors. CRP is a nonspecific marker for inflammation, but has been used to risk stratify subjects for cardiovascular disease. Furthermore, Zhang et al. has shown CRP promotes expression of angiotensin II-induced collagen type I and type III in mice , reflectA limitation of the study is that the MESA subjects were asymptomatic healthy subjects at baseline and thus at relatively low cardiovascular risk. Nevertheless, 49% of men and 55% of women had hypertension, and 14% had diabetes. However a low prevalence of disease in asymptomatic individuals likely results in considerable error and thus variation in the risk prediction models Table . In addi1 times suggest the clinical potential for CMR indices of fibrosis to be used in complement with risk scores, thereby adding prognostic value to patient care. However, future studies relating T1 time or ECV measurements to cardiovascular events will help to further refine the role of T1 mapping by CMR in asymptomatic individuals.Cardiovascular risk scores are routinely implemented to help identify patients who are at risk for subsequent cardiovascular events. The risk factors that comprise the risk scores have also been implicated in the development of myocardial fibrosis. Using CMR, diffuse myocardial fibrosis can be quantified, and greater CMR fibrosis correlated with greater CVD risk in men. These relationships were most prominent in the more contemporary risk scores derived from MESA and the Reynolds study using 25 minute T1 gadolinium time as an outcome measure. These moderate agreements between risk scores and T


A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species  on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component. For example, ethanol can be produced by fermentation of glucose obtained from enzymatic hydrolysis of cellulosic biomassIt is known that enzymes hydrolyze amorphous cellulose faster than crystalline cellulose2453In order to improve the accessibility of enzymes to the cellulose substrate, lignocellulose biomass is typically pretreated via various methods such as dilute acid, autohydrolysis, alkaline, or steam explosion pretreatment9111214In this study, three lignin-free model cellulosic substrates  were analyzed to monitor changes in crystallinity, crystal size, chain length, meso-scale packing, and surface contamination of substrates over the course of enzymatic hydrolysis. Although their compositions and physical structures were different, all three substrates showed a similar behavior in hydrolysis rate reduction during the enzymatic reaction. Therefore, it is hypothesized that there must exist common features or changes that could be correlated with such decreases in hydrolysis rate. This study uses a suite of analytical techniques covering various structural aspects of cellulose at multiple length scales17182018202324252627Avicel, a highly-crystalline pure cellulose Iβ substrate, was commercially purchased. Bleached softwood pulps were obtained from a local mill near North Carolina State University. The bleached softwood consisted of ~80% cellulose and ~20% hemicellulose. Bacterial cellulose was prepared as described in a previous paperCellulase (NS 50013), xylanase (NS50014), and β-glucosidase (NS 50010) were received from Novozymes  and used as a mixture at a ratio of 1:0.3:0.3 by weight. An enzyme dosage of 5 FPU/g substrate was used for bleached softwood, 10 FPU/g substrate for bacterial cellulose, and 20 FPU/g substrate for Avicel. The enzyme loading for each substrate was adjusted so that similar levels of carbohydrate conversion were obtained for the three model biomass samples. All enzymatic hydrolysis were performed in 100 mL of 100 mM acetate buffer (pH 5.0) at a 5% (w/v) solid loading. The substrate and enzyme mixtures were incubated in a shaking water bath at 50 °C and 150 rpm. The hydrolysis yield was measured after reactions for 12, 24, 48, 72, and 96 hours. All samples retrieved from the reaction vessel were thoroughly washed with deionized water, freeze-dried, and stored for further analytical analysis.The sugar and lignin contents of the sample were determined according to the standard procedure suggested by the National Renewable Energy Laboratory (NREL)w).The DP of cellulose was measured by gel permeation chromatography (GPC) after the carbanilation treatmentXRD experiments were performed using a Rigaku SmartLab x-ray diffraction with a Cu tube (λ = 1.5405 Å). The diffraction angle of 2θ from 9° to 41° was measured at a step size of 0.05°. Cellulose crystallinity was calculated based on the peak height method−1 interval in the CH stretch vibration region  and an 8 cm−1 interval in the OH stretch vibration region . Each data point was an average of 100 laser shots, and the measured SFG signal intensity was normalized with the IR and visible input laser intensities at each shot. Each spectrum reported here represents an average of 10 different locations of the sample pellet. The SFG probe volume has been estimated to be 200 μm × 150 μm on the surface and up to 20 μm deep from the external surface3240/(A3240 + A3270) where A3240 and A3270 are peak areas of the ~3,240 cm−1 and ~3,270 cm−1 peaksPellets were made from the powdered samples retrieved at various hydrolysis time points. The instrumental details of SFG have been described elsewhereXPS analysis was performed using a Kratos Axis Ultra, equipped with a monochromatic Al Kα radiation. Pristine samples, as well as samples retrieved after 12 h hydrolysis and the final hydrolysis residue  were analyzed. All spectra were acquired from a sample area of 1 mm × 1 mm.−1 with a 4 cm−1 resolution and averaged over 100 scans. All the spectra were normalized to ~1,031 cm−1 for representation purposes.ATR-IR spectra were collected using a Nicolet 8700 Research FT-IR Spectrometer (Thermo Scientific) equipped with a smart iTR diamond ATR unit, a KBr beam splitter, and a deuterated triglycine sulfate detector. All spectra were collected from the pressed sample pellet in the region of 650–4,000 cmThe experimental results are discussed in the following order. First, the enzymatic hydrolysis yield, as a function of reaction time, is described in Section 1. Then, it is discussed if the decrease in enzymatic hydrolysis rate of cellulose can be correlated with changes in cellulose chain length from DP measurements (Section 2), crystallinity, and crystal size from XRD (Section 3), meso-scale arrangement of cellulose microfibrils from SFG (Section 4), and surface contamination from XPS (Section 5). Finally, the polymorphic structure change of bacterial cellulose during enzymatic hydrolysis is discussed from SFG, ATR-IR, and XRD analyses (Section 6).To assess if the decrease in hydrolysis rate is related to cellulose digestion, the average DP of cellulose in the samples retrieved at various time points of the enzymatic hydrolysis process was measuredIn our study, the hydrolysis rate did not seem to correlate with changes in DP observed for all three cellulose substrates. As shown in In the case of bleached softwood, the DP decreased substantially from ~1,700 to ~275. When enzymes penetrate into the bleached softwood cell walls through pores and preferentially consume amorphous cellulose and hemicellulose4The DP of bacterial cellulose was initially ~3,000, which was the highest among the three samples analyzed in this study; it then reduced to ~560 after enzymatic hydrolysis. Bacteria synthesize cellulose in the absence of hemicellulose which could affect crystalline packing of cellulose chainsThe crystallinity of cellulose has been considered one of the primary factors that influence the rate of enzymatic hydrolysis6364i.e., after 12 hours of hydrolysis reaction) (5eaction) . The cryOne could hypothesize that the crystal size may affect the enzymatic hydrolysis activity since surface area varies with the crystal size. The crystal size estimation using the Scherrer equation  showed n11On the other hand, the crystal size of bacterial cellulose decreased slightly with increasing hydrolysis duration. This could be interpreted as thinning of the cellulose microfibrils by hydrolysis19The structural changes of cellulose microfibrils at the meso-scale (between ~10’s nm to a micron) could provide further information on how enzyme complexes and reaction products are transported inside the biomassThe surface morphology of biomass samples retrieved during enzymatic hydrolysis reactions has been investigated with atomic force microscopy (AFM), which reported opening of pores and fissures with increasing hydrolysis time412324182326−1 decreased drastically, and the shoulder peak at 3,450 cm−1 disappeared completely after the first 12 hours of enzymatic hydrolysis. The peak at 3,320 cm−1 is assigned to OH groups in the crystal interior−1 peak was speculated to originate from the surface hydroxyl groups of cellulose microfibrils, which can interact with hemicelluloses24−1 shoulder peak is absent in the SFG spectra of Avicel (−1 peak (f Avicel . Althoug(−1 peak  inset ma2) to hydroxyl (OH) peaks could be an indicator of cellulose meso-scale packing  and long microfibrils of cellulose Iβ embedded in a hemicellulose matrix. Both crystalline cellulose amount and apparent crystal size increased after enzymatic hydrolysis of bleached softwood . However1112The overall SFG intensity of bacterial cellulose was lower than those of cellulose in Avicel and bleached softwood. This is mainly due to the difference in cellulose packing in these samples. Cellulose crystals in Avicel and microfibrils in woody cell walls are predominantly cellulose Iβ and laterally-packed in an antiparallel fashionThe enzyme accessibility to cellulose substrate could be divided into two factors: (i) the transport of enzymes to cellulose surface through the matrix polymers in biomass and (ii) the availability of enzyme binding sites at the cellulose crystal or microfibril surfaces. The results shown in the previous section suggest that although the transport behavior of enzymes would be different for the three biomass samples studied here, all samples showed the same temporal behavior of hydrolysis rate. Ruling out the parameters considered so far, the remaining factor is the change in enzyme binding sites on the cellulose substrate over time. This hypothesis was tested by comparing the surface compositions of cellulose substrates before and after enzymatic hydrolysis reactions using XPS.47For all three substrates, XPS analysis results showed that the cellulose surface was covered by non-cellulosic substances after enzymatic hydrolysis reactions. In the case of Avicel, the nitrogen peak at 397 eV and the non-cellulosic component (285 eV) in the C1s region increased substantially, indicating the presence of enzymes. It was shown that solid substrates retrieved over the course of hydrolysis reactions are coated with proteinaceous species4949The nitrogen content in bleached softwood showed a small increase after the hydrolysis; at the same time, the Si2p peak increased noticeably. In plant cell walls, silica is deposited in intimate association with the organic componentsHence, among all parameters considered in this study, only the surface contamination revealed with XPS appears to be common for all three types of biomass and correlates well with the decrease in hydrolysis rate . The adsThe crystalline portion of bacterial cellulose contains 60–80% cellulose Iα, and the rest is cellulose Iβ−1 and 3,270 cm−1 are characteristic to cellulose Iα and Iβ, respectively. Thus, the ratio of these two components can be used to estimate the polymorphic ratio of cellulose. The deconvolution result showed that the Iα fraction had progressively decreased from 55% at 0 h to ~40% in 72 h sample (−1)−1 belongs to cellulose Iα and the peak at 710 cm−1 to cellulose Iβ−1 and 3,240 cm−1 became smaller compared to the cellulose Iβ peaks at 710 cm−1 and 3,270 cm−1.In the current study, the change in cellulose allomorph fraction (Iα/Iβ ratio) in bacterial cellulose was evidenced from the SFG and ATR-IR analysis results . In SFG,23h sample  inset. IIn XRD , it was Combining all SFG, ATR-IR, and XRD analysis results of bacterial cellulose, it can be concluded that the cellulose Iα component is more susceptible to enzymatic hydrolysis than the cellulose Iβ component. Discussing the origin of this difference is beyond the scope of this work. In the literature, this difference for cellulose polymorphs has been attributed to variances in crystallographic facets of two allomorphs or the ‘core-shell’ structure of microfibrils containing both allomorphs5459To better understand the factors affecting the decrease in enzymatic hydrolysis activity over time, a number of structural characterizations were performed. The accessibility of the cellulose microfibril surface was found to be a common factor for the tested substrates: Avicel, bleached softwood, and bacterial cellulose. The XRD crystallinity, crystal size, degree of polymerization, and changes in meso-scale packing did not directly correlate with temporal changes in enzymatic hydrolysis. In addition, the allomorphic change in bacterial cellulose over the course of enzyme hydrolysis suggested a preferential hydrolysis of cellulose Iα over cellulose Iβ.How to cite this article: Kafle, K. et al. Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis. Sci. Rep.5, 15102; doi: 10.1038/srep15102 (2015).


It could potentially be used as a tracer of N metabolism in plants under changing environmental conditions, where primary N metabolism may be complex, and losses and gains of N fluctuate over time. In order to test the utility of δ15N as an indicator of plant N status in N2-fixing plants grown under various environmental conditions, alfalfa (Medicago sativa L.) plants were subjected to distinct conditions of [CO2] (400 vs. 700 μmol mol−1), temperature (ambient vs. ambient +4°C) and water availability (fully watered vs. water deficiency—WD). As expected, increased [CO2] and temperature stimulated photosynthetic rates and plant growth, whereas these parameters were negatively affected by WD. The determination of δ15N in leaves, stems, roots, and nodules showed that leaves were the most representative organs of the plant response to increased [CO2] and WD. Depletion of heavier N isotopes in plants grown under higher [CO2] and WD conditions reflected decreased transpiration rates, but could also be related to a higher N demand in leaves, as suggested by the decreased leaf N and total soluble protein (TSP) contents detected at 700 μmol mol−1 [CO2] and WD conditions. In summary, leaf δ15N provides relevant information integrating parameters which condition plant responsiveness  to environmental conditions.The natural  However, analyses of the effect of CO2 and its interaction with other environmental conditions are of great relevance. In the field, multiple stresses, such as high temperature and drought periods in semi-arid or drought-stricken areas, often occur simultaneously. Studies of field crops and model plants have shown that the combination of heat and drought stresses has a stronger detrimental effect on plants growth and productivity than either stress alone. Furthermore, many reports indicate that it is not possible to extrapolate plant responses to combined stresses based on the responses to single stresses  have been used as tools that provide useful information on parameters conditioning plant growth, such as transpiration efficiency, the ratio of net photosynthesis to water transpired, etc., and that integrate the period during which CO2 is assimilated  has been used as a breeding criterion for increasing yield in crops exposed to low water availability and salinity stresses  have also been proposed as a useful trait for crop screening  morphophysiological differences (particularly in root systems); (ii) activity of principal enzymes involved in N assimilation and (iii) plant N demand and assimilation capacity. However, it should be remembered that further 15N fractionation might take place as a result of N recycling, transport, exudation or volatilization (through stomata as ammonia and nitrous oxide) by the plants . Thus, despite recent advances in the interpretation of plant δ15N, there is still a lack of knowledge of δ15N in plants where N2-fixation is the sole source of N.In recent decades, stable isotope techniques  and their reaction to environmental stresses may provide valuable information on the transport and metabolism of C-N compounds  plants, which are frequently exposed to high temperature and/or drought in field conditions, were studied. They were subjected to distinct levels of [CO2] (400 vs. 700 μmol mol−1), temperature (ambient vs. ambient +4°C) and water availability . In addition to growth, we characterized the N isotopic composition (δ15N) of whole plants and separate organs , and δ15N relationship with C-N related parameters.Given that atmospheric NMedicago sativa L. cv Aragon) plants were grown in 13 L plastic pots (five plants per pot) filled with 1:2 (v/v) vermiculite-perlite. At 2–4 weeks after planting, they were inoculated with Sinorhizobium meliloti strain 102F78 . One-month-old plants were transferred to the corresponding temperature gradient greenhouses . Within each TGG, one for each CO2 concentration (400- and 700-μmol mol−1), plants were separated into 4 treatments corresponding to all combinations of, temperature (ambient—around 19°C—and ambient +4°C) and water availability . After 1 month development, at the corresponding growth conditions, gas exchange measurements and harvest were carried out (60 days—old plants).Alfalfa  and air probes connected to another CO2 infrared gas analyser were placed at the center of each module 60 cm above the plants.Ventilated [CO2], concentration was monitored continuously at the outlet module by an infrared analyser  whose signal was fed into a proportional integrative differential controller that regulated the opening time (within a 10-s cycle) of a solenoid valve that injected CO2 into both inlet fans because otherwise lateral mixing of CO2 in the chambers was not complete. The data were continuously recorded by a computer through analog-digital converters  using Windmill software with the Test-Seq programming tool (Biodata Ltd). A subroutine of this software controlled solenoid valves that kept one of two sets of CO2 cylinders open or closed  thus supplying the gas to the elevated CO2 tunnel. When CO2 concentration decreased below a fixed level, signaling that one of the cylinder sets was exhausted, the corresponding valve was closed and that of the other set opened.The  increased the time to reach a particular water stress , whereas partially irrigated plants (WD) were watered at 50% θv of WW plants. These θv levels were maintained throughout the experiment by daily measurement of transpired water  and replenishing the lost water. In order to reduce evaporation from the soil, pots were covered with a plastic sheet perforated with very small holes to allow stems to pass through. In order to supply all treatments with the same amount of nutrients, WW plants were alternately watered with Evans N-free nutrient solution and distilled water, while WD plants were always watered with Evans solution. Pots were rotated weekly in each module to avoid edge effects. In order to avoid differences due to chamber effects, the plants were moved from one greenhouse to another every month. All the determinations listed below were made at the end of the experiment, when the plants were 60 days old, in apical fully expanded leaves.When analysing the interaction between [CO2], temperature and water availability conditions was determined by harvesting after 1 month of growth. Twenty plants were collected per treatment combination. The plants were divided into leaves, stems, roots and nodules, and the fresh weight of these components was recorded. After drying at 60°C for 48 h, their dry weight was determined. Leaf area was analyzed with an electronic planimeter . Total dry matter (DM) comprised leaf, stem, root and nodule DM.Plant growth in the TGGs under the aforementioned . This was kept on ice for 20 min and then centrifuged at 12,000 g and 4°C for 25 min. The total soluble protein content of the supernatant was determined according to the Bradford method  was estimated from net photosynthesis and conductance measurements according to Farquhar and Sharkey (−1 [CO2] (A400 and A700 respectively), at the corresponding growth temperature and with a photosynthetic photon flux density of 1200 μmol m−2 s−1.Fully expanded apical leaves from 50-day-old plants were individually enclosed in a leaf chamber , and the gas exchange rate was measured with a portable photosynthesis system  under growth conditions. Net photosynthesis (A) and leaf conductance (g) were calculated as described by von Caemmerer and Farquhar . The lea Sharkey . Fully eA subsample of frozen leaf, stem, root and nodule from each plant was dried at 60°C for 48 h in small tin capsules and weighed. The nitrogen and carbon isotope composition of the samples was determined using a Flash 1112 Elemental Analyzer  coupled to an IRMS Delta C isotope ratio mass spectrometer through a Conflo III Interface .15N) using international secondary standards of known 15N/14N ratios (IAEA N1 and IAEA N2 ammonium sulfate and IAEA NO3 potassium nitrate) relative to N2 in air:R is the 15N/14N ratio.Nitrogen results were expressed in parts per thousand (‰) in the δ notation  and stored in tin capsules. N and C content were determined at the Serveis Cientifico-Técnics of the University of Barcelona  using an elemental analyser .2-fixing alfalfa plants grown under various environmental conditions and Regression analyses of axial patterns of δ15N) and Statistica 10, data analysis software system, version 10 .Statistical analyses were performed with the programs SPSS for Windows, version 15.0 , and whether these results varied according to the organ tested. Besides analysis of whole plants , each organ  was analyzed separately. Homoscedasticity was determined using the Levene test  to test for effects and interactions of the various combinations of three environmental factors . Correlation and simple regression models for leaf parameters  were used to determine R2 and p-values for each analysis.Following an exploratory-inferential approach, data analysis revealed that leaves were the organs that were the most influenced by environmental factors, so several descriptive statistical analyses were conducted only on data from leaves. Simple regression models were estimated for δThe results of this study were obtained for plants cultured in several independent series, at least one sample was analyzed for each of three independent series. Sample size varied depending on the analysis carried out, from 32  up to 192 .15N reflects the 15N abundance of plant main N source(s): available soil N for non-N2-fixing plants and atmospheric N2 for N2-fixing plants  the physiological partition of the N metabolism between shoot and root; (2) the N efflux; and (3) on the exudation of metabolites. The δ15N values of the distinct plant organs  show that leaf δ15N is the one more responsive to environmental factors . The combined effect of increased [CO2] and WD caused the more significant changes in leaf δ15N  did not show differences between the δ15N values of nodules, roots or stems (δ15N ≈ −1.0), while leaves presented δ15N values closer to zero  influenced the correlations between leaf δ15N and biomass and several physiological parameters   and ecosystem types  Figure . However15N to the combination of [CO2] and water availability, together with the observed low correlations between leaf δ15N and plant transpiration associated with high [CO2] and water deficiency  and C/N ratios  from bacteroids to the plant . This observation, together with further investigation of isotope fractionation during transport and metabolic processes, may provide useful information on the metabolism, transport and allocation of N in N2-fixing plants exposed to combined environmental stresses.Leaf δThe 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.


Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development. There is a well described and established clinical phenomenon that correlates increasing efficacy of disease treatment with decreasing time interval between disease inception and treatment application12While biomarker assays promise to provide exquisitely sensitive screening tools capable of early disease detection, thus shortening the time interval between onset and treatment, such tools generally offer little information regarding the spatial characteristics of tissue pathology within the living organism. Accurate information regarding the spatial characteristics of diseased tissue is not trivial in the case of patient care as clinicians must be able to distinguish between healthy and unhealthy tissues for a variety of applications including diagnostic confirmation, post-treatment disease monitoring, and surgical planning for tumor excision6Clearly, clinical imaging methods must develop in conjunction with improvements made in molecular methods of disease detection if we are to realize the full potential of either methodology to improve patient health. Identifying the MR characteristics of both healthy and diseased tissues at the cellular level will provide insight into how such early stage—often asymptomatic—disease conditions affect MR signal and contrast characteristics. Thus, while tissue microstructure currently is—and may remain—inaccessible to direct observation in the clinic, possessing a thorough understanding of how specific disease states affect MR contrast parameters in the microenvironment will lead to an understanding of how pathological changes occurring at the microscopic level present themselves in clinical scans. Such cellular-resolution data (<10 μm isotropic) must be collected using ultra-high-field imaging equipment as these are the only systems capable of quantifying MR contrast parameters at the relevant physical scale. Hence, there is a current need for MR imaging systems capable of resolving mammalian cellular architecture for use in healthy and diseased tissue characterization studies.Preliminary MR imaging studies of mammalian cellular structure have been conducted exclusively in fixed tissue samples due to the lengthy collection time needed to produce cellular-resolution scans8101112MR microscopy studies conducted on living tissue explants have traditionally suffered from low—i.e. non-cellular—resolution, as well as imprecise control of perfusate conditions at the site of the isolated tissue. The inability to directly visualize cellular structure has since been overcome through vast improvements in radio-frequency (RF) microcoil technology1415161718192021232425To address the need for reliable, MR compatible microperfusion hardware for use with living explants, we have designed and fabricated a tissue retention/perfusion system specifically for use in cellular-resolution studies. The prototype system interfaces to a modified, commercially available micro surface coil . In addition to detailed schematics and fabrication procedures, we report the results of tests quantifying the dissolved oxygen content and pH of our artificial cerebrospinal fluid (aCSF) as well as MR signal stability over time in experiments where our in-bore oxygenator is in use. In the case of dissolved gas and pH quantification, results from tests using the in-bore oxygenator are compared to equivalent experiments employing an external membrane oxygenator device of earlier designA 500 μm diameter micro surface-coil  was modified to interface with the micro perfusion rig. A channel (15 mm LN × 3 mm HT × 4 mm D) was made in the back of the coil’s plastic assembly. Two nylon spacers (6 mm LN × 4 mm HT 0.5 mm W) were glued flanking the channel and filed down to the coil’s semicircular contour. Two narrower, bilateral groves (3 mm HT × 1.5 mm D) were extended from the channel until they wrapped around the coil assembly. A hole (2 mm Dia. × 14 mm D) was drilled in the top of the coil assembly centered along its width and located directly behind the chip plate.o to accommodate a silicone gasket  that was adhered using silicone sealant. A horizontal channel (2.5 mm HT × 1 mm D) was milled into the perfusion well’s closed end to accommodate a cable tie . Inflow and outflow lines  were held in place with urethane and were offset (3 mm) within the well’s interior to maximize turbulent flow and minimize metabolite gradients. Nylon retention ring  and mesh  were fashioned by hand from a nylon washer  and woven nylon sheet .High gas-retention perfusion lines  were employed between the gassed perfusate reservoir and oxygenators. In the external oxygenaor setup, this line also bridged the oxygenator and perfusion well. A peristaltic pump  drove perfusion (2 ml/min). For the perfusion chamber (150 μm volume), acetal rod was machined into a single, open-ended configuration . The open end was beveled 30A 5 mm diameter NMR tube  was modified into a glass pipe 16.5 cm in length to serve as the gas inlet (interior) chamber. A hole (1.5 mm Dia.) was cut in the top of the 5 mm tube cap to accommodate the carbogen supply line. Next, a 10 mm NMR sample tube  was modified into a glass pipe 18 cm long to form the gas exchange (exterior) chamber. A hole (6 mm Dia.) was cut in the center of the 10 mm tube’s cap to nest the 5 mm tube assembly. Two additional holes (1 mm Dia.) were made in the top of the 10 mm tube’s cap. One served as the exchange-membrane inlet while the other remained open to vent incoming carbogen. Silicone exchange tubing  was introduced through the top 10 mm tube cap, coiled tightly around the nested 5 mm tube inside the gas-exchange compartment, and passed through a second 10 mm NMR tube cap located at the oxygenator’s base. This cap housed an acetal peg  at its center held in place by urethane. Externalized lengths of gas exchange tubing were minimized to two 10.0 mm segments joined via a 1/16” nylon coupler  to perfusion lines  offering slightly higher gas retention. The perfusion line exiting the bottom of oxygenator was coupled directly to the perfusion chamber’s inflow line. The lower 10 mm NMR tube cap was attached without urethane as a means to access the interior of the oxygenator device for membrane replacement.3, 1.5 mM KH2PO4, 1.4 mM MgSO4·7 H2O, 2 mM CaCl2·2 H2O, 3 mM KCl, 10 mM Glucose; osmolality = 300 mOsm) were collected using an oxygen meter  interfaced to a volume-limited microelectrode probe . Multiple trials (n = 6) were conducted in which dissolved oxygen measurements were taken from the perfusate reservoir which was directly bubbled with 95% O2, 5% CO2 carbogen (positive control) or from the perfusion chamber during trials in which the in-bore oxygenator was supplied with gases of varying oxygen content . In order to compare the in-bore oxygenator’s function to the external membrane oxygenator, additional measurements (n = 10) were made in the perfusion chamber while employing the external oxygenator device supplied with carbogen gas  during rig operation (2 ml/min).Measurements for percent dissolved oxygen of aCSF perfusate  while the same gas mixture was supplied to both external and in-bore oxygenators. Results from the eight trials were averaged and the means of both treatment groups compared to the target physiological pH range (7.3–7.4).pH readings of aCSF perfusate were assessed using an Accumet Basic pH meter  interfaced to a low-volume sample probe . Measurements were taken in the perfusate well (n = 8) with the operational perfusion rig (2 ml/min) interfaced to either the external or in-bore membrane oxygenator. The aCSF reservoir was directly bubbled with carbogen gas  in the available scan time. Data analysis was performed offline using a statistical toolbox (Microsoft Excel) and Matlab®. Fourteen consecutive diffusion-weighted, spin echo scans  were collected (n = 3) comparing signal stability over time . Manual shimming was employed to ensure homogeneous field conditionsAll imaging was performed on a 600 MHz spectrometer (Oxford) equipped with microimaging gradients . Sample temperature (23 °C ± <1 °C) was measured continuously and kept constant using a feedback loop from the thermocouple to a chiller unit  which controls air temperature flowing through the spectrometer bore. All data was collected using a diffusion-weighted, spin echo sequence. Diffusion weighting was kept constant at b = 1200 s/mm2, 5% CO2), 4 °C aCSF bath before being introduced into the microperfusion rig. Samples were acclimated to 23 °C under continuous perfusion for a period of 1 h prior to imaging. Diffusion weighted images  were collected at long and short imaging intervals  with variations in perfusion conditions . Nonperfused cortex data was generated with a live, acute slice placed in the rig and imaged  over a 15.5 h timecourse in the absence of aCSF perfusate exchange. Similarly, stable control data was generated using a fixed slice over an 18.5 h timecourse with identical imaging parameters in the absence of perfusion. Diffusion signal intensity (arbitrary units) was recorded over a timecourse lasting 15.5 h to 21.5 h depending on experimental group.Acute cortical slices  from rats were isolated via vibratome in a continuously gassed  and three mixtures of carbogen (5% CO2) with varying concentrations of oxygen  balance nitrogen were tested in the in-bore oxygenator. Average dissolved oxygen readings for the results of six trials were 23.0%, 96.2%, 59.1% and 19.2% for ambient air (20–22%), 95%, 60%, and 19% O2 gases respectively. Results are compared to positive control trials (n = 6) conducted in the perfusate reservoir during direct bubbling of 95% O2, 5% CO2 carbogen gas: 95.1% O2 measured. Repeat measurements indicate a 100% gas saturation effect (i.e. percent saturation at tissue well matches the % O2 content of supplied gas) when using the in-bore oxygenator. Conversely, when employing the external oxygenator, dissolved gas content at the site of tissue perfusion was not equivalent to the percent oxygen content of the supply gas  and the perfusion chamber (43.43% O2) as measured using the external membrane oxygenator.Dissolved oxygen content measured in aCSF at the tissue well (n = 6) as a function of gases with variable oxygen content as supplied by the in-bore oxygenator device are shown . Atmosphpply gas . In repe2, 5% CO2) measuring pH at the site of tissue perfusion are presented  as a function of experiment time  is reported for two experimental conditions: trials with continuous (2 ml/min) perfusion and static trials with no perfusion (control) . AverageTissue stability as indicated through the diffusion-weighted MR signal intensity was analyzed for all live slice data sets as a function of experiment time (15.5 to 21.5 h) . The repl slices  exhibit 2) saturation characteristics tested using multiple concentrations of oxygen in pre-mixed carbogen supply gas  of our aCSF’s dissolved O2 content was lost during travel from an external membrane oxygenator to the tissue well (5 m) highlights the need for additional MR-specific hardware development. That such results were obtained even when employing high-retention, low gas permeability perfusion lines  suggests that simple engineering solutions retrofitted to preexisting perfusion equipment will not be adequate to achieve appropriate experimental conditions in ex vivo MR microscopy studies.In the present study, we describe a newly designed and fabricated microperfusion system and oxygenator device for use in explant studies conducted in ultra-high-magnetic field environments. These components were designed specifically to conform to the intense spatial and material limitations imposed by high-field MR imaging equipment used in cellular studies and thus possess particular features not available in any existing commercial microperfusion equipment. Importantly, the rig materials are all compatible with the electromagnetic environment (static and time-varying fields) of the MR experiment so that they are both safe to handle and do not interfere with measurement quality (e.g. by deteriorating the main field homogeneity). What’s more, our in-bore oxygenator device exhibited 100% dissolved oxygen  supplied to bicarbonate-buffered tissue culture systems. Addition of CO2 into such a system results in an increase in carbonic acid and thus an increase in the bicarbonate and hydrogen ions this compound dissociates into ultimately resulting in a lowering of pH. Conversely, if conditions in the media become too acidic, excess hydrogen ions will be scavenged by bicarbonate and become incorporated in water molecules upon the release of CO2 resulting in an increase of pH. Maintaining physiologically relevant pH conditions in a bicarbonate buffered media thus depends on a constant supply of CO2 gas. The concentration of CO2 present in carbogen mixtures used in the current study (5%) is an industrial standard known to be sufficient to maintain these conditions; however, when this was used as a supply gas in conjunction with the external membrane oxygenator, pH measured at the site of tissue perfusion was 8.13: excessively alkaline compared to our target pH range of 7.3–7.4. These data were observed despite target pH conditions being maintained in the aCSF reservoir used to supply the rig with perfusate  ensured efficient metabolite delivery and waste removal5052In the case of our live tissue experiment performed on acute cortical slices, diffusion signal stability was maintained for a period of 15.5 h following time of death (T.O.D.) and subsequent sample preparation prior to imaging . Slice tIt is our hope that the current rig design described and tested here will be employed in cellular resolution MR studies of living tissue explants as well as aid in the future development of MR-compatible perfusion devices.How to cite this article: Flint, J. J. et al. A Microperfusion and In-Bore Oxygenator System Designed for Magnetic Resonance Microscopy Studies on Living Tissue Explants. Sci. Rep.5, 18095; doi: 10.1038/srep18095 (2015).


It demonstrates that such nanowire systems exhibit intriguing dipole orders, due to the combining effect of the anisotropic electrostatic interaction of the nanowire, the SrTiO3-layer-modified electrostatic interaction and the multiphase ground state of BaTiO3 layer. Particularly, a strong polar-toroidal coupling that is tunable by the SrTiO3-layer thickness, temperature, external strains and electric fields is found to exist in the nanowires, with the appearance of fruitful dipole states  and phase boundaries. As a consequence, an efficient cross control of the toroidal (polar) order by static (curled) electric field, and superior piezoelectric and piezotoroidal responses, can be achieved in the nanowires. The result provides new insights into the collective dipole behaviors in nanowire systems.The collective dipole behaviors in (BaTiO Ordering of the spin, charge, lattice or other degrees of freedom are fundamental phenomena in condensed matters, and endow materials with an incredible variety of properties. For example, the cooperative alignment of electric dipoles (spins) leads to ferroelectricity (ferromagnetism), which forms the basis of many device designs, such as memories, sensors and actuators. The coexistence of different orders in a material or structure, namely a multi-order state, is promising in developing multi-state devices. An even more fascinating point of a multi-order state lies in the coupling of different orders134Recently, interests have been attracted by complex dipolar states with toroidal order, particularly those found in zero-dimensional magnets6810111213141617183 and KbNO3) and under an anisotropic screening condition, PTMO states may be developed. Indeed, via a first-principles-based simulation, recently Louis et al. have found a PTMO state in nanocomposites made of periodic arrays of BaTiO3 nanowires embedded in a SrTiO3 matrix3 exhibit a PTMO state under axial screening conditionPTMO states are likely to be found in two types of ferroelectric nanosystems. The first type nanosystems are those composite structures consisting of polar components and toroidal components, such as nanodots embedded in polar matrix12213)m/(SrTiO3)n nanowires (3-layer-modified electrostatic interaction and the multiphase ground state of BaTiO3 layer (3)m/(SrTiO3)n, m and n refer to the thickness, in unit cells, of the (001) BaTiO3 and (001) SrTiO3 layers, respectively. In the nanowires, the polar and toroidal orders are intimately coupled as they are both carried by the same degree of freedom , similar with the ferroelectric-ferroelastic state in ferroelectrics where the polar and strain orders are both dipole related. As further revealed by the features of electrical and mechanical responses of the nanowires under external fields, such polar-toroidal coupling brings us an efficient cross control of the toroidal (polar) order by static (curled) electric field, as well as superior piezoelectric and piezotoroidal responses in the nanowires.Using first-principles-derived simulations, here we show that (BaTiOanowires  exhibit O3 layer . In the 3)10/(SrTiO3)n nanowires with n = 0, 2, 3 and 4 and a lateral size being Lx = Ly ≈ 4 nm during a cooling-down process is depicted in g and the net polarization P as shown in A, B, C, D and E) exist in the nanowire systems. They are (1) the paraelectric state A at high enough temperatures in all the nanowires, (2) a purely polar state B (Pz ≠ 0 and gz = 0) in nanowires with n ≤ 2 at moderate temperatures, (3) a PTMO state C (Pz ≠ 0 and gz ≠ 0) in nanowires with n ≤ 3 at low temperatures, (4) a purely toroidal state D (Pz = 0 and gz ≠ 0) at moderate temperatures in nanowires with n ≥3, and (5) a distorted toroidal state E with the dipoles forming a “polydomain” pattern in the x-y plane (Pz = 0 and gz ≠ 0) at low temperatures in nanowires with n > 3. To be clearer about the domain states, in B of (BaTiO3)10/(SrTiO3)2 at 250 K, state C of (BaTiO3)10/(SrTiO3)2 at 150K, state D of (BaTiO3)10/(SrTiO3)4 at 250 K, and state E of (BaTiO3)10/(SrTiO3)4 at 150 K. It is original and important to show such a variety of dipole states with distinct polar and toroidal ordering exists in composite nanowires.The collective dipole configurations of 10/(SrTiO3)n nanowires with a sufficiently thin SrTiO3 layer (n ≤ 2) at moderate temperatures adopt a z-directed polar state B that commonly found in many ferroelectric nanowire systems12223 layers, the SrTiO3 layer polarizes by electrostatic effect in a magnitude of polarization smaller than that of the BaTiO3 layer, similar to the behavior of (BaTiO3)m/(SrTiO3)n superlattices25B but adopt a PTMO state C with a polar order along the z-axis and a toroidal order in the x-y plane. Dipoles of the BaTiO3 layer tilt at low temperature due to the rhombohedral ground state of BaTiO3. Nevertheless, a state with net in-plane polarization is not allowed by open-circuit the condition. As a result, the tilted dipoles have to rotate around the z-axis to form a vortex-like pattern. Such a PTMO state is similar to that found in BaTiO3 nanowires embedded in a SrTiO3 matrix173 nanodots under an anisotropic screening3 nanowires273 layer also simultaneously exhibit polar and toroidal orders due to the proximity effect, although with smaller polarization and toroidization than the BaTiO3 layer.The appearance of fruitful dipole states results from the combining effect of the anisotropic electrostatic interaction of the nanowire, the SrTiO3/SrTiO3 composite structure in previous works173 nanowires periodically embedded in three dimensional (3D) SrTiO3 matrix. The SrTiO3 matrix surrounds the side surface of the BaTiO3 nanowires and imposes an open-circuit like condition to the nanowires at the side surface. In case of large distance between the nanowires, the dipole state of the individual nanowire is similar to a strained single BaTiO3 nanowire. In this work, we focus on an entirely new structure, i.e., a single nanowire system with BaTiO3 and SrTiO3 layers (or blocks) periodically alternating along the wire direction. The individual BaTiO3 and SrTiO3 layers are confined in all dimensions, yet interact with others via short-range interfacial interaction and long-range electrostatic interaction along the wire direction. It is an important point of our work to put forward the concept of such a composite nanowire structure to show a large and tunable polar-toroidal coupling.We notice that the BaTiO3 layer (n ≥ 3) adopts a purely toroidal state D at moderate temperatures rather than the purely polar state B, indicating that the SrTiO3 layer thickness strongly affects the dipole behavior of the nanowires. This is due to that a thicker SrTiO3 layer decreases the layer electrostatic effects that help stabilize polar order and imposes a more open-circuit-like condition to the BaTiO3 layer. At lower temperatures, as the dipoles tilt toward z-axis, the purely toroidal state is not stable and would change into either a PTMO state C 10/(SrTiO3)4 nanowire 10/(SrTiO3)2 nanowire under an external z-directed static electric field EH = Eaez at T = 50 K is depicted in C at T = 50 K. The important observations are following. (1) For such PTMO state with toroidal order, it would transform into a purely polar state under the action of electric field. Different from the complicated path predicted in nanoparticlesz-axis. (2) The system’s toroidization exhibits a large and linear response at low field region (see the gray area in 3 nanowires embedded in a SrTiO3 matrixEa within the coercive field (~1 MV/cm), the toroidization changes linearly with Ea (either increases or decreases depending on the direction of the field versus the polarization) in a law of gz(Ea) = g0 − χEa, where g0 is the zero-field toroidization and the toroidal susceptibility of static field χ is about 2.8 e/V by a linear fitting. Beyond the coercive field, an increase of Ea monotonously increases the polarization magnitude and decreases the toroidization, with a vanishing of toroidal order at a field of about 7 MV/cm.The sensitivity of the state stability to SrTiO3)10/(SrTiO3)2 nanowire on static field Ea at different temperatures. As expected, the vanishing field of toroidal order significantly decreases as the temperature increases. And more importantly, the susceptibility χ increases as the temperature approaches to the phase boundary between polar state B and PTMO state C. To quantify the susceptibility at small field, we calculate it by the statistical correlation between gz and Pz12χ of the PTMO state C monotonously increases with temperature and reaches about 12 e/V at around 210 K (see χ is observed in the PTMO state C of (BaTiO3)10/(SrTiO3)3 nanowire, with a magnitude of over 150 e/V found near the phase boundary between purely toroidal state D and PTMO state C . These results clearly demonstrate the large and tunable polar-toroidal coupling in our composite nanowire system. It is noteworthy that, in single-order states B, D and E, the calculated statistical correlation between toroidization and polarization is misleading due to the absence of stable polar or toroidal order. A strong coupling of the toroidal order to external static field can still be achieved in toroidal states D and E, since these states would transform into PTMO state C once the field is exerted.10 K see . Similar3)10/(SrTiO3)2 nanowire at T = 250 K, its evolution of dipole state under a curled electric field EC = Saez × r is depicted in B (with Pz of about 0.32 C/m2) transforms into a purely toroidal state when Sa exceeds 4 × 1016 V/m2. Such a large response of net polarization to curled field reflects the easy titling of dipoles. As indicated by the Sa dependence of the polarization of the (BaTiO3)10/(SrTiO3)2 nanowire at different temperatures 10/(SrTiO3)n nanowires with n = 2, 3 and 4 as a function of temperature under zero external fields. For each nanowire, a change of strain state is accompanied with the transition of dipole states. Particularly, a large change of axial strain happens near the phase boundary of paraelectric state and state B in the (BaTiO3)10/(SrTiO3)2 nanowire, and the phase boundary of state D and state C(E) in (BaTiO3)10/(SrTiO3)3(4) nanowire, indicating large electromechanical responses near these phase boundaries. 3)10/(SrTiO3)2 and (BaTiO3)10/(SrTiO3)3 nanowires. Two remarkable observations are as follows. (1) Both nanowires have an overall notable piezoelectric coefficient 3 nanodot, which has a large We now turn to the electromechanical responses of the nanowires when they are under external electric fields or strain constraint, which are related to the well known piezoelectric and the less reported piezotoroidal effectstion see .3)10/(SrTiO3)2 nanowire in polar state A 10/(SrTiO3)2 nanowire as a function of curled field at T = 250 K, from which a large inverse piezotoroidal effect is indeed seen. The nanowire exhibits a sharp response peak at small field, similar with the strain response at small field of the BaTiO3 nanodot undergoing vortex rotation (see the insert). Importantly, the strain response to the curled field of the nanowire exhibits a much more linear feature than that of the nanodot. As the field reaches 5 × 1016 V/m2, a strain change of about 0.95% is achieved in the nanowire, which is much larger than the 0.45% strain change of the nanodot. Strong direct piezotoroidal responses are also found in the nanowires. As a typical result, for the (BaTiO3)10/(SrTiO3)2 nanowire at T = 250 K m/(SrTiO3)n composite nanowires. We demonstrate that a large polar-toroidal coupling exists in this entirely new composite system. In this composite nanowire system, the toroidal/polar coupling can be well tuned by the SrTiO3-layer thickness, temperature, external strains and electric fields, and brings fruitful dipole states and phase boundaries in the nanowires. Importantly, efficient cross control of the toroidal (polar) order by static (curled) electric field, and superior electromechanical responses can be achieved in the nanowires. We hope that the intriguing dipole behavior of the composite nanowires and their striking features in electrical and electromechanical responses will be experimentally confirmed soon.First-principles-derived simulations are preformed to investigate the dipole state in  order by static (curled) electric field. Secondly, it demonstrates that fruitful dipole states can be achieved in the composite nanowires by making use of the anisotropic electrostatic interaction of the nanowire, the layer-modified electrostatic interaction and the multiphase ground state of bulk ferroelectric. The layer-modified electrostatic interaction provides an alternative control of the nanowire’s anisotropic screening condition, and indicates fruitful controllability of the dipole behavior in ferroelectric nanosystems under mixed electric boundary conditions3 solid solutionsu i, inhomogeneous strain η I, alloy species σi , local strain ηloc resulting from the difference in ionic size between alloy species, and homogenous strain ηH,We perform the simulations using a first-principles derived effective Hamiltonian of TiOEave describes the total energy associated with TiO3 system in the virtual crystal approximation (VCA)Eloc gathers energy associated with alloying effects going beyond the VCA, and the last term takes into account the effect of external electric field E (Z is the effective charge). Explicit expression of Eave and Eloc can be respectively found in ref. 3)m/(SrTiO3)n structureswhere the first term Note that the surface polarization might have a significant impact on the collective dipole behavior of the nanowire. To describe this surface effect, an additional energy term has to be added to the Hamiltonian as36S referring to the free surfaces of the nanowire and index i running over the B sites closest to the surface. α|| runs over the axes along the surface. α|| component of the local mode that is the closest from the i site along the positive (negative) direction of the α|| axis. The p and t parameters quantify how vacuum affects the out-of-plane components of the local modes and inhomogeneous strains near the surface, respectively. s characterizes the change, with respect to the bulk, of the short-range interaction between the in-plane components of the local modes near the surface. The p, t, and s parameters can be determined from first-principles calculations on BTO (BaO-terminated) and STO surface (SrO-terminated).The three terms of 3)m/(SrTiO3)n nanowires with a sufficiently long length, which is mimicked by means of periodicity along the longitudinal z axis. The wires are chosen to be in rectangular shape, with their x, y, and z axes along the pseudocubic [100], [010] and [001] directions of the perovskite structure, respectively. The lengths of simulated supercells along x and y axes are Lx = nxa and Ly = nya, respectively, while the z-axis periodic length Lz is Lz = (m + n)a, with a ≈ 4 Å being the lattice constant of the primitive five-atom unit cell. We use the energy in We consider  reported in literature3 nanodots is not much affected by the surface. We have also conducted simulations on nanowires with different sizes . The result further indicates that the dipolar orders found in this work are not sensitive to the surface polarization when the size of the nanowire is not too small .