Circaeaster agristis, a narrowendemic and threatened species, in 2010 and 2011. We also examined the effects ofNND (nearest neighbor distance) and LNS , within radii of0.1 m, 0.2 m and 0.3 m, on reproduction in two of the populations in 2011.Habitat fragmentation and the resulting decline in population size and densitycommonly reduce the reproduction of rare and threatened species. We investigatedthe impacts of population size and density on reproduction in more than 30populations of Population size did not affect fruit (seed) number and fruit set in eitheryear studied. Population density had an indirect negative effect on fruit numberand fruit set as a consequence of a negative effect on plant size in 2010, but hadno effect on fruit number and fruit set in 2011. Within populations, individualfruit number did not change, and individual fruit set increased independent ofplant size, in response to increasing NND. Both individual fruit number andindividual fruit set increased, independent of plant size, with increases in LNSwithin a 0.1 m radius, but did not change with increases in LNS within radii ofbetween 0.1 m and 0.2 m radii or between 0.2 m and 0.3 m.C. agristis is scale-dependent. In contrast to the generallyaccepted idea that fragmentation reduces plant reproduction, reproductive successmay increase in sparse populations or increase in response to decreases in LNS inC. agristis.The effect of habitat fragmentation on reproduction of The online version of this article (doi:10.1186/s40529-015-0095-5) contains supplementary material, which is available to authorizedusers. Habitat fragmentation is a worldwide phenomenon, and is considered to be one ofthe major threats to the persistence and viability of plant populations . If fruWhen assessing the effects of habitat fragmentation on reproductive success,some researchers have focused on the population level: population size  and/or mean population density  andindividual (NND and LNS) levels, in 2010 and 2011. The data obtained here may assistin understanding the effects of fragmentation on plant reproductive success, andprovide useful guidelines for the management and conservation of C. agristis.In this study, we explored the effects of habitat fragmentation on reproductivesuccess in the endangered annual Circaeaster agristis Maxim.is the onlyspecies of the genus Circaeaster, which is amember of Circaeasteraceae together with another monotypic genus, Kingdonia  tall. The flowers are green,hermaphroditic and about 1 mm long, have two or three tepals, one or two stamens,and one carpel . Circaeand Jin, ). It rep et al., ).FlowerAbies faxoniana) andspruce-cypress forest , subalpine shrubs and meadow (3500–4400 m), and sparse alpinevegetation (4400–4900 m).Our experiment was conducted in the Wanglang Nature Reserve , southwestern China. The verticaldistribution of vegetation types in this region includes mixed forests of conifersand broadleaf trees, and broadleaf deciduous forest (2300–2600 m), fir forest. We then estimated themean density of each population by dividing its population size by its area. Thelog-transformed mean population density was significantly correlated to thelog-transformed population size in 2010  and 2011.In 2010, we identified 34 From early to mid-August 2010 and 2011 in each population, 30 individuals wererandomly marked, except in populations with less than 30 individuals, in whichcase all of them were marked. The number of leaves, flowers (flower scars) andmature fruits were counted on each of the marked individuals.In August 2011, we randomly marked 35 focal individuals in each of populations2 (intermediate population density) and 37 (low population density), with thefocal plants separated from each other by more than 1 m. For each focal plant, thenumber of leaves, flowers (flower scars) and mature fruits were counted. Theeffect of NND was quantified by measuring the distance from each focal plant tothe nearest conspecifics. The effect of LNS was quantified by counting the numberof conspecifics within radii of 0.1 m, 0.2 m, and 0.3 m around each focal plant.We included areas with multiple radii because the effects of LNS can bescale-dependent .rp = 0.865, P < 0.001, n = 34; 2011:rp = 0.861,P < 0.001, n = 35), fruit number  and fruit set in 2010 , we useda multiple regression analysis to analyze whether population size and meanpopulation density had an influence on mean population fruit number in 2010 and2011 or on fruit set in 2010, when accounting for mean population plant size.All variables except mean population fruit set were natural-log-transformed tostabilize variances. For all analyses, we examined the residuals for departuresfrom normality (Shapiro-Wilk test).To understand the effects of population size and mean population density, wefirst calculated the population means of leaf number per plant (plant size),flower number per plant, fruit number per plant, and fruit set (fruits/flowers)per plant, in both 2010 and 2011. We first used linear regression analysis toevaluate the effects of population size and mean population density on meanpopulation fruit number or fruit set for each year separately. Because thepopulation means of plant size were highly correlated with mean populationflower number , individual fruit number  andindividual fruit set , we then included individual plant size as a covariate in eachof the analyses to evaluate whether NND had effects on fruit number or fruit setwhen differences in individual plant size were accounted for. All variables exceptfruit set were natural-log-transformed to stabilize variances. For each analysis,initial models included all possible two-way or three-way interactions involvingcovariates. Non-significant interactions were dropped from the model usingbackwards elimination. For all analyses, we examined residuals for departures fromnormality.We first used analyses of covariance to analyze fruit number and fruit set ofselected individuals as a function of population (random factor) and NND(covariate). Because individual leaf number (plant size) was highly correlatedwith individual flower number  and 0.1 m LNS (covariate), 0.2 mLNS (covariate) and 0.3 m LNS (covariate). We considered 0.2 m LNS and 0.3 m LNSas the number of additional individuals beyond 0.1 m LNS within 0.2 m, and thenumber of additional individuals beyond 0.2 m LNS within 0.3-m, respectively.Multicollinearity was assessed by inspection of variance inflation factors, whichwas always ≤ 2.1, indicating that the level of collinearity was not problematic. We theP < 0.001; all variables except fruitset were log-transformed). All correlations were conducted using theselog-transformed variables, where applicable.Population means for leaf number per plant varied from 6.5 to 13.0 in 2010 and7.1 to 11.7 in 2011; population means for flower number per plant varied from 6.5to 22.2 in 2010 and 9.4 to 32.8 in 2011; population means for fruit number perplant varied from 1.7 to 13.1 in 2010 and 3.0 to 11.9 in 2011. Finally, populationmeans for fruit set per plant varied from 0.14 to 0.59 in 2010 and 0.19 to 0.57 in2011. All measured traits differed significantly among populations within the sameyear  or 2011   or 2011 . Meanwhile, mean population fruit number was negativelycorrelated to mean population density in 2010 , and was not significantly correlated to mean populationdensity in 2011   or 2011 . Furthermore, mean population plant size was negativelycorrelated to mean population density) in 2010 .Mean population fruit number was not correlated to population size in either2010  or 2011  . Mean population fruit set was negativelycorrelated to mean population density in 2010 , and not correlated to mean population density in 2011 .Mean population fruit set was not related to population size in either 2010. Mean NND was 0.020 m and 0.044 m  in populations 2 and 37 respectively, and differed significantly betweenpopulations .Individual plants of populations 2 and 37 respectively produced an average of10.3  and 10.6  leaves, and10.5  and 11.8  fruits. Meanfruit set of individual plants was 0.45  and 0.47 in populations 2 and 37 respectively. All ofthese measured traits did not differ significantly between populations .Individual fruit number did not vary with NND irrespective of whether or notindividual plant size was included in the analysis of covarianceTable a. IndiviP < 0.05).In populations 2 and 37, the mean 0.1 m LNS was 15.8 and 9.5 , respectively. Similarly, the mean 0.2 m LNS was24.3  and 12.3 , respectively,while the mean 0.3 m LNS was 28.3  and 18.6 , respectively. All pairwise contrasts between populations weresignificant , and was not correlated to 0.2 m LNS  or 0.3-m LNS .Individual fruit production was negatively correlated to 0.1 m LNS , suggesC. agristisvaried between years, from negative in 2010 to neutral in 2011. As indicated inTable C. agristis may bedetectable only when dense populations are surveyed. Our findings support the ideathat a 1-year study may not provide a realistic view of the effects offragmentation on plant reproduction . Howeved Yates, ).C. agristis in ourstudy increased in response to increasing NND and decreased with increasing LNS atthe 0.1 m spatial scale, and these relationships remained even when individualplant size was accounted for. These results are highly suggestive ofintra-specific competition for resources: competition for resources decreased withincreasing NND and increased with increasing LNS at the 0.1 m spatial scale duringfruit maturation. Individual fruit production in C.agristis decreased with increasing LNS at the 0.1 m spatial scale butdid not change in response to NND when individual plant size was accounted for;this indicates that competition for resources between the focal individual and thenearest conspecific individual may have been too weak to affect fruit productionin the focal plants. Similarly, individual plant size of C. agristis decreased with increasing LNS at the 0.1 m spatial scalebut did not change in response to NND, suggesting that intraspecific competitionfor resources between neighboring plants during growth was not strong enough to bedetected when only the nearest conspecific was considered. These results alsosuggest that plants of C. agristis with fewerneighbors within a 0.1 m radius would benefit from not only a direct negativeeffect of LNS on reproductive success, but also from an indirect negative effectof LNS on reproductive success, namely via its effect on plant size. This isbecause large individuals typically have a higher fruit set than smallindividuals. However, these effects were not evident at the 0.2 m and 0.3 mspatial scales, suggesting that competition for resources in C. agristis occurs on very local scales.Many studies have shown that the reproductive success of individual plants maydecrease in response to increasing NND or decreasing LNS due to pollen limitation, our st.C. agristis. First, our finding that C. agristis populations are distributed patchily onforest floors after fragmentation suggests that the maintenance of C. agristis habitat should be a priority. Second, smalland sparse populations are as important as large and dense populations from aconservation perspective, because the former will be at least as successful, if notmore, at reproduction, compared to large and dense populations. Third, a programaimed at re-establishing populations of this species should consider increasing thespacing between plants (compared to populations in the wild), which may reducecompetition for resources and thereby increase fruit production.Our study has implications for the conservation and management of


The isolated coliforms grew successfully on a mineral medium with crude oil vapor as a sole source of carbon and energy. Those strains also grew, albeit to different degrees, on individual n-alkanes with carbon chains between C9 and C36 and on the individual aromatic hydrocarbons, toluene, naphthalene, phenanthrene, and biphenyl as sole sources of carbon and energy. These results imply that coliforms, like other hydrocarbonoclastic microorganisms, oxidize hydrocarbons to the corresponding alcohols and then to aldehydes and fatty acids which are biodegraded by β-oxidation to acetyl CoA. The latter is a well-known key intermediate in cell material and energy production. E. coli cells grown in the presence of n-hexadecane (but not in its absence) exhibited typical intracellular hydrocarbon inclusions, as revealed by transmission electron microscopy. Raw sewage samples amended with crude oil, n-hexadecane, or phenanthrene lost these hydrocarbons gradually with time. Meanwhile, the numbers of total and individual coliforms, particularly Enterobacter, increased. It was concluded that coliform bacteria in domestic sewage, probably in other environmental materials too, are effective hydrocarbon-biodegrading microorganisms.Raw, domestic sewage of Kuwait City contained about 10 Environmental pollution with spilled oil and waste hydrocarbons has become a problem of major concern on the global scale. Those compounds contaminate water and terrestrial and atmospheric ecosystems. Early reports estimated that about 0.08 to 0.4% of the internationally produced crude oil is spilled into the environment . ProbablIn bioaugmentation, an important question regarding the choice of hydrocarbonoclastic microorganisms to be inoculated should be considered. Essential prerequisites are that those organisms should easily succeed in colonizing the new environment and in standing competition with the already existing inhabitants , 17. The Enterobacter, Klebsiella, Escherichia, and others. The literature includes several reports by other investigators on hydrocarbon-degradation by such coliforms [ Klebsiella and Enterobacter [In the course of our work on bioremediation of desert and aquatic ecosystems that had been heavily polluted with oil during the greatest man-made oil spill at the end of the 1991 Gulf-war, we used to isolate hydrocarbonoclastic bacteria that according to their 16S rRNA-gene sequences were coliforms. Such bacteria belonged predominantly to the generaoliforms –23. Evenrobacter –26. Alsorobacter  and consrobacter –31 were  E. coli, for example, makes up about 10% of the domestic sewage microflora worldwide.Coliform bacteria belong to the family Enterobacteriaceae, which comprises facultative anaerobic bacteria naturally inhabiting digestive tracts of warm-blooded animals including man. Therefore, such microorganisms are continuously discharged with feces, andThe major objective of this study was to investigate the hydrocarbonoclastic potential of coliforms from local domestic sewage samples. For this, microbiological, biochemical, and cytological techniques of analysis were integrated to obtain a more comprehensive evidence for the hydrocarbonoclastic potential of the obtained isolates. Should coliforms prove to be effective oil-biodegraders, domestic sewage would be a cost-effective, accessible source of microorganisms to be used in oil-bioremediation via bioaugmentation. Within this context, domestic sewage frequently receives waste hydrocarbons as constituents of disposed plant and animal wastes. Enterobacter colonies were pink with dark centers, Klebsiella colonies mucoid pink, and Escherichia coli colonies black with green metallic sheen The isolates were further characterized according to their potential for acid and gas production from lactose fermentation at 37°C and 44°C, following established routine procedures. Representative strains were isolated, purified, and characterized by comparing the sequences of their 16S rRNA-genes with sequences of type strains in the GenBank database. The method was essentially as follows: The total genomic DNA was extracted with PrepMan Ultra Sample Preparation Reagent  following the manufacturer's description. The 16S rRNA-genes were amplified by polymerase chain reaction (PCR) using a Veriti Thermal Cycler  and a standard protocol with initial denaturation at 95°C for 5 min and 32 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and primer extension at 72°C for 1 min. The mixture PCR contained puReTaq Ready-To-Go PCR Beads , 25 ng of DNA template, 1 μL of each of the universal primer combinations GM5F (5′-CCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCAATTCMTTTGAGTTT-3′) [μL with molecular water . Partial sequencing of the 16S rRNA-genes was done using a BigDye version Terminator Kit . The mixture contained 20 ng of the DNA template, 2 µl of the BigDye version 3.1 terminator, 2 µl of the 5x sequencing buffer, l μL of either 907R, or GM5F and the final volume was brought up to 10 μL with molecular water. Labeling was completed in the Veriti Thermal Cycler  using one cycle of 96°C for l min then 25 cycles of l min at 96°C, 5 s at 50°C, and 4 min at 60°C. The pure template DNA samples were processed in a 3130xl genetic analyzer . Sequencing analysis version 5.2 software  was used to analyze the results. Sequences were subjected to basic local alignment search tool analysis with the National Center for Biotechnology Information  GenBank database [∗ v.4 [Three local raw domestic sewage samples were collected from Al-Jahra station and used to count and isolate their indigenous coliforms. The conventional dilution-plating method was adopted, and the coliform-selective medium, Eosin Methylene Blue Agar (EMB) , 33, wasGTTT-3′) . The readatabase . A phyloe [∗ v.4 . Bootstr−1): 5.0 NaNO3, 0.56 KH2PO4, 0.86 Na2HPO4, 0.17 K2SO4, 0.37 MgSO4·7H2O, 0.007 CaCl2·H2O, and 25 ml l−1 of a trace element solution consisting of (g l−1) 2.32 CuSO4·5H2O, 0.39 Na2MoO4·2H2O, 0.66 KI, 1.0 EDTA, 0.4 FeSO4·7H2O, and 0.004 NiCl2·6H2O. The pH was adjusted to 7. Strains capable of growth on that medium were considered “presumptive” hydrocarbonoclastic.All the coliform strains were tested for their ability to grow on a mineral medium  with oiln-alkanes ranging in chain lengths between C9–C20, C23–C26, C28, C30, and C36. The aromatics toluene, naphthalene, phenanthrene, and biphenyl were also investigated. Cultures were incubated at 30°C for 15 d and examined for growth.To study the range of pure hydrocarbons that could be utilized by the individual strains, a common inoculum (loopful in 5 ml sterile water) was prepared for each strain, and one-loopful aliquots were streaked on the solid mineral medium containing 0.5% of individual μl was analyzed by GLC. The percent loss of the peak areas based on the peak areas of the control samples  was calculated as a quantitative measure of the oil-removal. For GLC, we used an Agilent 7890A GC (USA) system equipped with FID, a DB-5 capillary column , and He as a carrier gas. The oven temperature started at 50°C for 3 min, then rising at 3°C/min to 80°C, then rising at 8°C/min to 256°C, then rising at 30°C/min to 330°C, and holding at this temperature for 11 min.The crude oil and pure-hydrocarbon consumption by individual isolates was measured quantitatively by gas liquid chromatography (GLC). Aliquots, 100 ml, of the mineral medium  in 250 m E. coli, one from domestic sewage and the other from wheat straw [n-hexadecane [4 (at 4°C for 2 h), and washed three times in the same buffer. Cells were centrifuged at 5,000 ×g for 10 min and the pellet was mixed with an equal volume of 2.0% noble agar. The agar was spread on a slide, left to solidify, and cut into small cubes. The cubes were dehydrated in an ascending series  of acetone in water and rinsed twice in 100% propylene oxide. Finally, the samples were embedded in Epon resin. Ultrathin sections [In this experiment, two strains ofat straw , were inxadecane  for intrxadecane . The celsections  were obtsections , and vien-hexadecane, and 1.0 g portions of phenanthrene. The flasks were sealed to avoid hydrocarbon volatilization and incubated at 30°C on an electrical shaker, 200 rpm, for 20 d. At time zero and in 5 d intervals, cultures were harvested for quantitative oil and coliform analysis, as already described. Three replicate samples were analyzed throughout.Aliquots, 100 ml of freshly harvested raw sewage (not previously sterilized) in 250 ml flasks, were provided separately with 1.0 and 5.0 g portions of crude oil, 1.0 g portions of A similar parallel experiment was run but using previously autoclaved raw sewage aliquots, as controls.Three determinations for each analysis were done and the mean values ± standard deviation values were calculated using Microsoft Excel 2007. Also, Statistical Package for Social Sciences, version 12, was used to assess the degree of significance, where the analysis of variance (ANOVA) was used to differentiate between the means of the tested parameters.5, 9.8 × 105, and 9.6 × 105 ml−1 colony forming units (CFU) of total coliform bacteria as counted on the selective coliform medium. Based on their 16S rRNA-gene sequences, those coliforms consisted of Enterobacter hormaechei subsp. oharae, 56–58%; Klebsiella variicola, 23–25%; Klebsiella pneumonia, 10-11%; and Escherichia coli, 7–10%. The results in https://doi.org/10.1155/2017/1838072) were those expected according to their generic identities.The three raw sewage samples studied were found to contain 9.5 × 10A total of 313 pooled coliform colonies (on the selective EMB medium) were subcultured and purified. All the pure cultures grew on a solid mineral medium  with crun-alkanes, C9–C20, C23–C26, C28, C30, and C36, and the individual aromatic hydrocarbons, toluene, naphthalene, phenanthrene, and biphenyl. The growth on such a wide range of aliphatic and aromatic hydrocarbons reflects a high potential for hydrocarbon utilization. Earlier investigators also found that the same coliform species isolated elsewhere could grow on oil and hydrocarbons as sole sources of carbon and energy [β-oxidation to acetyl CoA units. The latter could then be used for synthesis of cell material and for the production of ATP through the tricarboxylic acid cycle.Five haphazardly selected strains for each coliform species were found to grow, albeit with different intensities, on the solid mineral medium  suppliedd energy –23. Succd energy –44. More16) and aromatic (phenanthrene) hydrocarbons in 15 days. Those hydrocarbons were provided in the mineral medium [The results in l medium  as sole  E. coli, one from sewage and the other from wheat straw [n-hexadecane [16 also showed clear morphological deformations.For this experiment, two strains of the typical coliform,at straw , were usxadecane . Cells i6 to 26.5 × 106 CFU ml−1 in 3% crude oil, to 52.7 × 106 CFU ml−1 in 1% crude oil, to 18.9 × 106 CFU ml−1 in C18, and to 15.8 × 106 CFU ml−1 in phenanthrene.  Enterobacter was the coliform that exhibited the highest increase in number, which may reflect a main role of this coliform in oil consumption. Other hydrocarbonoclastic, noncoliform bacteria indigenous to sewage are also probably involved in the measured crude oil removal. However, the significant increase (p < 0.05) in numbers of coliforms parallel to the significant decrease (p < 0.05) in the concentration of the remaining oil provides an experimental evidence for a major contribution of indigenous coliforms to self-cleaning of oil in sewage. This finding implies that the indigenous domestic sewage microflora is capable of controlling potential oil spills in this waste product. Furthermore, raw sewage is an accessible, cost-effective source of hydrocarbonoclastic microbial cocktails that can be used for bioaugmenting oil-contaminated areas.The results in  Klebsiella spp. and Enterobacter spp., are diazotrophic. In other words, nitrogen fertilization is also guaranteed in sewage for the hydrocarbonoclastic coliform communities.The results of this paper provide experimental evidence for the role of coliforms in biodegradation of oil spilled in the environment. This group should be particularly effective in cleaning oil spills in sewage, where those organisms are autochthonous (indigenous). Conditions prevailing during conventional sewage treatment probably bioenhance the coliforms potential for oil-bioremediation. On one hand, sewage treatment involves the routine practice of extensive aeration to reduce the sewage BOD values . In view of the fact that the initial step of microbial attack on hydrocarbons is catalyzed by oxygenases , aeration should bioenhance the spilled-oil bioremediation. Oil-bioremediation is also bioenhanced by nitrogen-fertilizers. During sewage treatment, nitrates and other nitrogenous compounds are continuously produced and in addition several coliforms, for example,Fresh cultures of the isolated coliforms were inoculated into tubes containing lactose peptone water and Durham tubes. Two batches were prepared; one was incubated at 37°C and the other at 44°C for 24 h. The tubes were inspected for acid (yellow color) and gas (in the Durham tubes) production.


Incidences of immune thrombocytopenic purpura occur in 1 in every 1000–10,000 pregnancies accounting for 3% of all thrombocytopenic pregnancies. A pre-existing immune thrombocytopenic purpura is known to be a risk factor for developing thrombocytopenia during pregnancy. We present here the treatment regime and management of a patient with known immune thrombocytopenic purpura who developed postpartum thrombotic thrombocytopenia with atypical response to traditional therapy.Pregnant women are more vulnerable to immune thrombocytopenic purpura or thrombotic thrombocytopenia. Pregnancy or postpartum thrombotic thrombocytopenia accounts for 10–25% of all thrombotic thrombocytopenia.in vitro fertilization, she delivered a healthy baby through caesarean section. Two days post-surgery, she had persistent thrombocytopenia, ecchymoses, bruises, and hemolysis. Her blood film revealed leukoerythroblastic anemia. Her blood tests also revealed a very low level of haptoglobin, and low level of ADAMTS13. A diagnosis of thrombotic thrombocytopenia was suspected. Plasma exchange therapy was started with partial response. We showed that rituximab in conjunction with mycophenolate mofetil following plasma exchange therapy was effective in controlling the low platelet count in our patient.This case report deals with the treatment regime and management of a patient with known immune thrombocytopenic purpura who developed postpartum thrombotic thrombocytopenia. A 30-year-old Middle Eastern woman, with a prior diagnosis of chronic immune thrombocytopenic purpura had remained off-the-treatment for many years. After primary unexplained infertility for 8 years, for which she underwent six failed trials of Rituximab in conjunction with mycophenolate mofetil following plasma exchange therapy was effective in controlling the low platelet count in our patient. Only two doses of rituximab were sufficient to normalize our patient. We present here a case of safe and effective use of rituximab in pregnancy-induced thrombotic thrombocytopenia. The occurrence of immune thrombocytopenic purpura (ITP) or thrombotic thrombocytopenia (TTP) is greatly increased during pregnancy. Incidences of ITP occur in 1 in every 1000–10,000 pregnancies accounting for 3% of all thrombocytopenic pregnancies . We pres9/L. She had primary unexplained infertility for 8 years for which she underwent six failed trials of in vitro fertilization (IVF). She conceived spontaneously and remained asymptomatic with whole blood platelet counts > 50 × 109/L. At 26 weeks of gestation, she was diagnosed as having glucose intolerance and started on metformin 850 mg twice daily. At 30 weeks of gestation, her whole blood platelet count was 30 × 109/L and she was still asymptomatic. She was started on 15 mg orally administered prednisolone. One week later, her whole blood platelet count dropped to 28 × 109/L, her prednisolone dose was increased to 30 mg daily. She was screened for lupus anticoagulant, anticardiolipin antibody (ACA), and anti-beta-2 glycoprotein 1 (BTIIGLYI) antibody and the results were normal: partial thromboplastin time (PTT) 27 seconds, ACA-immunoglobulin G (IgG) < 14 GPL, ACA-IgM < 12 MPL, BTIIGLYI-IgM 16 U/mL, and BTIIGLYI-IgG 17 U/mL. Two weeks later, her whole blood platelet count was 56 × 109/L. Her prednisolone dose was tapered gradually down to 15 mg at 34 weeks of gestation. At 35 weeks, her whole blood platelet count was 45 × 109/L, her blood hemoglobin (Hb) level was 125 g/L with an A-positive blood group, and she was asymptomatic with normal blood pressure (BP) and urine analysis.A 30-year-old married Middle Eastern woman, a housewife living in a big city in Jordan, with a prior diagnosis of chronic ITP remained off-the-treatment for many years. She was not known to have any other medical illness. There was no family history of ITP or TTP. She had never smoked tobacco, drunk alcohol, or used illicit drugs. She was investigated for other causes of thrombocytopenia including systemic lupus erythematosus (SLE)-antiphospholipid antibody syndrome (APS) but, unfortunately, the laboratory-specific results were not available, however, our patient stated that they were negative. Initially she was prescribed a low dose of orally administered prednisolone but this was discontinued years ago. She remained asymptomatic with approximate whole blood platelet counts of 50 × 109/L, and 33 × 109/L, respectively. Her liver function tests were normal: lactate dehydrogenase (LDH) 3.0 ukat/L and bilirubin total 8 umol/L. She insisted on caesarean section delivery despite thorough counselling regarding the mode of delivery. She was given intravenously 500 mg of methylprednisolone and intramuscularly 1500 IU of anti-D immunoglobulin in an attempt to quickly raise her platelet count. Six hours later, an uncomplicated caesarean section was performed under spinal anesthesia . A healthy baby was delivered weighing 2.3 kg with normal platelet count. No instance of bleeding was noted during the surgery. The operative blood loss was estimated to be around 1000 ml. Recovery after surgery was smooth. She was given low molecular weight heparin (LMWH) (40 mg of enoxaparin sodium), subcutaneously 12 hours after operation.By 36 weeks of gestation, she presented with ruptured membranes, with no uterine contractions. Her BP, oral temperature, and pulse rate were 120/70 mmHg, 37.1 degrees Celsius and 90 beats/minute, respectively. Her general examination was unremarkable. An obstetric examination revealed a cephalic presentation and a fundal height that was corresponding to a 36-week pregnancy. There were no skin lesions. Her neurological examination was normal with no focal neurological deficits. A cardiotocography (CTG) was performed and was normal. Her Hb, white blood cell (WBC) count, and platelet count were 130 g/L, 12 × 109/L, 18.35 × 109/L, and 96 g/L, respectively. She was started on 30 mg of orally administered prednisolone. On the second postoperative day, ecchymosis started to appear around the wound. Her BP and urine analysis were normal. She had a platelet count of 52 × 109/L. Although she was reassured about the safety of breast feeding, she was not happy to breast feed and she had no breast pain or engorgement. In the afternoon, the bruises and ecchymosis spread across her lower abdomen and down to her upper thighs. A hematologist was consulted who advised observation only.Postoperatively on day 1, she was doing well with stable vital signs and no abnormal vaginal bleeding. No incidence of bruises or ecchymosis around the wound or anywhere else was noted. Her platelet count, WBC, and Hb were 46 × 109/L, and platelet count was 50 × 109/L. We suspected a hemolytic process so a liver function test, haptoglobin, and blood film were requested. These revealed total serum bilirubin 17.07 umol/L, direct serum bilirubin 5.97 umol/L, total serum protein 55.6 g/L, serum albumin 34.4 g/L, serum alanine aminotransferase (ALT) 0.47 ukat/L, serum aspartate aminotransferase (AST) 0.80 ukat/L, serum gamma glutamyltransferase (GGT) 0.55 ukat/L, and serum LDH 21.1 ukat/L. Her serum haptoglobin level was low, 100 mg/L, with negative direct and indirect Coombs tests. Her urine was normal. Blood film showed normocytic red cell anemia, polychromasia, few schistocytes, one to two nucleated red blood cells (RBCs)/100 WBCs, neutrophilic leukocytosis, occasionally left shifted neutrophils, and thrombocytopenia with large forms. Three ampoules of iron sucrose complex were given intravenously over 3 hours and she was started on orally administered iron.On the third postoperative day, the ecchymosis expanded bilaterally over her suprapubic area, vulval area, flanks, and thighs. She complained of occipital headache which was relieved by simple analgesia, there were no other neurological symptoms or signs. Her BP was normal. An abdominal ultrasound (U/S) scan revealed an empty uterus with no free fluid in her abdomen or pelvis and no evidence of presence of hematoma. Her whole blood Hb was 88 g/L, WBC was 12.89 × 10Four days after surgery, she was doing well apart from recurrent attacks of headache. Her BP was normal and she had no focal neurological signs. Her serum vitamin B12 level, serum iron, serum folate level, serum ferritin level, plasma prothrombin time (PT), whole blood PTT, and plasma fibrinogen were normal. Her newborn’s platelet count was normal. Total as well as direct bilirubin and LDH were elevated: 26.4 umol/L, 7.2 umol/L, and 26.7 ukat/L, respectively. Five days after surgery, there were severe and extensive bruises as well as ecchymosis. No abnormal vaginal bleeding or epistaxis was seen. Orally administered mycophenolate mofetil 360 mg was started twice daily in addition to the daily 30 mg orally administered prednisolone she was already taking.9/L, platelet count 50 × 109/L, and LDH 28.7 ukat/L, and she had normal total bilirubin, direct bilirubin, random serum glucose, sodium, potassium, urea, and creatinine. Urine analysis showed + 1 proteinuria, no sugar, one to two WBC/high power field (HPF), and 2–4 RBC/HPF. Indirect and direct Coombs tests were negative. Two ampoules of iron sucrose were administered intravenously in 2 hours. Prednisolone dose was increased to 1 mg/kg daily.On the sixth postoperative day, ecchymosis was seen all over her abdomen, flanks, thighs, legs, and low back area. Her Hb was 83 g/L, WBC 16.6 × 109/L; total bilirubin, direct bilirubin, ALT, AST, and urine analysis were normal. TTP was suspected; hence, ADAMTS13 test was requested. The result indicated 11%. The next day, her bruises and ecchymosis were extensive and spreading. She was doing well with no neurological symptoms and stable vital signs. Her urine was normal. Blood film revealed plenty of schistocytes. TTP was strongly suspected; she was given 5 units of fresh frozen plasma (FFP). Eight hours after the FFP infusion, her platelet count was 76 × 109/L.Bruises and ecchymosis increased 1-week post-surgery. Blood film revealed leukoerythroblastic anemia, anisocytosis, nine nucleated red cells/100 WBC, 8% left shifted neutrophils, and thrombocytopenia with few large forms. Her LDH level was 28.4 ukat/L. Her platelet count was 56 × 109/L and Hb was 99 g/L with normal liver and kidney functions. Her LDH was 13.7 ukat/L, with normal PT, PTT, and fibrinogen level. Blood film showed leukoerythroblastic anemia, anisocytosis, polychromasia, schistocytes, and spherocytes. A third session of plasmapheresis was done. Following FFP infusion and plasmapheresis, ADAMTS13 level was > 19%.Bruises decreased the day after FFP infusion. Plasmapheresis  was started. The first session was done with 10 units of FFP and 4 units of cryoprecipitate. Investigations showed normal Hb, WBC, platelet count, liver function tests, and urine. On the successive day, bruises and ecchymosis decreased. LDH was 15.8 ukat/L with normal creatinine, calcium, phosphorous, magnesium, total bilirubin, direct bilirubin, ALT, AST, GGT, PT, PTT, and fibrinogen level. A urine examination showed + 1 proteinuria. A second session of plasmapheresis was done. On the next day, her platelet count was 60 × 109/L.On the 12th postoperative day, a fourth session of plasmapheresis was done. Her LDH was 10.8 ukat/L with normal kidney and liver functions. Her platelet count was 54 × 10After the fifth session of plasmapheresis, LDH was 8.56 ukat/L. PT, PTT, and fibrinogen level were normal. Ecchymosis decreased substantially. Our patient was asking for discharge.9/L; LDH was 13.4 ukat/L with normal liver and kidney functions. A sixth session of plasmapheresis was carried out. A urine examination showed + 1 proteinuria. During all these days, our patient was on 1 mg/kg orally administered prednisolone per day and 360 mg orally administered mycophenolate twice daily. Ecchymosis decreased further on the following day. She was given intravenously rituximab 500 mg in 5 hours. Her LDH level was 11.0 ukat/L, platelet count was 100 × 109/L, Hb 111 g/L, and WBC 7.92 × 109/L. She was discharged with a prescription for orally administered prednisolone 1 mg/kg daily and 360 mg of orally administered mycophenolate twice daily.The next day, her platelet count was 52 × 109/L and 60 × 109/L.One week later, she was given the second dose of rituximab as an out-patient. She continued to do well with no bleeding tendency and a platelet count swinging between 50 × 109/L, and 130 × 109/L, respectively. Her liver and kidney function tests were normal. Two weeks later, her platelet count was 80 × 109/L. After a further 2 weeks, she continued to receive the same mycophenolate dose but started to decrease her prednisolone dose until it was completely stopped 3 weeks later.At 4 weeks after surgery, her Hb, WBC, and platelet count were 128 g/L, 13.7 × 109/L. She was receiving only mycophenolate mofetil at a dose of 360 mg twice daily. She stopped mycophenolate mofetil and remained completely asymptomatic. Six months after delivery, she was asymptomatic with a platelet count of 85 × 109/L. One year after her delivery, she was doing well, asymptomatic, and her platelet count was 79 × 109/L. Table Ten weeks after surgery, she became asymptomatic with a platelet count of 92 × 10This was a case of pregnancy-triggered TTP in a patient with chronic ITP. Moreover, she underwent a caesarean section under a spinal anesthetic with severe thrombocytopenia and did not experience hemorrhagic complications. It unusually presented in the immediate postpartum period and was not a typical TTP; it did not respond to the traditional management pathways.TTP is a rare life-threatening disease which requires rapid intervention. Pregnancy is a known trigger for thrombocytopenia. A previous history of ITP aggravates thrombocytopenia in pregnancy . HoweverA corticosteroid, such as prednisolone, is the first line of therapy for ITP . It incrHowever, except for thrombocytopenia and microangiopathic hemolytic anemia, which occur frequently 50–75%), other symptoms of TTP are either not present or vaguely observed. Therefore, thrombocytopenia, microangiopathic hemolytic anemia, and absence of alternative etiology are sufficient to diagnose TTP , 7. This5%, otherSevere deficiency of ADAMTS13 is the underlying cause for developing TTP , 11. MorIdentifying the presence of hemolytic anemia and its underlying cause is of prime importance in diagnosing TTP. The presence of schistocytes, the fragmented RBCs injured by damaged endothelium, in blood film investigation is one of the strong indicators of hemolytic anemia , 13. HowEarly diagnosis of TTP and management of its symptoms acutely with advanced techniques such as plasma exchange therapy (PEX) is necessary as delay in initiating PEX is known to cause adverse outcomes , 19. Noret al., administration of rituximab in a woman with pregnancy-related TTP was found to be safe and effective [Rituximab is known to be a safe and effective treatment for newly diagnosed TTP and ITP; it decreases the number of PEX required for achieving remission and lowers the risk for recurrence by approximately 80%. In addition, unresponsiveness to steroids in chronic ITP is relatively common and fast responses to rituximab can be seen. Diagnostic consideration, therefore, cannot depend on the response to these therapies since they are both used effectively in both ITP and TTP , 24. Howffective . Applicaffective . IntraveThe ADAMTS13 level of our patient of 11% made the diagnosis of TTP difficult. Moreover, the PEX applications were ineffective in managing her platelet and Hb levels to normal. Therefore, the impact of ADAMTS13 levels and the presence of its inhibitors on overall survival, ultimate clinical outcome, responsiveness to plasma exchange, and relapse in pregnancy-related TTP are still controversial. Therefore, studies assessing this clinical correlation are recommended. We show that rituximab in conjunction with mycophenolate mofetil following PEX was effective in controlling the low platelet count in our patient. Only two doses of rituximab were enough to normalize our patient. We present here a case of safe and effective use of rituximab in pregnancy-induced TTP.


The endpoint was defined as eGFR <60 mL·min-1·(1.73 m2)-1 estimated through the chronic kidney disease epidemiology collaboration equation (CKD-EPI). The study included 1094 participants. The mean follow-up period was 5.05±1.05 years and 44 participants exhibited new-onset CKD. The prevalence of hyperuricemia was 4.2%. There was a significant inverse correlation between baseline serum levels of UA and baseline eGFR . Female gender  and age  but not UA levels  were associated with new-onset CKD. Diabetes mellitus and body mass index were independent factors for fast progression . These results did not support UA as an independent predictor for CKD progression in the studied population.Uric acid (UA) levels are increased in patients with kidney dysfunction. We analyzed the association between asymptomatic hyperuricemia and new-onset chronic kidney disease (CKD). A retrospective cohort study was designed to collect data from employees of an energy generation and distribution company in the city of Rio de Janeiro, Brazil, who had undergone the company’s annual medical checkup from 2008 to 2014. People with ≤2 years of follow-up, with baseline estimated glomerular filtration rate (eGFR) <60 mL·min Chronic kidney disease (CKD) is a worldwide public health problem and its management, by either dialysis or transplantation, imposes a considerable economic burden to health systems . Its preUric acid (UA) is the end product of purine metabolism in humans, and its high serum levels, hyperuricemia, are classically related to the precipitation of crystals in the joints, leading to arthritis . There iIn recent years, a large number of observational studies have examined the potential relationship between hyperuricemia and development of CKD. These studies have shown conflicting results. Feig examined 12 epidemiology studies, finding a positive correlation in 8 and absence of association in 4 . Two met–1·(1.73 m2)–1 in the first exam or with incomplete data were also excluded.This was a retrospective cohort study. Data was collected from employees of an energy generation and distribution company in the city of Rio de Janeiro, Brazil, who had undergone the company’s annual medical checkup from 2008 to 2014. The study population was composed of office workers in non-laborious activities during work hours. To certify that only cases of asymptomatic hyperuricemia were studied, participants with a history of gout attacks were excluded. Those with ≤2 years of follow-up, with estimated glomerular filtration rate (eGFR) <60 mL·minSociodemographic information, physical examination data and biochemical analysis data were based on the participant’s first year medical examination. A second result of serum creatinine was obtained from the last annual checkup.–1·(1.73 m2)–1 (Hyperuricemia was defined as a UA of >6.0 mg/dL in women and >7.0 mg/dL in men . CKD was73 m2)–1 . The eGF73 m2)–1 , as foll–0.329×0.993age;If female and serum creatinine (sCr) ≤0.7 mg/dL: eGFR=144 × (sCr/0.7)–1.209×0.993age;If female and sCr >0.7 mg/dL: eGFR=144 × (sCr/0.7)–0.411×0.993age;If male and sCr ≤0.9 mg/dL: eGFR=141 × (sCr/0.9)–1.209×0.993age.If male and sCr >0.9 mg/dL: eGFR=141 × (sCr/0.9)Hypertension was defined as systolic blood pressure of ≥140 mmHg, diastolic blood pressure ≥90 mmHg or current use of antihypertensive medication, according to the Seventh Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure . The diaBlood pressure was measured in the left arm in the sitting position, after 5 min at rest, using an aneroid sphygmomanometer with an accuracy of 5 mmHg. The first and fifth Korotkoff sounds were recorded, and the mean value of 3 measurements was used for analysis. Body weight was measured with light clothing and without shoes by an adjustable scale . Height was measured without shoes using a stadiometer to the nearest 0.1 cm. The body mass index (BMI) was calculated as the weight (in kilograms) divided by the square of the height (in meters). All measurements were taken by trained nurses.Higher education was defined as a complete post-secondary education. Sedentary lifestyle was defined as doing irregular or no physical activity.The biochemical analyses were performed in samples obtained after at least 12 h of fasting. The serum levels of fasting plasma glucose, creatinine, total cholesterol, HDL-C, and triglycerides were measured by enzymology using the automated chemistry analyzer Selectra-E  or the Labimax 240 premium . Serum UA was measured by an enzymatic method  with intra- and total variation coefficients of 0.82–0.97 and 1.14–1.48%, respectively.t-test in case of normal distribution or, alternatively, by its nonparametric equivalent, the Mann-Whitney test. Pearson or Spearman correlation tests were used as appropriate. Logistic regression was used to test for association of independent variables with eGFR <60 mL·min–1·(1.73 m2)–1. P values less than 0.05 were considered significant.Statistical analyses were conducted using the Statistical Package for Social Sciences version 18.0 . The results of continuous variables are reported as mean and standard deviation if distribution was Gaussian, and alternatively as median and range if the distribution was not normal. Categorical variables are reported as frequencies. Comparisons between two groups were carried out by the unpaired The study was conducted with the approval of the Research Ethical Committee of the Faculdade de Medicina, Universidade Federal Fluminense, Niterói, RJ, Brazil (CAAE: 44061515.7.0000.5243).–1·(1.73 m2)–1. The mean follow-up period of the study population was 5.05±1.05 years. Their baseline characteristics are shown in From a total of 1413 participants, 1094 were selected for the final analysis . Of thos–1·(1.73 m2)–1 (P<0.001) resulting in a mean decline of 17.7±18.8 mL·min–1·(1.73 m2)–1. The mean annual rate of progression was 3.5±4.3 mL·min–1·(1.73 m2)–1. Patients were classified according to the rate of progression, following a previous study (–1·(1.73 m2)–1, characterizing a mild progression; in 30.0%, the progression was moderate [>1 to 4 mL·min–1·(1.73 m2)–1]; and in 45.6%, fast [>4 mL·min–1·(1.73 m2)–1]. Serum levels of uric acid of each group were 4.8±1.3, 4.6±1.3, 4.5±1.3, and 4.5±1.3 mg/dL, respectively, without statistically significant differences between groups. At the end of follow-up, 44 cases (4%) exhibited eGFR <60 mL·min–1·(1.73 m2)–1.The eGFR varied along the study from 105.6±14.8 to 87.9±17.8 mL·min–1·1.7 m2–1 (P<–1·(1.73 m2)–1, we resorted to a multivariate logistic regression model –1 were excluded from the analysis, the follow-up period may not have been long enough to detect the development of CKD in patients with a high baseline eGFR. This subject was additionally explored in the second multivariate logistic regression model in which diabetes was confirmed as a strong independent factor associated with a fast progression rate. It should also be pointed out that an adequate control of diabetes and hypertension is well recognized as a factor that attenuates progression to CKD  evaluating the effects of UA lowering therapy on renal outcomes were reviewed in two meta-analysis. Zhang et al. reviewed seven RCT involving 451 cases and identified that UA lowering therapy could delay the progression of CKD . OtherwiThere are a number of mechanisms by which high levels of UA increases the risk for CKD development. In a study in rats, hyperuricemia increased systemic blood pressure, proteinuria, renal dysfunction, progressive renal scarring and induced vascular disease via a COX-2-dependent pathway . AnotherThe understanding of the biological function of UA is far from elucidated. UA is recognized as a powerful antioxidant and is responsible for approximately half of the antioxidant capacity of human plasma. On the other hand, the molecule can also function as a pro-oxidant, either by generating free radicals during its degradation or by stimulating NADPH oxidase . HyperurOur study involved a relatively large sample. Participants had uniform access to health services and ease of follow-up, since the annual medical checkup was standardized. For legal reasons, medical records and laboratory results need to be kept for 20 years allowing precise monitoring. Nevertheless, some limitations must be considered. First, a single baseline measurement of UA was used to predict events several years later. However, a number of previous studies have resorted to the same approach. Second, the subjects were limited to employees of one company in Rio de Janeiro, predominantly composed of whites and not representative of the general Brazilian population. Third, the definition of renal progression was based on changes in eGFR, rather than more precise measurements of renal function, such as proteinuria or iothalamate clearance. Finally, the prevalence of hyperuricemia was very low in the cohort.In conclusion, our findings indicate that UA had a weak, but significant, cross-sectional association with baseline eGFR and was not correlated with new-onset CKD in a specific group of Brazilian workers. These results did not support UA as an independent predictor for CKD progression in the studied population. The question of whether a study addressing poorer segments of the Brazilian population would lead to a similar conclusion should be investigated.


Substantial evidence indicates that children’s physical fitness levels are markers of their lifestyles and their cardio-metabolic health profile and are predictors of the future risk of chronic diseases such as obesity, cardiometabolic disease, skeletal health and mental health. However, fitness reference values for ethnic children and adolescents have not been published in a Latin-American population. Therefore, the aim of the study was to provide sex- and age-specific physical fitness and anthropometric reference standards among Colombian-Indian schoolchildren.2max indirect. Centile smoothed curves for the 3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles were calculated using Cole’s LMS method.A sample of 576 participants (319 boys and 257 girls) aged 10 to 17 years old was assessed using the FUPRECOL test battery. Four components of physical fitness were measured: 1) morphological component: height, weight, body mass index (BMI), waist circumference (WC), triceps skinfold, subscapular skinfold, and body fat (%); 2) musculoskeletal component: handgrip and standing long jump test; 3) motor component: speed/agility test (4 × 10 m shuttle run); and 4) cardiorespiratory component: course-navette 20 m, shuttle run test and estimation of maximal oxygen consumption by VOOur results show that weight, height and BMI in each age group were higher in boys than in girls. In each groups, age showed a significant effect for BMI and WC. Boys showed better than girls in cardiorespiratory fitness, lower- and upper-limb strength and speed/agility and girls performed better in low back flexibility.Our results provide for the first time sex- and age-specific physical fitness and anthropometric reference values for Colombian Nasa Indian children and adolescents aged 10–17.9 years. Physical fitness is a multi-dimensional construct that includes skills and health related components, of which cardiorespiratory fitness (CRF) and muscular fitness in particular are powerful determinants of health in youth . SubstanThe inclusion of physical fitness within health surveillance systems is therefore clearly justifiable, and schools may be an ideal setting for the monitoring of youth fitness. Various health-related fitness test batteries are used to assess young people internationally, including the FUPRECOL battery , FITNESSHowever, race and ethnicity are also thought to be important determinants of health . Both arIndigenous populations have disparities in health along with disparities in modifiable risk factors, including a low participation in physical activity . For insTherefore, the aim of the study was to provide sex- and age-specific physical fitness and anthropometric reference standards among Colombian-Indian schoolchildren.Indigenous populations have disparities in health along with disparities in modifiable risk factors, including low participation in physical activity . The preA sample of 576 (319 boys and 257 girls) healthy, Nasa Indian children and adolescents (10–17.9 years old) participated in the study. All the participants of the selected indigenous households were invited to participate in the study. The participation rate was greater than 95 %. A convenience sample of volunteers was included in groups by sex and age with 1-year increments . The sample size was estimated at 15 to 30 participants per group. A power analysis showed that this sample size was sufficient to estimate the proportion of subjects with an aerobic capacity indicative of future cardiovascular risk (7 to 11 %) with a precision of 11.4 % and a power of 80 % by data from the FUPRECOL study . Here, “V” accounts for velocity (in km/h−1) of the last completed stage and “A” accounts for the subject’s age (in years)  were repeated by undergoing the tests again 1 week later. The same inter-trial period has been used earlier in similar reliability studies conducted with healthy young individuals [course-navette shuttle run test (ICC = 0.967)], with the exception of the 4 × 10 m shuttle run, which obtained a moderate agreement (ICC = 0.685).All fit measurements in a subsample of , and the L, M and S curves were estimated [All variables were checked for normality of distribution before analysis using histograms and Q-Q plots. None required transformation. The participants were divided into 8 age groups: 10 to 10.9, 11 to 11.9, 12 to 12.9, 13 to 13.9, 14 to 14.9, 15 to 15.9, 16 to 16.9 and 17 to 17.9 years. The age- and sex-specific values were reported as the mean ± the standard deviation (SD). We analyzed sex-group differences in the fitness variables by a one-way analysis of variance. To provide percentile values for the sample, we analyzed waist circumference, BMI, triceps skinfold, subscapular skinfold, handgrip, standing long jump test, 4 × 10 m shuttle run and course-navette shuttle run test data by maximum penalized likelihood using the LMS statistical method for boys and girls separately . We deristimated . Except 2, waist circumference of 70.0 ± 7.3 cm, subscapular skinfold of 10.3 ± 3.7 mm, triceps skinfold of 12.5 ± 6.1 mm and body fat of 21.8 ± 5.5 %. The prevalence of overweight and obesity were significantly higher in girls (p = 0.001). Girls had a significantly higher % body fat and a significantly higher WC (p = 0.001). Handgrip strength, standing broad jump and VO2max were significantly different between sexes, and boys had significantly higher scores in the musculoskeletal component (p = 0.001), the cardiorespiratory component (p = 0.001) and the motor component (p = 0.001).The characteristics for the four components of the FUPRECOL health-related fitness test according to the sex and age of the study sample are shown in Table course-navette shuttle run test in the Nasa Indian Community, classified according to sex and age and expressed in percentiles from 3 to 97. In boys, the BMI and WC 50th percentiles ranged from 16.8 to 23.5 kg/m2 and 62.3 to 74.2 cm, respectively. In girls, the BMI and WC 50th percentiles ranged from 16.1 to 20.1 kg/m2 and 64.0 to 76.5 cm, respectively  ranged from 51.2 to 52.8 ml•kg•min−1 in boys and from 37.2 to 49.1 ml•kg•min−1 in girls. Nevertheless, values show a decreasing trend with age in girls  or age- and gender-specific BMI cut-off values of the 85th percentile but <95th percentile or equal to 30  were suggested. In this study, girls had a significantly higher % body fat and a significantly higher WC (p = 0.001). Similar mean body fat values between age groups were observed in American [th percentile to the 75th percentile. Naturally, because of the action of sexual hormones, a progressive increase in body fat was observed in girls with maturation; however, their lower involvement in physical activities [Expert panels have recommended measuring triceps and subscapular skinfold thicknesses as part of the in-depth medical assessment of children and adolescents; age- and gender-specific BMI cut-off values of the 95American  and BrazAmerican  girls, wtivities  and inadtivities  may conttivities , this inth percentile to the 25th percentile of levels of physical fitness was identified as the “pathological fitness level” or as a “warning sign”; thus, youth in or below the 25th percentile should be examined to determine whether they have a cardiovascular risk factor. Reports have shown that low levels of self-reported physical activity or low cardiorespiratory fitness are associated with insulin resistance in other indigenous groups [In 2009, Ruiz et al.  systemats groups , 44. Hows groups .50 of the standing long jump was 172.0 cm vs 120.5 cm for Colombian Nasa Indian and European boys, respectively, and 127.0 cm vs 110.7 cm for Nasa Indian and European girls, respectively. The P50 of handgrip was an average of 21.5 kg vs 11.8 kg for Nasa Indian and European boys, respectively, and 16.2 kg vs 10.8 kg for Nasa Indian and European girls, respectively. In addition, the P50 of the 20-m shuttle run (stage) was 8.0 vs 2.0 for Nasa Indian and European boys, respectively, and 6.0 vs 2.0 for Nasa Indian and European girls, respectively. Likewise, Tremblay et al. [th percentile was reported. For example, in the study of Tremblay et al. [2max in ml•kg•min−1) at the 50th percentile of 11–14-year-olds was 54.9 ml•kg•min−1 and 48.9 ml•kg•min−1 in boys and girls, respectively. In the present study, the performance in VO2max at the 50th percentile of 11–17.9-year-olds was 51.9 ml•kg•min−1 and 43.3 ml•kg•min−1 for boys and girls, respectively. In addition, in the study of Tremblay et al. [th percentile was 25 kg and 22 kg in boys and girls, respectively, which was calculated as the sum of the best right- and left-hand attempts. In our study, performance in the handgrip test at the 50th percentile of 10–17.9-year-olds was 21.5 kg and 16.2 kg for boys and girls, respectively. In terms of speed/agility, the values for boys were similar to those of three studies , whereaPrevious research indicates that such low fitness levels can linger on into adulthood in which low cardiorespiratory fitness or low muscular strength is associated with increased mortality risk , 51. HowCompared to Nasa Indian adolescents in another studies, our sample had a significantly better performance in the musculoskeletal (SBJ), cardiorespiratory and motor components. Therefore, explaining the differences between urban and rural adolescents remains speculative and studies on the topic from other countries are inconsistent. One possible explanation for the differences in physical fitness profiles among indigenous adolescents may be the differences in moderate to vigorous physical activity  or diffePhysical activity and fitness have been found to be independently associated with certain cardiovascular risk factors among children and adolescents . Contrar2max from the FITNESSGRAM standards of the 20 m-shuttle run is known to vary with the equation used. A previous study [2max and the actual the VO2max. We were unable to confirm, for example, that the observed associations remain after control for physical activity or pubertal stage as others have shown due to lack of such data in this study. However, such limitations do not compromise the results obtained when validating these results. Finally, the small number of studies on the indigenous population did not allow us to make comparisons with the results of this work. The majority of the published fitness reference values are for children from high income countries in North America [There are a few limitations of this study. Firstly, the cross-sectional nature we cannot discern the direction of the observed associations between physical fitness and future cardiovascular risk, which may indeed be reciprocal. Adolescents with healthy body composition and healthy physical fitness may be more likely to engage in physical activity, which may lead to healthier cardiorespiratory and muscular fitness and contributes to the prevention of obesity , 53. Furus study  has test America –62, Asia America –68, wher America  and othe America  making i America  for exam America , 72. Pre America –76. Like America , only 13 America . These dOn the other hand, our decision to categorize physical fitness according to health predictive value instead of using continuous variables can be considered a strength of the study as it allowed for greater public health interpretability. Another potential strength of the study was the use of four health-related, valid, and reliable field tests recommended for Latin-American youth fitness assessment . Finallyth percentile). Also, these cut-offs are especially interesting in educational setting due to school could play a major role in helping to identify adolescents with low physical fitness. Simultaneously, this study allows for more accurate categorization, which considers a youth age and gender, and enables comparisons among normative values from other countries. In addition, they are important because monitoring health-related physical fitness early in life might contribute to substantial improvements in life expectancy and reduced risk of chronic diseases such as obesity, cardiometabolic disease, skeletal and mental health in the indigenous population.In summary, indigenous boys showed better than girls in cardiorespiratory fitness, lower- and upper-limb strength and speed/agility and girls performed better in low back flexibility. These values may be useful in identifying adolescents being at higher risk for developing unfavourable health outcomes owing to their low fitness level (<10


Here we present a comprehensive analysis of the electrical and thermal conductivities of polycrystalline Pb-doped p-type bulk Bi0.48Sb1.52Te3. Pb doping significantly increased the electrical conductivity up to ~2700 S/cm at x = 0.02 in Bix0.48-PbxSb1.52Te3 due to the increase in hole carrier concentration. Even though the total thermal conductivity increased as Pb was added, due to the increased hole carrier concentration, the thermal conductivity was reduced by 14–22% if the contribution of the increased hole carrier concentration was excluded. To further understand the origin of reduction in the thermal conductivity, we first estimated the contribution of bipolar conduction to thermal conductivity from a two-parabolic band model, which is an extension of the single parabolic band model. Thereafter, the contribution of additional point defect scattering caused by Pb substitution (Pb in the cation site) was analyzed using the Debye–Callaway model. We found that Pb doping significantly suppressed both the bipolar thermal conduction and lattice thermal conductivity simultaneously, while the bipolar contribution to the total thermal conductivity reduction increased at high temperatures. At Pb doping of x = 0.02, the bipolar thermal conductivity decreased by ~30% from 0.47 W/mK to 0.33 W/mK at 480 K, which accounts for 70% of the total reduction.Cation substitutional doping is an effective approach to modifying the electronic and thermal transports in Bi The zT value can be improved by reducing the κ and enhancing the S while maintaining the σ. Low κ maintains the temperature difference between the hot and cold sides of a material, and high S associates the high voltages generated at a certain temperature gradient.The thermoelectric (TE) effect offers the direct conversion of a temperature gradient into electrical energy, and vice-versa. Among the many TE materials, perature ,2. HowevzT can be enhanced by reducing the lattice thermal conductivity (lattκ) caused by interface phonon scattering in nanostructured materials, which effectively scatter low-frequency phonons. For example, Poudel et al. [zT of 1.4 at 100 °C in nanograin composite Bi0.5Sb1.5Te3. Additionally, it has been recently found that dense dislocations formed at grain boundaries can intensify phonon scattering by the additional scattering of mid-frequency phonons [lattκ by introducing point defects for phonon scattering, which target high-frequency phonons. It has been experimentally found that certain elements, such as Al, Ga, In, Cu, Ag, and Fe, reduce lattκ effectively [σ·S2) and bipolar conduction.l et al.  reportedectively ,7,8. How0.48Sb1.52Te3 can reduce lattκ and increase power factor (σ·S2) [S, σ, and lattκ of Pb-doped Bi0.48Sb1.52Te3 polycrystalline samples, were further analyzed by using the single parabolic band (SPB) model [0.48Sb1.52Te3 polycrystalline sample in order to closely examine the effect of Pb substitution on bipolar conduction and lattκ reduction. The S and σ were fitted to experimental values by adjusting the deformation potentials and the effective masses of both the conduction and valence bands by the two-parabolic band model based on the SPB model. Through this model, the bipolar conduction (bpκ) was estimated, while the lattκ was analyzed using the Callaway model.We have recently demonstrated that the substitutional doping of Pb in Bir (σ·S2) . In thisB) model  and CallB) model , and comσ and S of the Pb-doped samples  and undoped Bi0.48Sb1.52Te3 (x = 0) is shown in σ of Bi0.48Sb1.52Te3 at 300 K is about 640 S/cm, which increased significantly to 2700 S/cm (x = 0.02) with an increase in Pb content. The hole concentrations (pN) at 300 K were 2.47, 3.54, 4.96, 6.60, 8.76, and 11.96 × 1019/cm3 for x = 0, 0.0025, 0.005, 0.01, 0.015, and 0.02, respectively. Therefore, the substitution of Pb for Sb induces significant hole carriers by the introduction of acceptor defects. The value of S decreased from 215 μV/K to 117 μV/K, revealing a clear trade-off relationship with the σ value. It is noteworthy that while the maximum value of S is obtained at 360 K for x = 0, it shifts to a higher temperature of 480 K for x = 0.02; this implies that the charge compensation from bipolar conduction is reduced due to the significant increase in pN as Pb is added. The temperature dependence of totκ and (totκ-elecκ) of the Pb-doped samples  and undoped Bi0.48Sb1.52Te3 , where totκ is the measured total thermal conductivity and elecκ is the electronic thermal conductivity without considering the bipolar conduction. Thus, (totκ-elecκ) is the thermal conductivity excluding the contribution of the increased pN. The elecκ was estimated using the Wiedemann–Franz law, and the Lorenz number was calculated using Equation (1) [tion (1)  which istotκ increased with Pb content due to a significantly enhanced pN value (totκ-elecκ) value decreased by over 14% to 0.55 W/mK for x = 0.02, as compared to 0.64 W/mK of the undoped sample at 300 K. Furthermore, the amount of reduction in the (totκ-elecκ) value increased with increasing temperature and reduced up to 22% for x = 0.02 at 480 K. For x = 0.02, which is only a 1% substitution for the cation site, a significant reduction of 14–22% in (totκ-elecκ) was achieved.Indeed, the Np value a. As see2 for x = 0.01 from 2.97 mW/mK2 for x = 0. The pN value of 4.96 × 1019/cm3 at x = 0.01 seems to be the optimum value for the highest power factor. For x = 0.015 and 0.02, the power factor decreased due to a significant reduction in the S value. zT values of the samples. The zT values at temperatures above 400 K increased as the substitution increased to x = 0.01, as shown in the inset of x = 0.02, the zT value reduced up to 440 K due to a significant increase in the σ value, which results in a significant rise in totκ. The optimal σ range is 700–1200 S/cm for a high zT in this case.σ due to an increase in pN and reduces the (totκ-elecκ) value, which includes the bipolar thermal conductivity (bpκ) and lattice thermal conductivity (lattκ). In order to further understand the origin of (totκ-elecκ) reduction, we first estimated the bpκ from a two parabolic band model, which is an extension of the SPB model that considers acoustic phonon scattering. Thereafter, the contribution of additional point defect scattering to lattκ was closely analyzed using the Debye–Callaway model and compared with the point defect contribution from its native cation disorder. The Pb substitutes are additional point defects in the native cation disorder of (Bi0.48Sb1.52) in their mother compound Bi0.48Sb1.52Te3.Pb substitutional doping significantly increases In the two-band model, the thermoelectric parameters of valence and conduction bands computed from the Boltzmann transport equations  can be siσ, iS, and HiR in Equations (2)–(5) are the electrical conductivity, Seebeck coefficient, and Hall coefficient of an individual band, respectively. The parameters with subscript “total” include contributions from all the participating bands (these variables can be measured directly). Equation (5) describes the contribution of elecκ, lattκ, and bpκ to the totκ. In order to estimate bpκ, the calculated totalσ and totalS of one sample were fitted to the experimental σ and S of the sample by adjusting the deformation potentials and density-of-states (DOS) effective masses (m*) of its valence and conduction bands  in Bi2Te3 reported in the literature can elucidate the band structure of Bi0.48Sb1.52Te3 when two bands  are assumed to participate in the transport. Due to the high crystal symmetry of BixSbx2-Te3, more than one pocket of Fermi surface contributes to m* as m* = VN2/3b*m, where VN and b*m are the valley (pocket) degeneracy and band mass of a single valley, respectively. The VN of the highest valence band of BixSbx2-Te3 is 6, while that of the lowest conduction band is 2, as listed in 2Te3 reported in the literature along with m* can help us estimate reasonable deformation potentials. Moreover, HiR was calculated from the measured Hall carrier concentration ) and other band parameters computed above . The Fermi level of each band obtained from HiR was used to crosscheck that it calculated from iS. Because the band gap between the valence and conduction bands are given, we only needed to calculate the Fermi level of either band.Here, on bands . Since ibpκ is plotted in bpκ) vs. (1/T) reveals that the bipolar conduction increases exponentially with temperature. It is clearly seen that the estimated bpκ decreases systematically with Pb doping, as inferred from bpκ decreases by ~30% from 0.47 W/mK to 0.33 W/mK at 480 K for x = 0.02. The Pb doping of Bi0.48Sb1.52Te3 effectively suppresses the bpκ by increasing the concentration of the major carrier (holes) and reducing the concentration of the minor carrier (electrons), which partly accounts for the reduced lattκ values at high temperatures in Pb-doped Bi0.48Sb1.52Te3.The estimated bpκ for each sample was added to the theoretical lattκ, which is calculated below, to compare with the experimentally determined lattκ. Heat flow (q) is defined as a product of thermal conductivity (κ) and temperature gradient (ΔT). The negative sign of the product (q = −κ ΔT) indicates that heat flows from hot to cold. κ can be expressed as arising from the heat capacity (VC), velocity (v), and distance between the collisions (l) according to the kinetic gas model presented in Equation (6).The estimated l in Equation (6) is a product of v and the relaxation time, τ. Here, κ can also be described in terms of VC, v, and τ (Equation (6)). By applying Equation (6) to the phonons in solids instead of gas particles, we obtain the Callaway equation for lattκ (Equation (7)).The ω), the product of each parameter is integrated over the frequency. Here, gv refers to the phonon group velocity (gv = dω/dk). By treating the gv and phase velocity (pv = ω/k) as equivalent to the speed of sound, v, Equation (7) can be written as Equation (8) .kB, ћ, θ, and z are the Boltzmann constant, reduced Planck’s constant, Debye temperature aθ, and ћω/BTk, respectively [aθ (94 K) [lattκ of a material can be calculated using Equation (8) once its τtotal (z) is determined from the individual relaxation times (iτ) for different scattering processes, according to Matthiessen’s rule (Equation (9)),Because each parameter is dependent on frequency   and avera (94 K)  were obtUτ), boundary scattering (Bτ), and point-defect scattering (PDτ). Here, point-defect scattering arises from atomic disorders in alloys, and is described in terms of a scattering parameter (Г) within the PDτ formula as:The parameter for relaxation times is associated with Umklapp scattering (Г is a scattering parameter related to the difference in mass (ΔM) and lattice constant (Δa) between two constituents of an alloy. The parameters f, ΔM, G, , r, and Δa are the fractional concentration of either constituents, difference in mass, parameter representing a ratio of the fractional change of bulk modulus to that of local bond length, Grüneisen parameter, Poisson ratio, and the difference in lattice constant, respectively. The parameter (G) represents material dependent (ΔK/K) (R/ΔR), where ΔK and ΔR are the changes in bulk modulus and local bond length, respectively. We used the same parameters used in reference [Uτ) and boundary scattering (Bτ).In Equations (10) and (11), P is the fitting parameter and eference  for estiPDτ), we regard P and f (or lattκ varying with f for the (Bif1-Sbf)2Te3 alloy by Stordeur and Sobotta [lattκ of the undoped sample in f = 0.48/2 = 0.24 (Bi fraction in the cation site). The P and −41 s3 and 5.142 × 10−41 s3 and the result is represented as a black solid line in PDτ(Pb), using the fractional concentration of Pb in the cation site, which is fPb = x/2 . The fitting parameter for the scattering of Pb defects, PPb, is 208.8 × 10−41 s3, and thus, the −41 s3 for x = 0.0025, 0.005, 0.01, 0.015, and 0.02, respectively . Therefore, the substitutional doping approach should be considered not only for the introduction of additional point defects for phonon scattering, but also for the suppression of bipolar conduction in BixSbx1-Te alloys, especially at high temperatures.Although no meaningful enhancement in the maximum −4 Torr. The stoichiometrically mixed elements were vacuum sealed and the contents were melted in a box furnace for 10 h at 1273 K, and then water quenched. The ingots were ground in a ball mill and consolidated by spark plasma sintering (SPS) using a graphite die (diameter = 10.4 mm) under a dynamic vacuum with the application of 50 MPa uniaxial pressure at 753 K. Room temperature Hall measurements were carried out in a constant magnetic field (1 T) and electric current (50 mA) using a Keithley 7065 system. The S and σ values were measured using a ZEM-3 system  in the temperature range from 300 K to 480 K. The κ values (κ = ρsCpλ) were calculated from separated measurements of sample density (ρs), heat capacity (Cp), and thermal diffusivity (λ). The Cp values obtained using a Quantum Design physical property measurement system were almost constant at ~0.187 J·g−1·K−1, and the λ values were collected by a laser-flash method . The TE properties were measured perpendicular to the SPS press direction for polycrystalline Bi0.48Sb1.52Te3 and Pb-doped Bi0.48Sb1.52Te3 samples.High-purity elemental Bi , Sb , Te , and Pb  were used as starting materials. According to the formula, the mixed elements were loaded into a quartz tube and vacuum-sealed under 100.48Sb1.52Te3 were comprehensively analyzed using the single parabolic band model and Debye–Callaway model, and compared with that of Bi0.48Sb1.52Te3 polycrystalline alloys to closely examine the effect of Pb substitution on bipolar conduction and lattice thermal conductivity. A two-band model based on the single parabolic band model showed that Pb doping effectively suppressed the bipolar thermal conduction. The bipolar contribution to thermal conductivity decreased by ~30% from 0.47 W/mK to 0.33 W/mK at 480 K for x = 0.02 in Bix0.48-PbxSb1.52Te3. By using the Debye–Callaway model, it was found that the phonon scattering effect of Pb substitutes was much greater than the native alloy disorder in Bi0.48Sb1.52Te3. The interaction of disorders with the matrix, for example, bonding nature and structure, appeared to be important in inducing strong point-defect scattering. Therefore, we conclude that Pb doping is effective in suppressing both the bipolar thermal conduction and lattice thermal conductivity simultaneously in Bi0.48Sb1.52Te3 alloys. In addition, the bipolar contribution to thermal conductivity reduction increased at high temperatures in Pb doping. At 480 K, the contribution of bipolar conduction suppression to thermal conductivity reduction increased up to 0.13 W/mK, which is 70% of the total reduction of 0.19 W/mK, due to Pb doping (x = 0.02).In this study, the electrical and thermal conductivities and Seebeck coefficient of a series of polycrystalline bulk Pb-doped p-type Bi


In this study, we found significant relationships of bulk leaf carbon isotopic signature (δ13CBL) and bulk leaf oxygen enrichment above source water (Δ18OBL) with gas exchange and TE in the model C4 grasses Setaria viridis and S. italica. Leaf δ13C had strong relationships with E, gs, water use, biomass, and TE. Additionally, the consistent difference in δ13CBL between well-watered and water-limited plants suggests that δ13CBL is effective in separating C4 plants with different availability of water. Alternatively, the use of Δ18OBL as a proxy for E and TE in S. viridis and S. italica was problematic. First, the oxygen isotopic composition of source water, used to calculate leaf water enrichment (Δ18OLW), was variable with time and differed across water treatments. Second, water limitations changed leaf size and masked the relationship of Δ18OLW and Δ18OBL with E. Therefore, the data collected here suggest that δ13CBL but not Δ18OBL may be an effective proxy for TE in C4 grasses.Leaf carbon and oxygen isotope ratios can potentially provide a time-integrated proxy for stomatal conductance ( Ci/Ca is a measure of the supply of CO2 to photosynthesis, and as Ci/Ca increases, discrimination decreases , b4 is the combined fractionation of PEP carboxylation and the preceding isotopic equilibrium during dissolution and hydration of CO2 (–5.2‰ at a leaf temperature of 30 °C) as described in b3 is the fractionation by Rubisco (30‰), s is the fractionation during the leakage of CO2 out of the bundle sheath cells (1.8‰), and ϕ is the leakiness of CO2 from the bundle sheath  and stomatal conductance (gs). For example, increasing Anet can draw down Ci relative to Ca and a reduction in gs can decrease the supply of atmospheric CO2 to the intercellular air space for photosynthetic assimilation. Since TE is also related to Anet and gs, this means that TE and Δ13C are linked through their relationship with Ci/Ca, which makes δ13CBL a potential proxy for TE (The COressures . Therefo18OBL) comes partly from oxygen isotopic enrichment in leaf lamina water, a component of leaf water enrichment (Δ18OLW), and where organic compounds are synthesized within the leaf. At these sites of carbonyl oxygen isotope exchange, the leaf water oxygen isotope signal is passed on to photosynthetic intermediates and consequently passed on to bulk leaf tissue  is influenced by the isotopic enrichment above source water of ambient vapor (Δ18Ov), temperature-dependent gradient in the molar ratio of ambient to intercellular water vapor (ea/ei), temperature-dependent equilibrium fractionation (ε+), and kinetic fractionation (εk), which is a function of stomatal and boundary layer conductances (Eq. 3 and more in-depth explanation in Appendix).where isotopic enrichment of water at the evaporation sites  and the mean effective path length (L) through which water passes from the xylem to the stomates  P. Beauv. and S. italica (L.) P. Beauv. These species are part of the C4 panicoid grass clade and are closely related to important food and biofuel crops, such as sugar cane, maize, miscanthus, and sorghum  map for quantitative trait locus analysis  P. Beauv. (accession A-10) was grown in the greenhouses at Washington State University, Pullman, WA, USA between June and July of 2013. Day and night temperatures were 26–30 and 21–25 °C, respectively. Daytime and night-time relative humidity were 30–57% and 59–89%, respectively. Plants received 500–1500 µmol m−2 s−1 photosynthetic photon flux density (PPFD) over 14 h. Pot distribution in the greenhouse was randomized every day to minimize the influence of lighting heterogeneity. Fifteen S. viridis seedlings per treatment with one seedling per pot were transplanted into 11.3-liter pots using a commercial potting soil mix (Sunshine LC 1). Plants received 20-20-20 NPK with micronutrients  twice weekly at 2.8 g l−1 water.−1, respectively. In GWC calculations, fresh biomass would be considered soil water weight, causing GWC to be overestimated, so throughout the experiment daily transpiration was used as a proxy for changes in fresh weight over time to accurately calculate the soil water volume and estimates of GWC. All pots and lateral holes were covered with plastic sheeting to minimize soil evaporation. Daily transpiration was measured as the difference in pot weight between dawn and dusk minus soil evaporation (difference in a covered pot weight without a plant). All of the plants could not feasibly be measured and harvested in one day, so the plants were randomly divided into three collection groups of five plants from each treatment. Gas exchange measurements and harvesting of plant material of collections 1, 2, and 3 took place on 39, 43, and 44 d after germination. At all collection times, panicles had begun to emerge.After the initial watering at transplanting, pots in the well-watered and water-limited treatments were maintained at a gravimetric water content (GWC) of ~4 and 1 g water g soilSetaria italica (L.) P. Beauv.  were transplanted into 7.5-liter pots at 15 d after germination. The potting soil was the same as was used with S. viridis. Plants received 15-5-15 CalMag  twice weekly at a rate of 2.5 g l−1 water, Sprint 330 iron chelate (0.25 g l−1) weekly, and Scott-Peters Soluble Trace Element Mix, 10.0 mg l−1 biweekly .n B-100;  was grow−1, respectively. Pots were covered with plastic similar to S. viridis. Six and five plants from each treatment were randomly selected to be harvested in the first and second collections, respectively. Leaf gas exchange measurements were made at six time points . Plant material for stable isotope analysis was only collected at the final harvest, immediately following gas exchange measurements. At all collection times, panicles had begun to emerge in all plants.After initial watering after transplanting, the GWC of the well-watered, moderately and severely water-limited treatments was maintained at 4.0, 0.9, and 0.5 g water g soil−2 s−1 PPFD, leaf temperature of 29 °C, flow rate of 300 µmol s−1, 21% O2, 35 Pa CO2 for S. viridis. The same conditions were used for S. italica except that light intensity was 900 µmol m−2 s−1 PPFD to reflect the light intensity of their growing conditions. Relative humidity (RH) in the LI-COR chamber was within 10% of the RH under growth conditions, and the implications of this difference in RH is explained in the next section.Measurements were made on the uppermost fully expanded leaf between 11:00 h and 15:00 h. Leaves were placed in a 2 × 3 chamber of an LI-6400XT open gas exchange system . The leaf was allowed to acclimate at 1500 µmol m18O of leaf water (δ18OLW) from S. viridis, an aggregate of five to eight leaves (including the leaf used to measure gas exchange) was collected from each plant at the time of harvest. However, in S. italica the leaf used for gas exchange measurements was sufficient to analyse δ18OLW. The same leaf samples that were analysed for δ18OLW were also analysed for leaf carbon isotopic composition (δ13Cleaf) and oxygen isotopic composition of bulk leaf tissue (δ18OBL). All leaves collected for stable isotope analysis were the youngest, fully expanded leaves, which developed 15–20 d after soil water content reached the treatment set point. For both species, leaves were removed from the plant and photographed to measure leaf area using ImageJ software  could differ between growth and gas exchange chambers . Bags were flushed several times with air before filling, and δ18O of water vapor was immediately measured on the cavity ringdown spectrometer .The δ18OIW.Following the gas exchange measurements, root crowns were cleaned of soil and stored at −20 °C in air-tight tubes. Root crowns are considered the bottom 1 cm of the culm or tiller, but no actual roots were collected. Additionally, two soil samples were collected at the top and bottom of each pot, and stored using the same method as with the root crowns. Leaves, soils, and root crowns were distilled using a cryogenic vacuum distillation method . AdditioAfter collection of plant tissue for stable isotope analysis, the entire aboveground biomass was collected and weighed for fresh weight. Samples were dried at 65 °C for 3 d before weighing dry biomass.18O and δ13C, respectively) were reported in δ notation in parts per thousand (‰),The stable isotope composition of carbon and oxygen (δRsample and Rstandard are the molar ratios of heavy to light isotope (18O/16O and 13C/12C) of the sample and international standard, respectively. The international standard used for oxygen was Vienna Standard Mean Ocean Water (VSMOW) and for carbon was Vienna Pee Dee Belemnite (VPDB).where 2 with an elemental analyser  and analysed with a continuous flow isotope ratio mass spectrometer  and analysed with a continuous flow isotope ratio mass spectrometer . CO2 was analysed with a continuous flow isotope ratio mass spectrometer  connected to a vaporization chamber (V1102-i). The mean δ18O was calculated of the last three of six consecutive analyses on each sample. Three laboratory standards, calibrated to the VSMOW scale, were interspersed among the samples and were used to correct the sample δ18O values to the VSMOW scale. Water vapor was analysed for at least 15 min, but the mean of the last 5 min was used for δ18O and corrected to the VSMOW scale.Soil and irrigation water were measured by isotope ratio infrared spectroscopy (model L1102-instantaneous (Anet/E) and TEintrinsic (Anet/gs) are derived from gas exchange measurements, but they are independent of both TEplant and TEw, which are independent of each other. TEplant was derived from whole-plant measures of biomass and transpiration. TEw was calculated using Ci/Ca derived from δ13CBL, and the calculations are independent of the gas exchange measurements (described below).TE13CBL) and Ci/Ca are related in Eq. 2 and were used to calculate the integrated Ci/Ca over the life of the leaf , defined as the ratio of dry matter produced per unit of water transpired, was calculated from the δ13CBL as described in Discrimination . This parameter was calculated from gas exchange measurements made during the experiment. The parameter ϕw was calculated as the measured ratio of whole-plant night to day transpiration  measured in this experiment. Both night-time and daytime whole-plant transpiration were measured on S. viridis. Both ϕr and ϕw were measured for S. viridis and assumed to be the same for S. italica. Ci/Ca was calculated from δ13CBL using Eq. 2.where E, we used the method described by f) of Δ18OLW from Δ18Oe plotted against E where f is calculated as:To test the applicability of the Péclet model and its relationship to L) was calculated using the equations described in Effective path length , normality was tested by plotting residuals on quantiles–quantiles plots. In all cases, where normality was questionable, transforming the data did not change the statistical results, so the data were not transformed. One- and two-way analysis of variance (ANOVA) was used to determine differences across treatments in the experiment with S. italica and between treatments and collection periods for S. viridis. Two-sample Student’s t-tests were performed to determine the difference between δ18OSW and δ18ORC values. One-sample t-tests were performed to determine if the difference of both δ18OSW and δ18ORC with δ18OIW was significantly different from 0. Repeated measures ANOVAs were conducted on the following parameters: total plant water use and GWC for S. viridis and on daily water use, GWC, Anet, E, and gs for S. italica.In both experiments statistical analyses were conducted in R v. 3.3.0 , using cS. italica were similar to S. viridis in that the GWC was reduced by 72% and 82%, and total water use by 70% and 80% in the moderately and severely water-limited treatments, respectively , rates of transpiration (E), and the net rate of CO2 assimilation (Anet) were generally higher in the well-watered treatment compared with the water-limited treatment. Gas exchange measurements of S. viridis were made during the three biomass collections, and E was 33% and 67% greater in the well-watered treatment in collections 2 and 3, respectively, but did not differ between treatments in collection 1  to water limitations was similar for both species. For example, δ13C values in S. viridis were consistently lower in the water-limited treatment for all collections by 1.1‰ . The difference in leakiness would account for 0.22 ± 0.03‰ and 0.24 ± 0.01‰ of the observed difference in Δ13CBL between treatments over the observed range of Ci/Ca in S. italica and S. viridis, respectively.Using the simplified equation (Eq. 2) of Δplant; grams of aboveground dry biomass per liter water transpired); (ii) instantaneous TE ; (iii) intrinsic TE ; and (iv) δ13CBL-derived TE . For S. viridis the water-limited plants had higher TE regardless of how it was estimated, except in collection 3 where there was no difference in TEinstantaneous between treatments  long term TE (TEeatments . In S. iand δ13C .18Ov) in both the greenhouse for S. viridis and in the growth chambers for S. italica were relatively stable. For S. viridis, δ18Ov values (mean±SE) were −17.4 ± 0.2, −21.9 ± 0.4, and −24.2 ± 0.3 for the three collections, respectively. For S. italica, δ18Ov values were −25.8 ± 0.1 and −20.1 ± 0.8 for the two collections, respectively.The gas exchange measurements and leaf samples were collected between 11:00 and 15:00 h. During this time the vapor oxygen isotope ratios . The mean δ18OSW values (average of δ18O of soil samples from the top and bottom of the pot) of the well-watered and water-limited treatments were 0.8 ± 0.1‰ and 1.9 ± 0.2‰ higher than irrigation water for S. viridis (P<0.0001), respectively. For S. italica, δ18OSW values were 0.8 ± 0.2‰, 2.7 ± 0.2‰, and 3.9 ± 0.4‰ higher than irrigation water in the well-watered, moderately and severely water-limited treatments, respectively (P<0.0001). In S. viridis, the δ18ORC values were 0.6 ± 0.1‰ and 2.0 ± 0.1‰ higher than δ18OIR in the well-watered and water-limited plants (P=0.0002 and P<0.0001), respectively. In S. italica, δ18O values were 1.1 ± 0.1‰, 3.1 ± 0.2‰, and 4.3 ± 0.3‰ higher than the irrigation water in the well-watered, moderately and severely water-limited treatments (P<0.0001), respectively. The δ18O of root crown and soil water was not significantly different in either S. viridis or S. italica (P>0.05).Plants were top irrigated in covered pots with irrigation water (−17.0 ± 0.1‰ and −16.9 ± 0.1‰ for 18OLW) showed a significant treatment effect in S. viridis independent of which water was considered source water , but the treatment effect on Δ18OLW was greatest with irrigation water. Likewise the strength of the correlation between parameters of gas exchange, water use, and growth depended on which source water was used to calculate Δ18OLW. However, independent of the source water, Δ18OLW negatively correlated with Anet, E, and gs and positively correlated with TEintrinsic and δ13CBL  when negative correlations were expected.In ce water . The onlreatment . This di18OLW from Δ18Oe  with E (see JXB online). This relationship was significant for S. viridis  but not for S. italica (P=0.93) suggesting that in S. viridis Δ18OLW deviated more from Δ18Oe as E increased. For S. italica, the Δ18OLW was larger than Δ18Oe , causing Δ18OLW to be more enriched than would be expected based on the evaporative environment. For S. viridis the estimated effective leaf length (L) was small but significantly different between treatments , the calculated ϕ from δ13CBL and Ci/Ca produced similar values (0.17–0.21) to what has been published previously . Additionally, the relationship between Δ13CBL and Ci/Ca was similar for both species under all treatments, potentially falling on the same line where ϕ controls the slope  calculated from δ13CBL correlated strongly with leaf-level gas exchange measurements of gs and TEintrinsic (Anet/gs). Therefore, calculating TEw based on Ci/Ca estimated from δ13CBL accurately reflected differences in gs between treatments. However, in S. viridis, TEplant was more highly correlated with TEintrinsic and TEinstantaneous than in S. italica. This may be because the short and bushy S. viridis has proportionally more leaf biomass than the upright S. italica, so leaf characteristics would have a greater influence on plant-level estimates of TE (e.g. TEplant) in S. viridis than in S. italica. Nonetheless, for both species, δ13CBL reflected differences in both whole-plant and leaf-level estimates of TE.Transpiration efficiency  with E, showing evidence that the Péclet model best describes leaf water isotopic composition  are temperature-dependent  may be because Δ18OLW increases with leaf length in C4 grasses  should reflect the isotopic signature of leaf water in which bulk tissue is synthesized  used to calculate Δ18OLW and Δ18OBL. In this study, we measured the isotopic signature of three possible source waters: (i) irrigation water, (ii) mean soil water, and (iii) root crown water. Isotopically soil and root crown water were statistically indistinguishable, confirming previous studies that there is little fractionation of oxygen isotopes upon uptake by roots, so root crown water is a good representation of δ18OS at the time of water collection  from evaporative site water (Δ18Oe) oxygen isotopic enrichment (f) and transpiration rate (E).Fig. S1. The relationship between proportional deviation of leaf water , denominator degrees of freedom (ddf) and P values from two-way ANOVA of the effects of a differential irrigation treatment and collection period on plant water use and growth, leaf water relations, and isotopic composition for S. viridis.Table S1. S. viridis grown under well-watered and water-limited conditions and harvested during three collection periods.Table S2. Plant water relations, growth, and isotopic composition of F values, numerator degrees of freedom (ndf), denominator degrees of freedom (ddf) and P values from one-way ANOVA of the effects of a differential irrigation treatment on plant water use and growth, leaf water relations, and isotopic composition for S. italica.Table S3. S. italica grown under well-watered, moderately and severely water-limited treatments and harvested during two collection periods.Table S4. Plant water relations, growth, and stable isotopes of 18OLW), bulk leaf enrichment (Δ18OBL), and δ13C for S. viridis.Table S5. Correlations between measured parameters of both well-watered and water-limited plants and leaf water enrichment (Δ13C, Δ18OLW, and Δ18OBL for S. italica.Table S6. Correlations of measured parameters with δSupplementary Tables S1-S6 and Figure S1Click here for additional data file.