Cytotoxicity of Auranofin was evaluated using MTS assay and the Trypan blue dye exclusion method. With fluorescent dyes SR-FLICA and 7-AAD apoptotic death and necrotic death were differentiated by Flow cytometry. A concentration dependent decrease in the viability occurred and cells were shifted to the apoptotic phase. Intracellular calcium ([Ca2+]i) was recorded using florescence microscopy and a calcium sensitive dye (Fluo-4 AM) with a strong negative correlation (r = −0.713) to viability. Pharmacological modulators 2-APB (50 μM), Nimodipine (10 μM), Caffeine (10 mM), SKF 96365(20 μM) were used to modify calcium entry and release. Auranofin induced a sustained increase of [Ca2+]i in a concentration and time dependent manner. The use of different blockers of calcium channels did not reveal the source for the rise of [Ca2+]i. Overall, elevation of [Ca2+]i by Auranofin might be crucial for triggering Ca2+-dependent apoptotic pathways. Therefore, in anti-cancer therapy, modulating [Ca2+]i should be considered as a crucial factor for the induction of cell death in cancer cells.Auranofin, a transition metal complex is used for the treatment of rheumatoid arthritis but is also an effective anti-cancer drug. We investigate the effects of Auranofin in inducing cell death by apoptosis and whether these changes are correlated to changes of intracellular calcium concentration ([Ca Cancer is a result of uncontrolled cell-proliferation, invasion and destruction of healthy tissue. A defective apoptotic program is characteristic of all cancers. One of the main anticancer treatment strategies is to induce apoptosis in cancer cells. To achieve this goal, metal-based anti-cancer drugs have been developed  . Today, The mechanism of antitumor activity of gold compound is “DNA-independent”, and different from those of platinum drugs . Marzano2+]i) and there are considerable amounts of evidence on the central role of [Ca2+]i in proliferation and cell death. An increase in [Ca2+]i was shown to induce cell death in HeLa-S3 treated with cisplatin [2+]i, which could occur through an influx of Ca2+ through Ca2+-selective channels at the plasma membrane or by release from intracellular stores, is a key factor to trigger apoptosis. Boehning and coworkers emphasized the role of InsP3R-mediated calcium release from the endoplasmic reticulum in inducing apoptosis in response to specific stimuli [2+-response by specifically activating TRPA1 channels expressed in human differentiated neuroblastoma cell lines (nIMR-32). This mechanism is independent of the inhibition of the thioredoxin reductase [2+]i by mobilizing calcium from multiple storage sites, in a study done in neutrophils [2+]i in the induction of apoptosis by Auranofin in the breast cancer cell line MCF-7.Apoptosis is also regulated by intracellular calcium signaling , the control value and the values for low concentrations  were significantly different (p < 0.001) from the data taken with higher concentrations . In addition, a significant difference was found between concentrations of 3.1 μΜ and 12.5 μΜ or 25 μΜ (p = 0.002).Viability of MCF-7 cells were significantly impaired with increasing concentrations of Auranofin . Concentp > 0.05). At concentrations of 12.5 μM and 25 μM, the “live” population was found to be almost eliminated. The live population dropped significantly with concentrations higher than 6.25 μM (53.5 ± 31.6%). As it was not possible to do a morphological analysis of the cells by FACS, a clear distinction could not be made between debris and necrotic population which is a limitation of this method, nevertheless considering the total cytotoxicity (apoptosis + necrosis) in the two groups, untreated versus e.g., 12.5 μM of Auranofin, the total cytotoxicity was 17% and 97% respectively. A comparison of different concentrations for the different groups is illustrated in Increasing concentrations of Auranofin (in a single experiment) resulted in more apoptotic cells A. Under 50 of Auranofin calculated from FACS analysis was 5.1 μM. Pearson’s correlation was tested for the live and apoptotic population and showed a strong reciprocal (r = −0.964) link with respect to concentration, which means, the apoptotic population increased proportionally as viability decreased. Hence a concentration dependent increase in apoptotic population was detected.The IC2+]i  of Auranofin showed a significant increase in [Ca2+]i for the last three concentration within two hours as compared to initial [Ca2+]i .Auranofin triggered increase of [Ca2+]i A. The up [Ca2+]i B in more2+]i imaging experiments and the viability (MTS) assay, showed that the degree with which the viability was decreasing is closely related to increase in [Ca2+]i , caffeine (10 mM), nimodipine (10 μM) caffeine (10 mM) and SKF 96365 (20 μM)) did not change [Ca2+]i significantly . Thapsigargin did not change the effect caused by Auranofin significantly. The rise of [Ca2+]i was significantly different compared to the drug free condition (Control with thapsigargin only). p < 0.05 for all time points. SERCA was found to play no major role in Auranofin induced [Ca2+]i rise  based anti-arthritic drug. Here we examine its ability to induce cell death in a human breast cancer cell-line (MCF-7) and whether its cytotoxicity is related to a drug induced modulation of i, also plays a vital role in modulating pathways involving any of the above mentioned targets. Multiple studies support the central role for [Ca2+]i in the initiation of apoptosis in cancer cells. Splettstoesser and coworkers have reported that cisplatin induces concentration dependent [Ca2+]i by IP3-receptor mediated pathway in HeLa-S3 [2+]i elevation resulted in an increase of cytotoxicity and apoptosis in neuroblastoma cells treated with cisplatin and/or arsenic trioxide [2+ shift was regarded as an earlier marker of cytotoxicity in a study conducted with a panel of toxins tested in different mammalian cell lines [2+]i is modulated by Auranofin, as the strong reciprocal relation of “[Ca2+]i” versus “viability” indicates an important role of [Ca2+]i in inducing apoptosis.As calcium is an important signaling ion, which exerts effects on many enzyme and protein functions, it is likely that [Ca HeLa-S3 . A studytrioxide . Cytosolll lines . Our stu2+-conductance at the cell membrane [2+ from intracellular stores [2+]i and cell death [2+]i but the additional release of calcium from TG-sensitive calcium stores of the endoplasmic reticulum that triggers apoptosis [2+]i i .2+]i elevation directly caused an activation of caspases. Our studies with the blockers did not reveal the exact mechanism of [Ca2+]i elevation. Taking into consideration the complexity of Ca2+ signaling, at this juncture it remains difficult to elucidate the exact mechanism behind the apoptosis induced by elevation of [Ca2+]i. In addition the amount of calcium released from the ER or the mitochondria and the calcium entry through the plasma membrane for the observed elevations in [Ca2+]i needs further investigation in order to distinguish whether their functions are interconnected.With our analysis, we could not conclusively answer the question whether Auranofin induced apoptosis was a direct effect of the rise of intracellular calcium or an indirect effect and whether the i level for at least 30 min (control). The baseline for [Ca2+]i was obtained by averaging 20–25 frames at the beginning of the experiment (before drug application) and was considered as 100%. For each experiment at least 15–25 cells were recorded. Results were analyzed offline. For each experiment the Regions of Interest (ROI) were selected and only cells which were intact over the entire experiment were selected for analysis. Data (averaging the intensity of the fluorescent in the ROI) were exported to excel. Background values corresponding to each image were subtracted. The values obtained from the control conditions were averaged and set as control value (100%) to calculate the percentage change in intensity over time by the equation = (F/F0) × 100. Where, F is the intensity at a given time and F0 is the average intensity of the control window. The results were represented as 3D graph with time and concentration plotted against percentage increase in [Ca2+]i. Images were represented in “rainbow” scale  to highlight the difference in fluorescent intensities. Blue color represents lowest [Ca2+]i and yellow-red shows regions of high [Ca2+]i).Cells were plated in a 35 mm “eazygrip” BD Falcon tissue culture plates . The plates were washed with Tyrode’s buffer  for emptying the ER stores, 2-APB (50 μM) for inhibiting store-operated calcium (SOCE) release and a IP3-receptor blocker i and viable cells to apoptotic cells. Differences were considered significant when p-values were smaller than 0.05.Post hoc analysis using Bonferroni correction (2+]i in MCF-7 cell line. Prolonged elevation of calcium might be a key factor for triggering a Ca2+-dependent apoptotic pathway. To the best of our knowledge, this is the first study which analyzes the modulation of [Ca2+]i by Auranofin in a breast cancer cell line.Results indicate that the Auranofin induced an elevation of [Ca2+]i is a crucial factor to induce cell death in cancer cells. An answer to the mechanism of calcium modulation by Auranofin, by studying the calcium channels and pumps will help to improve the efficiency of drug action. To understand the exact mechanisms of [Ca2+]i modulation, further experiments are needed.We conclude that modulating [Ca


Two hundred and six patients, diagnosed with primary breast or prostate cancer completed self-report questionnaires on two occasions: before treatment (T1) and 12 months later (T2). The questionnaires included: the Hospital Anxiety and Depression Scale; Impact of Events Scale; the Metacognitions Questionnaire-30 (MCQ-30) and the Illness Perceptions Questionnaire-revised. A series of regression analyses indicated that metacognitive beliefs at T1 predicted between 14 and 19 % of the variance in symptoms of anxiety, depression and trauma at T2 after controlling for age and gender. For all three outcomes, the MCQ-30 subscale ‘negative beliefs about worry’ made the largest individual contribution with ‘cognitive confidence’ also contributing in each case. For anxiety, a third metacognitive variable, ‘positive beliefs about worry’ also predicted variance in T2 symptoms. In addition, hierarchical analyses indicated that metacognitive beliefs explained a small but significant amount of variance in T2 anxiety (2 %) and T2 depression (4 %) over and above that explained by demographic variables, T1 symptoms and T1 illness perceptions. The findings suggest that modifying metacognitive beliefs and processes has the potential to alleviate distress associated with cancer. Survival rates in cancer continue to improve. It is estimated that there are over 10 million cancer survivors in the USA  .From February 2010 to May 2011, 229 patients were consecutively recruited through two pre-treatment cancer clinics at a National Health Service (NHS) teaching hospital in North-West England. Inclusion criteria were a diagnosis of primary breast or prostate cancer and at least 18 years of age. Patients with recurrent or metastatic disease and those judged by the clinical team or researcher to be too distressed or confused to give informed consent were excluded. T1 data was obtained from self-report questionnaires and medical records shortly after diagnosis before primary treatment. Follow-up data (T2) was collected 12 months later through self-report questionnaires mailed to participants’ homes.All Measures were Assessed Both at T1 and T2The Hospital Anxiety and Depression Scale [HADS, ; and beliefs about causality. As the IPQ-R was included to assess the relative importance of patients’ illness appraisal, the emotional representation subscale was disregarded. The final section of the IPQ-R assesses patients’ causal attributions. These items are typically not summed as a single scale but may be analysed as separate items or as groups devised on the basis of theory  to .89 (‘positive beliefs about worry’) at T1 and from .79 (‘need to control thoughts’) to .91 (‘positive beliefs about worry’) at T2, indicating adequate to excellent internal consistencey.Metacognitive beliefs were measured using the Metacognitions Questionnaire 30- [MCQ-30 , the relevant variables were entered as demographic control variables in the first step of subsequent regression analyses.As not all scales were normally distributed, nonparametric statistics  were used to compare participants who completed both assessments with those who only completed T1 assessment on age group (divided above and below the median age), gender, educational level, perceived emotional social support, stage of disease, T1 HADS and IES scores. They were also used to examine the relationship of each T2 outcome with demographic, clinical and social support variables. Where significant associations with T2 outcomes were found  after controlling for demographic variables . These analyses were then repeated, using just the significant IPQ-R subscales from Analysis 1, and also controlling for T1 symptoms of anxiety, depression or trauma . As we had no a priori theory about which subscales would independently predict T2 outcomes, the IPQ-R subscales were included in each analysis using stepwise rather than forced entry. The subscales identified as independent predictors in Analysis 2 for each outcome were then entered as variables in Analysis 3 for that outcome (see below).This sequence of analyses was repeated for the MCQ-30, thereby testing hypothesis 1. We first identified the T1 MCQ-30 subscales that independently predict T2 outcomes after controlling for demographic variables , and then entered these in a further analysis also controlling for T1 symptoms of anxiety, depression or trauma . As with the IPQ-R analyses, as we had no a priori theory about which subscales would independently predict T2 outcomes, MCQ-30 subscales were included in each analysis using stepwise rather than forced entry. The subscales identified as independent predictors in Analysis 2 for each outcome were then entered as variables in Analysis 3 for that outcome (see below), which tested hypothesis 2.Final hierarchical multiple regression analyses  assessed whether the T1 MCQ-30 subscales which had been identified as significant predictors in Analysis 2 (see above) were able to predict variance in T2 outcomes over and above that explained by demographic variables, T1 symptoms and the negative content of thoughts about cancer at Time 1 . This final analysis used forced entry and bootstrapped sampling to ensure findings were robust.p = .034).Of the 229 participants who completed T1 questionnaires 206 (90 %) also completed the assessment 12 months later. No significant differences between completers and non-completers were apparent on T1 HADS and IES scores, age, gender, education, or tumour grade. However non-completers were more likely than completers to report low levels of perceived emotional support at T1 , and reported more trauma symptoms  than did men with prostate cancer or older patients. Women with breast cancer also reported more symptoms of depression at T2 than did men with prostate cancer . No outcome was related to education, perceived emotional support or tumour grade. Therefore just age and gender were used as demographic covariates in subsequent analyses. The levels of anxiety, depression and trauma symptoms at both time points are shown in Table The baseline demographic and clinical characteristics of the final sample N = 206) are shown in Table 06 are shRegression of emotional distress on the IPQ-R subscales Table  indicateThe results of the hierarchical multiple regression analyses to test hypothesis 1 are shown in Table The results of the hierarchical multiple regression analyses to test the second hypothesis  are shown in Table In the case of trauma, ‘cognitive confidence’ did not make any significant contribution to the variance explained in T2 symptoms after controlling for demographic variables, T1 symptoms and T1 illness perceptions (‘illness coherence’). In fact, younger age and T1 symptoms were the only variables to make a significant individual contribution to the final model, which accounted for 39 % of the variance.This is the first study to explore whether metacognitive beliefs soon after cancer diagnosis, and before active treatment (T1), predict emotional distress 12 months later (T2). T1 metacognitive beliefs predicted T2 anxiety, depression, and trauma after controlling for age, gender and T1 symptoms, thus supporting hypothesis 1. This finding builds on previous research in non-clinical populations in which metacognitive beliefs prospectively predicted levels of anxiety and depression two (Weber and Exner 2013), three  could be considered to be markers for worry or rumination in that they may be the outcome of these processes. Of the five MCQ-30 subscales included in Analysis 1, two (‘negative beliefs about worry’ and ‘cognitive confidence’) independently predicted T2 anxiety, depression and trauma, with a third (‘positive beliefs about worry’) also significantly contributing to the variance in anxiety. In all three cases ‘negative beliefs about worry’ made the largest individual contribution, as would be predicted by the metacognitive model of emotional disorder Wells . These fOne possibility is that this finding is due to the limitations of using hierarchical regression in a prospective study design where baseline emotional distress inevitably predominates in predicting future distress. Previous research  were also able to explain additional variance in anxiety and depression over and above age, gender, T1 symptoms, and T1 illness perceptions . This supports hypothesis 2 for these two outcomes. However, for trauma, metacognitive beliefs (‘cognitive confidence’) no longer significantly predicted T2 symptoms after including T1 illness perceptions (‘illness coherence’) in the analysis . However, it should be noted that the proportion of variance in T2 trauma explained by ‘cognitive confidence’ is unchanged between Analysis 2 (controlling for T1 trauma) and Analysis 3 (controlling for T1 trauma and T1 ‘illness coherence’). Furthermore, there is little difference in the variance explained by ‘cognitive confidence’ in trauma (1 %) and in anxiety (2 %). Therefore this apparent discrepancy may be an artefact of the present data. To our knowledge this is the first study to explore the prospective relationship between metacognitive beliefs (as measured by the MCQ-30) and trauma symptoms, making it difficult to judge the reliability of this finding.One limitation of the study is the restriction of the sample to breast and prostate cancer patients. These populations were selected because they represent the largest tumour groups in each gender and have a broadly similar prognosis. However this means it is not possible, in this sample, to separate out any effects that may be due to tumour group or gender. Furthermore, we cannot assume that the predictive effects found in this study would generalise to other cancer populations. Further studies will be needed to test the stability of the observed predictive effect of metacognitive beliefs on persistent emotional distress across genders and different tumour and prognostic groups. In addition, despite the prospective design, it should be noted that causality can still not be assumed as the influence of unmeasured confounders cannot be ruled out. In order to provide more compelling evidence of a causal role for metacognitive beliefs, further studies are necessary that adopt different approaches to design, such as experimental manipulation.In summary, the findings of the current study provide promising first evidence that metacognitive beliefs can help to predict anxiety, depression and trauma 1 year after diagnosis of breast and prostate cancer. Furthermore they support the hypothesis that metacognitive beliefs add to the variance explained in persistent anxiety and depression over and above that explained by negative content of thoughts about cancer. Consequently, therapeutic approaches targeting metacognitive beliefs and processes, rather than the content of negative thoughts about cancer, may prove beneficial for preventing persistent emotional distress in these populations.


Over the past decade many works have focused on various aspects of the dynamics of liquids confined at the nanoscale such as e.g. water flow enhancement through carbon nanotubes (CNTs). Transport of room temperature ionic liquids (RTILs) through various nanochannels has also been explored and some conflicting findings about their translational dynamics have been reported. In this work, we focus on translational dynamics of RTILs confined in various CNTs. By means of molecular dynamics simulations we highlight a substantially enhanced diffusion of confined RTILs with an increase up to two orders of magnitude with respect to bulk-phase properties. This ultrafast diffusion of RTILs inside CNTs is shown to result from the combination of various factors such as low friction, molecular stacking, size, helicity, curvature and cooperative dynamics effects. Room Temperature Ionic Liquids (RTILs) are molten salts composed of large organic cations and inorganic or organic anions. This class of solvents has been widely studied over the past decades due to their important applications11234671314et al. evidenced a faster molecular motion of [dmim+][Cl−] confined between two parallel structureless walls, notably for ions located close to the wallset al. obtained an enhanced diffusivity for [bmim+][Tf2N−] and [H2NC(dma)2][BETI] confined into mesoporous carbons31+][TFMSI−] confined inside uncharged slit-like graphitic nanopores of different widths+][TfMSI−] was found heterogeneous and strongly dependent on the distance between ions and the pore walls. Dynamics of ions located in the center of a slit pore of 5.02 nm in width was found similar to that of the bulk RTIL. Recently, Li et al. carried out MD simulations of [C4mim+][Tf2N−] confined in silica and carbon mesopores and observed a slowdown in ion diffusion inside both mesopores compared with bulk valuesSeveral numerical and theoretical works have focused on RTILs confined into cylindrical nanopores, such as CNTs1416171819202123242127272127294mim+][Tf2N−], [C8isoq+][Tf2N−] and [Me3BuN+][Tf2N−] confined in (i) two armchair CNTs of radius 13.05 Å ) and 27 Å ), (ii) a zigzag CNT of radius 13.05 Å ) and (iii) a slit pore made of graphene sheets separated by a distance of 27 Å were carried out. Models, force fields and computational procedure are detailed in the Method Section. [C8isoq+][Tf2N−] and [Me3BuN+][Tf2N−] were chosen so as to investigate the impact of aromatic rings on RTIL translational dynamics.In the present study, we explore the interplay between translational dynamics and structure of RTILs confined into uncharged CNTs with the aim of addressing several fundamental questions: i) How is RTIL translational dynamics impacted inside hydrophobic CNTs? ii) Is there translational heterogeneity according to the distance between ions and the CNT wall? iii) Is there any connection between local structure and dynamics? iv) Are there some cooperative effects? v) What is the impact of the CNT topology on RTIL dynamics? To address these issues, MD simulations of three RTILs, [C4mim+][Tf2N−] confined in different nanopores. Details about diffusion coefficient calculation are provided in the Method Section. A substantial increase in the diffusion coefficient of [C4mim+] confined in the CNT was observed with respect to the bulk value. Indeed, as shown in 4mim+] at 300 K is tenfold higher in the CNT than in the bulk phase. Our results are qualitatively in line with those obtained by Pinilla et al. who showed a local faster molecular motion of [dmim+][Cl−] confined between two parallel structureless walls+][4mim+] with respect to [Tf2N−] in the bulk phase344mim+][Tf2N−] in the ) at 400 K so as to evaluate the impact of temperature. As indicated in 4mim+] diffusion coefficient is about 300% at 400 K against 1000% at 300 K). This impact of temperature on the increase in the diffusion coefficient of confined RTILs was also observed experimentally4mim+][Tf2N−].We report in 8isoq+][Tf2N−] and [Me3BuN+][Tf2N−]). Let us note that different force fields were used to describe the different RTILs, namely all-atom and united-atom force fields (see Method Section), which shows that the enhanced diffusion phenomenon was predicted independently of the force field. Moreover, 8isoq+] and [Me3BuN+] cations confined in the CNT is greater than for [C4mim+] (in the case of [Me3BuN+] the increase in the diffusion coefficient with respect to the bulk phase can be as high as 160 at 400 K). These findings suggest that the enhanced diffusion phenomenon is not correlated with the number of aromatic rings in the RTIL structure. Moreover, in this work we studied three RTILs with different sizes and ultrafast diffusion was always evidenced. Let us mention that very recent experiments highlighted an increase in diffusion of other RTILs confined in CNTs. Therefore, although diffusion is quantitatively impacted by the size of RTIL ions the enhanced diffusion phenomenon reported in this work is expected to occur irrespective of ion sizeAs shown in 4mim+] and [Tf2N−] close to the pore surface  see . Such an4mim+][Tf2N−] confined within several media. As shown in 4mim+] strongly depends on the confining medium. Notably, its value inside a silica nanopore of 13 Å in radius (referred to hereafter as SiOH) or between graphene slabs (separated by 27 Å) was found to be lower than the bulk value. On the other hand, the diffusion coefficient of [C4mim+] confined in CNT and CNT was found to be higher than in the bulk phase although this increase was less pronounced than for the CNT. Comparing these different nanopores with the CNT, as a reference system, enables to investigate the impact of various effects on RTIL translational diffusion, namely size , R = 27 Å), helicity , R = 13.05 Å), roughness  and curvature  effects.Over the past decade some works reported the ultralow friction between water27373839404141D = kbT/Sλ where D is the diffusion coefficient, kb the Boltzmann’s constant, T the temperature, S the surface area and λ the friction coefficient. 4mim+][Tf2N−] confined in the different nanopores . The friction coefficient was found to increase according to the sequence CNT < CNT < CNT < graphene, in agreement with the Einstein’s relation. Furthermore, the results obtained with the CNT, CNT and the graphene slit-like pore highlight a curvature effect already observed for both water and organic molecules confined in CNTs27Based on previous findings on low friction of confined water within CNTs3738414mim+] inside the SiOH nanopore exhibits several peaks, which highlights RTIL interfacial anchoring while a smooth profile is observed in the CNT as a result of surface smoothness and low friction, which promotes free sliding along the nanotube axis. We report in 4mim+] aromatic ring and that of the nanopore for both SiOH and CNT frameworks. As shown in 3BuN+][Tf2N−] confined within CNTs also exhibits an ultrafast diffusion  diameter). As shown in 4mim+] diffusion coefficient being reduced by about 96% with respect to the bulk phase (z) and azimuthal (Φ) positions of [C4mim+] inside the CNT as a function of time. An obvious correlation was found between (z) and (Φ) thus suggesting a spiral motion inside the nanopore. This correlated motion in both directions is likely to play a significant role in the RTIL enhanced diffusion phenomenon. Such a translational mechanism  within CNTs was already observed with small molecules such as water, ethane and ethylene and was attributed to armchair CNT topology inducing preferential helicoidal pathways444mim+] diffusion coefficient within the CNT was found to increase by about 70% with respect to the bulk phase against 1000% for the CNT). As shown in 4mim+] axial and azimuthal motions was evidenced in the CNT, thus excluding spiral diffusion. Instead, z while keeping azimuthal mobility. These circular motions  dramatically limit the enhanced diffusion phenomenon inside zigzag CNTs. It is worth noting that surface tension was found similar for both CNTs  and the CNT, respectively) and cannot therefore be invoked to explain the great difference between RTIL translational dynamics within armchair and zigzag nanotubes . Additionally, as shown in As shown in lk phase . This ouFrom a local viewpoint, diffusion coefficients of ions confined in the CNT were found identical in both inner and interfacial zones see . In othe+][TFMSI−] confined inside uncharged slit-like graphitic nanopores4mim+][Tf2N−] dynamics when confined in silica and carbon mesoporesTo sum up, we reported ultrafast diffusion of RTILs confined in CNT materials. We showed that this enhanced diffusion phenomenon is ruled by an interplay of low-friction, size, helicity, curvature and cooperative dynamics effects. This study clearly highlights the role of pore size that induces cooperative dynamics of confined ionic liquids and the role of pore helicity, curvature and smoothness on the translational dynamics of RTILs. Our results also allow understanding some results reported in the literature such as the local increase in the translational mobility of [Emim4mim+][Tf2N−]) and octyl isoquinolium bis(trifluoromethyl)sulfonimide ([C8isoq+][Tf2N−]). [C4mim+][Tf2N−] was modeled from a recent united atom force field8isoq+][Tf2N−] was described by means of the non-polarizable AMBER99 force fieldet al. have demonstrated that the generic non-polarizable AMBER force field is able to reproduce RTILs physical properties3BuN+][Tf2N−], was described by using a revisited OPLS force fieldet al.τt = 0.5 ps was considered. Periodic boundary conditions were applied in the three directions. MD simulations were performed in the canonical ensemble at T = 300 K and T = 400 K. MD simulations were performed using a time step of 0.002 ps to sample 200 ns (acquisition phase) after a 100 ns equilibration. Electrostatic interactions were truncated at 12 Å and calculated by using the Ewald sum with a precision of 10−6. Short range interactions were modeled by using a Lennard-Jones potential and a cutoff of 12 Å. Lennard-Jones interactions between inorganic framework and ionic liquids have been taken into account by means of the Lorentz-Berthelot mixing rule. Statistical errors were estimated using the block average method. Comparisons between bulk and confined properties were performed at All MD simulations were carried out with the DL_POLY package (version 4.0)54Density of confined RTILs was computed by means of an anisotropic barostatz component of the total Mean-Square-Displacement (MSD) of molecular center-of-mass56Given the radial anisotropy and the cylindrical symmetry the translational diffusion coefficient t0 the time origin, N the number of molecules and N0 the number of t0. Note that in the bulk phase the total MSD was considered and was then divided by 6 for determining the bulk diffusion coefficient.with λ) was computed from f)S is the surface, kB the Boltzmann’s constant, T the temperature, N the number of ions and Naf the number of framework atoms. We report in 4mim+] [Tf2N−] in the CNT. The λ values given in the manuscript correspond to the plateau values.Friction coefficient (γ) was calculated by using the non-exponential methodγ is expressed as F the free energy, A the surface area, N the number of molecules and V the volume. Let us note that the non-exponential method was recently developed with a rigorous theoretical background596162U is the configurational energy. This expression was approximated through a finite difference such that U is the energy difference between two states of different surface areas (ΔA = A1−A0 where 0 stands for the reference state and 1 stands for the perturbed state). Thus, to maintain the volume constant the following anisotropic transformations were used L the box length. The area of a cylindrical interface is Re is the radius of the equimolar dividing surface. Re was achieved by calculating the radial density profile (ρ(r)) from ρv and ρl are the vapor and liquid densities, respectively. Let us note that this choice is arbitrary and other definitions could be used such as a fit of the density profile by means of a hyperbolic function. Indeed, this definition was specifically developed for spherical interfaces. However, we compared the so-calculated Re with the radius obtained by means of a hyperbolic tangential fitLet us note that it was the first time that the surface tension of ionic liquids confined within a nanopore was calculated. Surface tension (U(0) (rN) and U(1)(r′N) are the configurational energies of the reference and perturbed states, rN and r′N are the configurational space for both states, <…>0 stands for that the average taken over the reference state. Surface tensions of [C4mim+][Tf2N−] confined in CNT, CNT, CNT and graphene-based slit pore were 6.1, 8,9 20.9 and 34.4 mN m−1 respectively. Error bars were between 1–3 mN m−1.where How to cite this article: Ghoufi, A. et al. Ultrafast diffusion of Ionic Liquids Confined in Carbon Nanotubes. Sci. Rep.6, 28518; doi: 10.1038/srep28518 (2016).


Paraoxonase-1, a high-density lipoprotein linked enzyme complex, was shown to be decreased in several cardiovascular diseases. We aimed to explore whether serum paraoxonase and arylesterase activities differ in dipper and non-dipper prehypertensive subjects compared to healthy controls.Sixty prehypertensive subjects and 30 controls were enrolled. All subjects underwent echocardiographic assessment and 24-hour ambulatory blood pressure monitoring (ABPM). According to the blood pressure (BP) course on ABPM, prehypertensive subjects were categorized into two: non-dipper prehypertensive (NDPH) and dipper prehypertensive (DPH) groups. Serum paraoxonase and arylesterase activities were detected spectrophotometrically.Paraoxonase and arylesterase activities were significantly lower in patients with NDPH compared to both DPH and control groups. Both paraoxonase and arylesterase activities showed significant negative correlations with BP and left ventricular mass index.We have demonstrated that NDPH subjects have lower paraoxonase and arylesterase activities compared to DPH subjects and normotensives. Further prospective studies are needed to clarify the role of paraoxonase and arylesterase activities in the development of overt hypertension in prehypertensive subjects. Ambulatory blood pressure monitoring (ABPM) provides valuable information about diurnal BP patterns. In the vast majority of the population, night-time BP values are 10% to 20% lower than the day-time BP values (dipper), whereas the nocturnal BP decrease is blunted or even absent in a certain group of people (non-dippers).6 The non-dipping BP pattern is also reported to be associated with increased rates of CV morbidity and mortality.7The term prehypertension, defined in the 7th Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7), is widely used to classify individuals whose systolic blood pressure (SBP) levels are in the range of 120 to 139 mm Hg and diastolic blood pressure (BP) between 80 and 89 mm Hg.9 This enzyme consists of paraoxonase, arylesterase and diazoxonase activities.10 Human serum PON-1 activity has been reported to be inversely related to the risk of CV diseases and percutaneous coronary intervention related complications such as no-reflow and contrast induced nephropathy.13 In addition, reduced PON-1 activities were observed in patients with coronary artery disease, HT, familial hypercholesterolemia and diabetes mellitus.18Paraoxonase-1 (PON-1) is an enzyme located on high-density lipoprotein (HDL) particles and was demonstrated to protect lipoprotein particles from oxidation.19 However, paraoxonase and arylesterase activities have not been investigated in prehypertension yet. The goal of this study was to evaluate whether or not paraoxonase and arylesterase activities are related to the diurnal BP profiles in prehypertensive subjects.Paraoxonase-1 activity was found to be at low levels in patients with non-dipper HT and white coat HT.1 None of the subjects had known coronary risk factors and cardiac symptoms. All participants had normal electrocardiographic and echocardiographic findings. Past medical histories of all participants were recorded and thorough physical examination was performed. The individuals with a history of established heart disease (n = 42), smoking (n = 36), diabetes mellitus  (n = 18), HT (defined as BP > 140/90 mm Hg or those who were receiving anti-hypertensive medication) (n = 29), inflammatory diseases (n = 5), cancer (n = 2), chronic renal disease (n = 14), cerebrovascular disease (n = 3), thyroid disease (n = 1), liver disease (n = 3), alcohol or drug abuse (n = 8), hypercholesterolemia (n = 26), steroid use (n = 3) and the ones who refused to participate the study (n = 6) were excluded from the study. The study was approved by the local Ethics Committee, and each subject provided written informed consent.A total of 286 individuals who admitted consecutively to cardiology outpatient clinic of Dicle University Heart Hospital and underwent 24-hour ABPM between September to December’14 were evaluated for this case–control study. One hundred ninety-six of them were excluded due to exclusion criteria and 60 of them were diagnosed as prehypertension and formed the study group. The remaining 30 healthy individuals having a day-time average SBP < 120 mm Hg and DBP < 80 mm Hg formed the control group. According to JNC 7 report, prehypertensive subjects were defined as those with BPs ranging from 120 to 139 mm Hg systolic and/or 80 to 89 mm Hg diastolic at office settings.Blood pressures of individuals were measured with a mechanical sphygmomanometer manually in the office settings. The average of two or more properly measured BP was recorded after the individuals were seated quietly for at least 5 minutes in a chair with feet on the floor and arm supported at heart level. Caffeine, exercise, and smoking were avoided for at least 30 minutes prior to BP measurement.22Twenty four-hour non-invasive ABPM was performed on a workday with a portable compact digital BP recorder . Automatic BP measurements were recorded at 20-min intervals for diurnal readings (08.00–22.00 h) and at 30-minute intervals for nocturnal readings (22.00–08.00 h), which yielded more than 60 BP recordings during the 24-hour period. Night-time and day-time periods were assessed based on the information declared by the subjects. The sleep BP was defined as the average value of the BP measurements from the time subjects went to bed until the time they got up. The day-time BP was defined as the average of the BP recorded during the rest of the day. The subjects were divided into 2 groups according to the amount of decrease in BP during night-time: prehypertensive patients with a nocturnal reduction in average day-time systolic and diastolic BP of less than 10% were classified as non-dipper prehypertensives (NDPH group); those with a ≥10% decrease during night-time were classified as dipper prehypertensives (DPH group).23 Left ventricular mass (LVM) was calculated according to the Devereux Formula24: LVM = (1.04 × [(LVEDd + IVSth + PWth)3 − (LVEDd)3] − 13.6). Then, LV mass index (LVMI) was derived by dividing LVM by body surface area.Transthoracic echocardiography was performed to all participants using an echocardiograph equipped with a broadband transducer . Left ventricle end-systolic (LVESd) and end-diastolic diameters (LVEDd), left atrial diameter, interventricular septal thickness (IVS) and ventricular posterior wall thickness (PW) were measured. Left ventricle (LV) ejection fraction (EF) was determined by Teichholz method.Blood samples were obtained to measure the glucose, creatinine, total cholesterol, triglycerides, LDL, and HDL cholesterol levels following an overnight fasting. Samples were withdrawn from an antecubital vein into dry blood tubes and centrifuged at 3000 rpm for 10 minutes. Serum samples were stored at −80°C and then dissolved and analyzed.25 The rate of hydrolysis of paraoxon was measured by monitoring the increase in absorbance at 412 nm at 25°C. Phenylacetate was used as a substrate to measure the arylesterase activity which is calculated as described by Haagen et al26 Both paraoxonase and arylesterase activities were expressed as U/L serum.Paraoxonase activity was determined as described by Eckerson et alt test or Mann–Whitney U test was used for the continuous variables between two groups and the chi-square test for categorical variables. Comparison between 3 groups was performed by one-way analysis of variance (ANOVA) or Kruskal–Wallis test. Post hoc analyses of the pair wise comparisons were conducted with Tukey's post hoc test in one-way ANOVA. Mann–Whitney U test with Bonferroni correction was used to conduct the pair wise comparisons when there is a significant difference between 3 groups in Kruskal–Wallis test. A p value <0.017 (0.05/3) was considered as the cut-off point to reject H0 hypothesis during the pair wise comparisons. Pearson or Spearman correlation test was used for correlation analysis. With the help of an online program, the needed sample size for the study was calculated as 30 subjects per group in order to achieve the power of test above 80%. Statistical significance was defined as p < 0.05.Data were analyzed with the SPSS software version 18.0 for Windows . The Kolmogorov–Smirnov test was used to verify the normality of distribution of continuous variables. Continuous variables were defined as mean ± standard deviation; categorical variables were given as percentages. According to the distribution pattern, the independent samples p < 0.001 and p = 0.006, respectively).Among 90 patients who were clinically evaluated with the ABPM, 32 patients had non-dipper prehypertension, 28 patients had dipper prehypertension and 30 patients were normotensive. Demographic, clinical, and echocardiographic characteristics of the groups were shown in Table p < 0.001, for both).The measurements of ABPM and paraoxonase and arylesterase activities were shown in Table p < 0.001 for all, Figure p < 0.001 for all) . Correlation analysis between enzyme activities and other parameters are presented in Table Serum paraoxonase and arylesterase activities of NDPH group were lower than both DPH and control groups  are significantly lower in patients presented with acute coronary syndrome and paraoxonase and arylesterase activities had a significant protective effect even after adjustment for HDL level, age, BMI, and PON1 polymorphism in the logistic regression analyses. Another study by Demirbag et al37 demonstrated reduced levels of paraoxonase and arylesterase activities in CAD patients who received bare-metal stent and developed in-stent restenosis (IRS) compared to those who did not develop IRS and healthy controls. Besides CAD, both paraoxonase and arylesterase activities were found significantly lower in patients with non-dipper HT than those of dipper hypertensives and healthy subjects.19 Our results were consistent with previous studies; the NDPH group had lower paraoxonase and arylesterase levels compared to DPH group and healthy subjects.Recent studies showed that oxidative stress plays an important role in the atherosclerosis pathogenesis and LDL oxidation has been shown to have a pivotal act in the development of endothelial dysfunction and atherosclerosis.39 Although the exact underlying mechanism of the enhanced oxidative stress in HT is not completely understood it has been widely accepted that increased vascular reactive oxygen species (ROS) forming enzymes, such as xanthine oxidase and NADH/NADPH oxidase, contributes to HT development.41 Increased ROS in the arterial wall induce smooth muscle contraction and proliferation, which causes aggravation of HT.42 Arterial stiffness, evaluated by pulse wave velocity, is an important component of isolated systolic HT in elderly and was shown to be a predictor for future cardiovascular events in several studies.44 Reduced paraoxonase activity was found to be associated with arterial stiffness in hypertensive and renal transplant patients.46Several studies have shown that humans with essential HT have a decreased antioxidant capacity and enhanced oxidative stress.48 In a study conducted by Sathiyapriya et al, prehypertensives had higher malondialdehyde and protein carbonyl levels, which are products of polyunsaturated fatty acid peroxidation and protein oxidation, respectively. In addition, erythrocyte glutathione was reduced in the prehypertensive subjects suggesting an imbalance in the oxidant/antioxidant ratio in prehypertensive state.47 The ATTICA study that was conducted with 3042 subjects and demonstrated lower total antioxidant capacity and higher oxidized LDL levels in prehypertensive subjects compared to normotensives. Besides, they reported an inverse correlation of total antioxidant status and a positive correlation of oxidized-LDL with BP.48 However, previous studies have not investigated paraoxonase and arylesterase activities in prehypertensive subjects. In the present study, paraoxonase and arylesterase activities were decreased in NDPH group compared with DPH and control groups. Moreover, both paraoxonase and arylesterase activities were negatively correlated with BP levels and LVMI.The presence of oxidative stress in prehypertensive subjects was reported in previous studies.10 Our results do not have capability of generalization due to rather small sample size. Further large scale, prospective studies are needed.The small sample size and the cross-sectional design were the main limitations of our study. Lack of other oxidative stress markers and PON-1 genotype evaluation was another limitation. However, serum PON-1 activity was shown to be better than PON-1 genotype for the prediction of future CV disease risk.In conclusion, with the present study we demonstrated that NDPH subjects have reduced paraoxonase and arylesterase activities compared to DPH subjects and healthy controls. Additionally, NDPH group had significantly higher LVMI compared to DPH and control groups, which may herald forthcoming cardiac structural alterations seen in hypertensive patients. An increase in the oxidant/antioxidant ratio may give an idea about the relation of non-dipping state with increased risk of HT development in the future. With prospective large-scale studies, the role of paraoxonase and arylesterase activities may be clarified in the development of HT in prehypertensive subjects.


SERT; SLC6A4), serotonin receptor 2A (HTR2A), and oxytocin receptor (OXTR) genes with ASD. We examined their association with high functioning autism (HFA) including siblings and their interaction.Autism spectrum disorder (ASD) is heritable and neurodevelopmental with unknown causes. The serotonergic and oxytocinergic systems are of interest in autism for several reasons: (i) Both systems are implicated in social behavior, and abnormal levels of serotonin and oxytocin have been found in people with ASD; (ii) treatment with selective serotonin reuptake inhibitors and oxytocin can yield improvements; and (iii) previous association studies have linked the serotonin transporter  and one in HTR2A (rs6311) as well as the triallelic HTTLPR (SERT polymorphism).In this association study with HFA children (IQ > 80), siblings, and controls, participants were genotyped for four single nucleotide polymorphisms (SNPs) in G alleles)  = 1.697, 95% CI 1.191–2.204)). Four polymorphisms  in combination conferred nominal significant risk for HFA with a genetic score of ≥4 . The resulting area under the receiver operating characteristic curve was 0.595 (p = .033).We identified a nominal significant association with HFA for the HTTLPR s allele  contains supplementary material, which is available to authorized users. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interactions and communication and by repetitive behaviors , 2. An AHTR2A gene (NCBI Gene ID: 3356) and the SERT gene  have suggested association with ASD [SERT contains a polymorphic region (HTTLPR) with a short allele (S) and a long allele (L) that is 44 bp longer and can contain an additional single nucleotide polymorphism (SNP) (rs25531), making the locus triallelic  [SERT expression, with the S and LG alleles  reducing transcriptional efficiency [HTR2A have concentrated mostly on three non-coding SNPs  [Both the serotonergic and oxytocinergic systems seem to play a role in ASD and social behaviors . The serwith ASD –29. In tG and S) , 31. Theficiency , 31. Stificiency . Associa rs6314) –28, 32.oxytocin gene with autism; however, several studies have reported an association with the oxytocin receptor gene , although inconsistently [OXTR polymorphism: rs2301261, rs53576, rs2254298 and rs2268494, were studied in ASD and social behavior [Similarly, the oxytocinergic system has attracted attention for similar reasons: (i) Low plasma oxytocin levels have been observed in autistic boys , 34;  and OXTR (up-regulation), which might further reveal downstream pathways mediating brain developmental disorders. Moreover, Dolen et al. [Recent reports have indicated some interactions among 5-HT, serotonergic components, and oxytocin , 45, 46.n et al.  could deSERT, HTR2A, and OXTR have led to controversial findings, but the mentioned genes seem to interact with one another, we attempted to replicate these associations analyzing ASD children (high functioning), their siblings, and controls with no clinical diagnoses. In contrast to other studies, we included only patients with HFA in our study, eliminating confounding parameters such as IQ. Sibships transmission analysis was included to enhance further the case–control findings. Additionally, we tested for interaction among the three genes, since these were reported to be involved in ASD and even further to interact with each other, on the assumption that neurodevelopmental disorders are polygenetic rather than monogenetic.Because previous association studies of The study was approved by the ethics committee of the Canton Zurich, Switzerland (E-36/2009). Parents of all participants gave their written consent after being informed about the aim of the study. All participants  were Caucasians between 5 and 17 years of age collected in the Department of Child and Adolescent Psychiatry at the University of Zurich. In all patients diagnosis was confirmed using the Autism Diagnosis Observation Schedule  and the The siblings of the ASD group  did not have an ASD diagnosis or other severe psychiatric disorders according to screening questionnaires (see below). For the control group, only children without any clinical diagnosis were included in the study .Additionally, all participants were screened for psychopathology with the following parent reports: Child Behaviour Checklist ; Social Saliva samples for DNA isolation were collected from all recruited individuals using the Oragene DNA kit . DNA was isolated from saliva according to the manufacturer’s protocol . For rs2301261, rs2254298, rs2268494, and rs6311 , genotyping was performed using TaqMan® SNP Genotyping Assays . PCR was carried out on a CFX384™ Real-Time System  in a 5 μl (10 μl for rs6311) volume using TaqMan® 2× Universal PCR Master Mix No AmpErase® UNG (Applied Biosystems) and 10 ng (22.5 ng for rs6311) of DNA. Initial enzyme activation was carried out at 95°C for 10 min, followed by 40 cycles at 92°C for 15 s and 60°C for 1 min.For rs25531 and HTTLPR analysis, the restriction fragment length polymorphism method was used. Amplification was carried out on a CFX384™ Thermal Cycler (Bio-Rad) in a 10 μl (rs25531) or 25 μl (HTTLPR) volume using GoTaq® Green Master Mix 2× .For rs25531, the same primers as described previously were used . PCR conFor HTTLPR, the primer sequences were 5′-TGC CGC TCT GAA TGC CAG CAC-3′ and 5′-GGG ATT CTG GTG CCA CCT AGA CG-3′. PCR conditions were similar to those for rs53576, but only 30 cycles were carried out at 95°C for 45 s, 66.5°C for 45 s, and 72°C at 1 min. Fifteen microliters of PCR product were run on a 3% agarose gel to distinguish the L allele (463 bp) and S allele (419 bp). The remaining PCR product was digested similarly as described above but with 20 U MspI . Visualization on 3% agarose gel allowed distinction of the G allele  from the A allele  of rs25531.2 test. A sibship disequilibrium test was performed for each polymorphism according to Horvath and Laird [Each study group and the total sample were tested for deviation from Hardy–Weinberg equilibrium for all polymorphisms, and no significant departures were found .Gene–gene interactions were studied by comparing each combination of two polymorphisms. For each combination, a three-dimensional contingency table with “polymorphism 1 × polymorphism 2 × study group” was built, and a three dimensional χAdditionally, a receiver operating characteristic (ROC) curve analysis was performed for the various combinations of genetic scores sums, simulating their polygenetic effects on the risk for ASD . GeneticThe nominal significance threshold was set to 5% and the adjusted significance according Bonferroni for multiple testing was set to 0.8%. Power analysis was performed using G*Power version 3.1.6 , 63.OXTR, HTR2A, and SERT. Genotype and minor allele frequencies for all six polymorphisms are summarized in Table To investigate whether oxytocinergic and serotonergic system genes are associated with HFA, 253 children were genotyped for polymorphisms in the HTR2A and OXTR with autism was observed.HTTLPR triallelic polymorphism allele frequencies were nominal significantly associated with Asperger diagnosis  = 1.697 (95% CI 1.191–2.204)) with the s allele as a risk allele Table . The absForty “mixed” sibships were included (sibships with affected and unaffected siblings). No significant transmission disequilibrium was detected for any of the tested polymorphisms Table .Table 42-test revealed nominal significant association of HTTLPR and HTR2A (rs6311) with ASD, as well as of the two OXTR SNPs rs53576 and rs2268494  and between rs2254298 and rs2268494 (p = .005); probably causing high transmission of these variant combinations.Testing for gene–gene interactions by a χTR2A rs631 with ASHTR2A rs6311, and OXTR rs2254298 and rs53576  was below 20% for polymorphisms with two genotypes and about 30% if all three genotypes were present.The limitations of our study include the relatively small minor allele frequency for three of the The strength of our study is the narrow phenotype regarding the intellectual and language ability and cognitive function. Most previous investigations have analyzed samples consisting of the complete spectrum of autism whereas in our investigation all individuals were diagnosed with HFA. In the past, 50–70% of autistic children were classified as intellectually disabled, but those with Asperger have, by definition, an IQ in the normal range  and typiOXTR in HFA, finding a weak association [oxytocin gene itself [CD38 gene [PITX, ATP2B2, SLC25A12, and EN2, we could show that a combination of four polymorphisms  confers a nominal significant risk for HFA. This result points to the possibility that these genes play a role in ASD, probably in combination with additional risk genes that should be further explored.As far as we know, only one study has investigated an association of ociation . Of the ociation . Furthere itself ; the CD3D38 gene , whose gD38 gene –80; and D38 gene , whose gD38 gene . These fD38 gene , who comOXTR, HTR2A, and SERT with ASD, but we could not confirm these with HFA except for a nominal association with the HTTLPR polymorphism. Our findings might be explained by the fact that HFA individuals have different symptoms from others with ASD and by the wide heterogeneity in the ASD population. Of interest, however, a combination of those polymorphisms resulted in nominal significant risk for HFA, pointing to the importance of a polygenetic rather than monogenetic context, in which each gene contributes to a very small fraction of the phenotype. Therefore, we suggest that future association studies should look into this aspect and examine various combinations of risk genes with HFA.In summary, many studies have found associations of The data sets supporting the results of this article are included within the article .Table S2. Allelic variation used in the calculation of genetic score under an additive model. Table S3. Gene–gene interactions and their associations with autism spectrum disorder (ASD). Table S4. Receiver operating characteristic curve analysis results for the different combinations of polygenetic risk for autism spectrum disorder (ASD). (DOC 152 KB)Additional file 1: Table S1: Results of testing for deviation from Hardy–Weinberg equilibrium.


Kplant). Leaves account for an important fraction of Kplant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (Kleaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, Kleaf recovered only 2 hours after plants were rewatered. Recovery of Kleaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in Kleaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in Kleaf.Gas exchange is constrained by the whole-plant hydraulic conductance ( High gas exchange rates can only be sustained when leaves are kept well hydrated. This, in turn, depends on the properties of the xylem pipeline and on the way in which water moves through living cells in roots and leaves Vitis viniferaArabidopsis thaliana, Picea glauca and Oryza sativaPopulus trichocarpainvitro and in plantaThis recovery of hydraulic function may be facilitated by the activity of aquaporin (AQP) water channels Populus trichocarpa plants were exposed to moderate drought and then rewatered. The objective was to study the recovery of Kleaf from water stress at both physiological and molecular levels. We hypothesized that leaves would quickly  recover from water stress, and that this would be associated with modulation of AQP activity. To test this hypothesis, we monitored Kleaf and Ψleaf during a dehydration-rehydration episode. We also explored the regulation of 12 leaf-expressed AQP isoforms as well as the tissue-specific location of PIP1, PIP2 and TIP2 proteins. Recovery of Kleaf was assessed in two ways: (i) intact plants were taken through a drying-rewatering cycle, and (ii) detached leaves were bench-dried and subsequently xylem-perfused with AQPs inhibitors.In this present study, P. trichocarpa clone 664042 cuttings . Rooted cuttings were produced and established in a greenhouse at the University of Alberta for 2 months in 3.8 L containers with sunshine mix 4 (Sun Gro Horticulture Canada Ltd.) under semi-controlled conditions  once a week).All experiments were carried out with 2 solution  was used for these measurements. Flow rate through leaves was measured with a balance , which logged data every 30 s to a computer. The air was well stirred by a fan as explained by Sack & Scoffoni −2 s−1 photosynthetically active radiation (PAR) at the leaf surface by an LED worklight . Leaf temperature was monitored by a thermocouple. Leaf water potential (Ψleaf) was measured using a pressure chamber . For hydrated leaves, the Kleaf was calculated as described previously leaf , which was determined at the end of the measurement of E, immediately after removing the leaf from the tubing system. The Kleaf was normalized by leaf area, which was determined with a scanner.Leaf hydraulic conductance was measured using the evaporative flux method (29) on six plants per treatment. A filtered (0.2 µm) 20 mM KCl+1 mM CaClKleaf was measured on leaves corresponding to leaf plastochron index (LPI) 9 leaf (ΨO) was measured using a leaf immediately above or below the leaf that was subsequently connected to the tubing system. Once a leaf was connected to the tubing system, a stable flow rate (E) was usually reached in less than 20 minutes; this value of E was subsequently used for calculating Kleaf. Leaves that did not provide stable E within 20 min were discarded. When dehydrated leaves are measured with the EFM, their Ψleaf may change because the petiole is connected to water at atmospheric pressure final and ΨO were compared. In most cases, ΨO and Ψfinal were similar and the more negative of these two values was used to calculate Kleaf. When ΨO and Ψfinal differed by more than 0.2 MPa, the leaf was discarded and Kleaf was not calculated. The leaf vulnerability curve (shown in Kleaf = a/[1+(Ψleaf/x0)b]. The curve fit was done using SigmaPlot v. 13 . The curve fit was used to calculate the maximum leaf hydraulic conductance as well as the leaf water potentials at 50% and 80% loss of hydraulic conductance.A leaf vulnerability curve was generated with plants experiencing different levels of water stress following methods of Sack & Scoffoni shown in  was fittKleaf in intact plants, plants were randomly assigned to different watering regimes in the greenhouse. One group of plants was kept well watered (control). Another group of plants was subjected to a drought treatment. Water was withheld for several days until plants reached a Ψleaf of −0.77±0.05 MPa . This Ψleaf was associated with a substantial reduction in Kleaf. A subset of drought-stressed plants was then rewatered, and Ψleaf and Kleaf were re-measured 2 h and 26 h after rewatering.To study the recovery of Kleaf, excised leaves were bench-dried for 1 h ) and then perfused for 2 h with AXS, AXS+0.2 mM HgCl2, AXS+50 mM H2O2 or AXS+50 µM ABA. Solutions were introduced into the transpiring leaf by immersing the petiole inside 50 mL containers. Leaves were placed near a fan; light was provided at a light level of ∼1,000 µmol m−2 s−1 PAR. Mercury chloride and H2O2 have been widely used as AQP inhibitors; ABA may also reduce AQP activity in leaves . Control leaves were always kept hydrated and were perfused with pure AXS for 2 h. Immediately after perfusion with these solutions, Kleaf was determined as described above. All measurements were conducted at the same time of day (10∶00–11∶30 h).To assess the effect of AQP inhibitors and abscisic acid (ABA) on the recovery of After perfusion with the different solutions, stomatal pore aperture of leaves was measured as described in Laur & Hacke −2 s−1 PAR . Dye (0.1% (w/v) safranin) was dissolved in pure AXS or AXS+0.2 mM HgCl2. Control leaves were excised from well-watered plants and then perfused for 2 h with 0.1% safranin-containing AXS without prior dehydration treatment. Images were recorded in six randomly selected fields of view of each leaf. Fields of view were located near the point of maximum leaf width on the abaxial leaf surface.The extent of dye uptake in excised leaves was used as an additional method to assess xylem refilling in leaf veins during the rehydration phase. We also used the dye uptake experiments in an attempt to study how embolism reversal in leaf veins is impacted by mercury. Excised leaves were bench-dried for 1 h and rehydrated for 2 h by immersion of the petioles in filtered safranin solutions. Transpiration during dye uptake was promoted by placing leaves near a fan at a light level of ∼1,000 µmol mFully expanded leaves corresponding to LPI 7–10 were collected, immediately frozen in liquid nitrogen and stored at −80°C until analyzed. Samples were always collected between 10∶00 h and 11∶30 h to minimize any diurnal effect on AQP expression. Total RNA was extracted from 3 plants per treatment following the CTAB method of Pavy et al. Samples were fixed in formaldehyde-acetic acid and embedded in paraffin as described previously P≤0.05.All statistical analyses were carried out using SigmaPlot software. Differences due to the effect of physiological treatments were analyzed after testing for normality and equal variance by using a one-way ANOVA followed by a Tukey’s test. A one-way ANOVA followed by Bonferroni’s post test was used for the gene expression analysis. Differences were considered significant at Kleaf declines as a function of Ψleaf, we first constructed a vulnerability curve. Water was withheld from plants in the greenhouse until plants reached different levels of water stress. Leaves were highly vulnerable with 50% and 80% loss of hydraulic conductance occurring at Ψleaf = −0.47 MPa and −0.71 MPa, respectively  are well within the range of water potentials that trees experience under natural conditions Kleaf is subject to substantial diurnal changes under natural conditions, similar to what has been observed in rice and other species To assess how ectively , insert.Kleaf would recover after a drought treatment when plants were left intact during the dehydration-rehydration episode. In this experiment, leaves of well-watered control plants had a Ψleaf of −0.33±0.03 MPa , which was associated with a Kleaf of 3.37±0.41 mmol m−2 s−1 MPa−1   and a six-fold drop of Kleaf to 0.55±0.12 mmol m−2 s−1 MPa−1  , indicating that leaves completely recovered their hydraulic function.We next tested whether , n = 6) , insert.Kleaf, AQP expression was measured in leaves at different stages during the dehydration-rehydration experiment. Three PIP1, three PIP2, and six TIP candidate genes were selected for analysis. Among them, PtPIP1;1, PtPIP1;2, PtPIP1;3; PtPIP2;4 and PtTIP2;1 exhibited the highest total number of mRNA molecules in leaves of control plants (To study the role of water channels in the recovery of l plants .PIP1s) remained down-regulated while the expression of a second group of genes matched or exceeded the transcript levels measured in control leaves. With the exception of PtTIP2;1, all tested TIPs were significantly up-regulated after 2 h. Among the PIPs, only the expression level of PtPIP2;3 increased to match the control level.The drought treatment resulted in a significant reduction in the expression of all tested genes . In leavKleaf. Control leaves exhibited a Kleaf of 8.49±0.57 mmol m−2 s−1 MPa−1 , which is higher than the values shown in Kleaf was measured while the control leaves in Kleaf. Hence, the absolute Kleaf values shown in Another set of experiments was conducted on leaves that were excised from the plant prior to the dehydration-rehydration treatment. Working with detached leaves allowed us to study the effect of AQP inhibitors and ABA on the recovery of Kleaf relative to fully hydrated control leaves , similar to the value of ∼10 µm previously reported for P. trichocarpa leaves P. deltoides and in P. trichocarpa x deltoides hybrids Kleaf in dehydrated leaves and in leaves that were perfused with AQP inhibitors is greater than that of changes in stomatal aperture. Guyot et al. Kleaf, and they discuss possible reasons for a mechanistic independence of stomatal and leaf hydraulic conductance.We next asked whether differences in 2 . Genes that were strongly down-regulated by dehydration, such as PtTIP1;6 also exhibited strong down-regulation after perfusion with ABA solution while the expression of other genes, such as PtPIP2;4, changed less in response to either of these factors .Notably, very similar degrees of down-regulation were found in bench-dried leaves and in dried leaves that were subsequently xylem-perfused with AXS + ABA . While our data is consistent with these observations, it is not clear yet which cells may perform the role of a ‘control center’ in P. trichocarpa leaves  were TIPs and were also found to be up-regulated when intact plants were rewatered after a drought  of the conserved the C-terminal region of PIP2s, and (c) TIP2s. Consensus amino acids are underlined in black.(a) Amino acid multiple sequence alignment of the N-terminal region of the (DOCX)Click here for additional data file.Figure S2Transverse section of a Populus trichocarpa leaf showing minor veins with (left) and without (right) bundle sheath cell extensions. Scale bar = 20 µm.(DOCX)Click here for additional data file.Table S1Primer sequences used for the gene expression study.(DOCX)Click here for additional data file.


This phenomenon rapidly increased soil moisture and stimulated microbial carbon (C) cycling, and the flux of water vapor to soil had a stronger impact than temperature on microbial activity. In a semiarid grassland, we also observed that non-rainfall water inputs stimulated microbial activity and C cycling. Together these data suggest that, during rain-free periods, atmospheric moisture in drylands may significantly contribute to variation in soil water content, thereby influencing ecosystem processes. The simple physical process of adsorption of water vapor to soil particles, forming liquid water, represents an overlooked but potentially important contributor to C cycling in drylands.Water drives the functioning of Earth’s arid and semiarid lands. Drylands can obtain water from sources other than precipitation, yet little is known about how non-rainfall water inputs influence dryland communities and their activity. In particular, water vapor adsorption – movement of atmospheric water vapor into soil when soil air is drier than the overlying air – likely occurs often in drylands, yet its effects on ecosystem processes are not known. By adding  In drylands, precipitation is so low relative to demand that the availability of water represents the primary control over biological processes4This significant role in global biogeochemical cycles and the central influence of water in regulating activity has generated interest in the relationships between precipitation and C cycling in dryland ecosystems7891113In the absence of precipitation, there are three primary mechanisms for the addition of water to surface soils: fog deposition, dew formation, and water vapor adsorption17181521222224252018O-enriched water vapor was added to the atmosphere of sealed jars containing field dry soil from a semiarid grassland. Temperature and relative humidity were monitored over a three-day period as soils experienced simulated diurnal temperature fluctuations  typical for an arid region. Through the addition of 18O-enriched water vapor to the atmosphere , we were able to trace the movement of water vapor into the soil. Addition of water vapor to the atmosphere increased the atmospheric relative humidity (18O atom percent excess (APE) confirmed the transfer of moisture from the atmosphere to the soil (18O APE) was linearly related to the gravimetric moisture content of the soil . Across all treatments, 35–50% of the water added to the atmosphere was adsorbed. Water vapor adsorption occurred at atmospheric relative humidity values ranging from 20–60% . These results suggest that temperature may have opposing effects on microbial activity in dryland soils. On one hand, higher temperatures, which increase molecular diffusion rates, will lead to greater respiration activity but also deplete soils of scarce moisture. On the other hand, colder temperatures will allow more water vapor adsorption, thereby increasing soil moisture and microbial habitat in soil. Maximum respiration rates may be observed when soils are warming up early in the morning, as they still contain moisture trapped through water vapor adsorption, yet are warm enough to support high microbial activity. Others have also observed peaks in respiration during the relatively cool morning hoursA second laboratory experiment was performed to examine the influence of temperature on water vapor adsorption and the extent to which it regulates soil microbial activity. Field dry soil was incubated for three days under the simulated diurnal temperature fluctuations previously described, or at a constant temperature of either 23 or 40 °C. Water vapor was added to the atmosphere of treatment jars (500 μg fully evaporated into ~1 L headspace), while experimental controls received no water addition. Water vapor adsorption was confirmed by an increase in the gravimetric water content of the soils over the course of the incubation. Relative to their moisture content at the initiation of the experiment (2.3%), the average moisture content of soils in the water vapor adsorption treatment increased by 65.9% for the simulated diurnal treatment, 63.3% for the 23 °C treatment, and 59.9% for the 40 °C temperature regime. Thus, water vapor adsorption occurred under all temperature regimes, and the concomitant increases in CO2 efflux  indicate2 = 0.42, p < 0.001). Relative humidity varied from 14 to 49%, gravimetric soil moisture content varied from 0.36 to 4.79%, and both were generally higher during the morning hours. These findings are consistent with other studies showing that diurnal fluctuations in water vapor adsorption by soil during the summer season follow fluctuations in atmospheric relative humidity2230We also explored atmospheric water inputs in the field. At a semiarid grassland in northern Arizona, USA , changesStipa tenacissima tussocks, as vegetation limits fluctuations in atmospheric relative humidity near the soilWhile we focused our study efforts on bare patches of soil within a grassland ecosystem, it is important to consider the microclimate that develops within the plant canopy2 efflux in the field . Triplicate jars for each treatment were then subjected to temperature cycling designed to simulate diurnal temperature fluctuations typical of an arid environment: 4 h at 23 °C, 8 h at 40 °C, 4 h at 23 °C, 8 h at 10 °C, repeated over a 72-h period. Immediately prior to each temperature transition, relative humidity and temperature values were recorded. Gravimetric moisture content was determined for each soil sample at the end of the experiment (105 °C for 24 h). Water was extracted from soil samples using a cryogenic vacuum line18O using a G1102-i isotopic H2O cavity ring down spectrometer . The amount of water vapor adsorbed (H2OWVA) was calculated using the known isotope compositions of the water added to the atmosphere (2 atom% 18O), the native soil water , and the isotopic mass balance equation where:In the first laboratory experiment, 30 g samples of sieved (2 mm) soil from the grassland were placed in 1000 ml sealed Nalgene jars also containing a hygro-thermometer  and a small reservoir with either 0, 50, 100, or 500 μg of 2 concentration using an infrared gas analyzer model L1-6262 . All samples showed a linear increase in CO2 concentration over time, and production rates (μg CO2-C g soil−1 day−1) were calculated using linear regression (median correlation coefficient = 0.92).For the second laboratory experiment, 30 g samples of sieved (2 mm) soil from the grassland were placed in each of 30 sealed Mason jars (946 ml). Half of the jars included a small reservoir with 500 μg of water, while the other half experienced no atmospheric water addition. The jars were then equally divided so that five replicate jars were incubated for 72 h at high (40 °C) or moderate (23 °C) temperature, or the temperature cycling previously described. Gas samples (5 ml) were collected from the headspace of each jar 0, 52, and 72 h after the initiation of the experiment using an air-tight syringe inserted through a septum on the lid of each jar. Headspace samples were analyzed for COBouteloua eriopoda, Bouteloua gracilis, Sporobulus cryptandrus, and Pleuraphis jamesii) with few shrubs . Soils are cindery and are classified in the U.S. Department of Agriculture Soil Taxonomic Subgroup of Typic Haplustolls. There is a Campbell Scientific  weather station located at this field site that collects temperature, moisture, and humidity data in addition to other environmental variables.The field site is a semiarid, high-desert grassland near Flagstaff, AZ, USA . The dominant plants are perennial grasses . Ten soil collars (50 cm diameter) were installed in plant interspaces throughout the site, and CO2 efflux measurements were taken at these locations every hour for 24 h. One soil core (5 cm diameter and depth) was collected every hour during the 24-h period for gravimetric moisture determination. Soil samples were sieved (2 mm) and weighed in the field, then carefully transported back to the laboratory where they were dried (105 °C for 24 h) and reweighed.In early July of 2013, prior to the arrival of monsoon precipitation and during a period when grasses were physiologically dormant, a 24-hour observational study was conducted in order to characterize diurnal soil moisture fluctuations and COhttp://www.R-project.org). Path analysis was performed with SPSS Amos 22, and the path model was a good fit to the data .Treatment effects in the laboratory experiments were assessed with one- or two-way ANOVA in JMP Pro 9.0.2 . Linear regressions were performed in R .


For bone collagen, we found a sizeable range of 1.8‰ for δ13Ccol and 1.9‰ for δ15Ncol among laboratories, but an interpretatively insignificant average pairwise difference of 0.2‰ and 0.4‰ for δ13Ccol and δ15Ncol respectively. For bone hydroxyapatite the observed range increased to a troublingly large 3.5‰ for δ13Cap and 6.7‰ for δ18Oap, with average pairwise differences of 0.6‰ for δ13Cap and a disquieting 2.0‰ for δ18Oap. In order to assess the effects of preparation versus analysis on isotopic variability among laboratories, a subset of the samples prepared by the participating laboratories were analyzed a second time on the same instrument. Based on this duplicate analysis, it was determined that roughly half of the isotopic variability among laboratories could be attributed to differences in sample preparation, with the other half resulting from differences in analysis . These findings have serious implications for choices made in the preparation and extraction of target biomolecules, the comparison of results obtained from different laboratories, and the interpretation of small differences in bone collagen and hydroxyapatite isotope values. To address the issues arising from inter-laboratory comparisons, we devise a novel measure we term the Minimum Meaningful Difference (MMD), and demonstrate its application.Over the past forty years, stable isotope analysis of bone (and tooth) collagen and hydroxyapatite has become a mainstay of archaeological and paleoanthropological reconstructions of paleodiet and paleoenvironment. Despite this method's frequent use across anthropological subdisciplines (and beyond), the present work represents the first attempt at gauging the effects of inter-laboratory variability engendered by differences in a) sample preparation, and b) analysis . Replicate analyses of a  The past thirty years have witnessed an explosive increase in the ubiquity of stable isotope analysis of osseous remains in the fields of archaeology, paleoanthropology, and paleoecology . Indeed,13C 13C signatures. Shortly after this first publication, DeNiro 13Ccol and δ15Ncol in controlled diet experiments with a variety of animals 13Ccol and δ15Ncol values could be used to discriminate between habitual consumers of marine versus terrestrial foodstuffs, and Ambrose documented the importance of both diet and environment on collagen isotope values The importance of stable isotopes for archaeology was first realized by Robert Hall in the late 1960s when he noted anomalously young radiocarbon dates produced by maize or any other species enriched in 13Cap), and realized the potential for the technique's application to specimens from “well back into the Pleistocene” [20∶335]. With Lee-Thorp and Van Der Merwe's 13Cap values of dental enamel preserved biogenic signatures, the die for such work was cast  dental and skeletal remains has become commonplace. Indeed, Curiously, however, while the pace of archaeological and anthropological applications of stable isotope analysis has increased, the validation of the technique's assumptions has lagged behind. There have been numerous studies of relevant methodological issues, including isotopic routing 13Ccol, δ15Ncol) derived from different laboratories are directly comparable. However, the direct comparison of isotopic data derived from bone hydroxyapatite  is more dangerous because variability engendered by differences in pretreatment, analysis, and standardization is of a far greater magnitude. To remedy this issue, we introduce what we have termed the Minimum Meaningful Difference (MMD) value, which serves as an empirically derived threshold by which the significance of values obtained in different laboratories might be judged. In the end, the results of this study have serious implications for choices made in the preparation and extraction of target biomolecules, the comparison of results obtained from different laboratories, and the interpretation of small differences in bone collagen and hydroxyapatite isotope values.The results of this study suggest that, in general, isotopic data from bone collagen  identification and sub-sampling of a suitable ancient human bone sample, 2) verification of this bone's antiquity, 3) recruitment of a representative cohort of participating laboratories, and 4) construction of a rigorous survey and reporting regime by which both laboratory methods and results could be compiled in a manner that would facilitate subsequent statistical analysis. The goal of this work is to characterize the amount of variation present among laboratories rather than comment on “better” or “worse” preparation methods or analytical facilities.In 2011, one of us (WJP) obtained a presumably ancient unprovenienced human femoral diaphysis from the Museo Gustavo Le Paige in San Pedro de Atacama, Chile. All necessary permits were obtained for the described study , which complied with all relevant regulations, and the field studies did not involve endangered or protected species. This specimen was judged to be appropriate based on its apparent excellent state of preservation , large size (>100 g), and likely ancient date.14C dated at the University of Arizona NSF-AMS facility following their established protocols for 14C dating of bone . The resulting AMS date for this specimen (laboratory #AA99865) is 1728±47 14C years before present (δ13C -17.3‰), This equates to a 2-sigma calibrated age range of 238–470 cal AD when calibrated using Calib 7.0 and the SHCAL13 southern hemisphere terrestrial curve The specimen was AMS 13C, δ15N, δ18O), participating laboratories would be expected to provide details on pretreatment and analytical methods, as well as sample preservation assessments . All potential participants were informed that while their participation in the project would be made known, laboratory attributions of individual results would be kept confidential and all publicly disseminated data would be presented using randomly generated designators. However, in order for each participating laboratory to be able to assess its results compared to those for other study participants, the respective PI's were informed that they would be provided, at the conclusion of the study, with a full complement of the study's results with their data indicated.Subsequent to radiocarbon dating, the authors solicited forty-six archaeological and paleoecological isotope laboratories in order to assess their willingness to participate in this study. Interested laboratories were informed that they would be provided with sufficient sample material to prepare and analyze at least three collagen and three hydroxyapatite replicates . While this study was intended to document variation in the analysis of both collagen and hydroxyapatite, participants were asked to perform both types of analysis only if this was routine for their laboratory. In addition to isotopic data  ultimately committed to participate . When laIn addition to receiving five bone samples, each participating laboratory was provided with an instruction letter requesting that they prepare all five replicate samples using the same standard laboratory method and four standardized survey forms –S4 to usSummaries of the collagen and hydroxyapatite protocols for each laboratory are provided (using anonymous identifiers) in Plus , and standardized in reference to NBS-19 (TS-Limestone). These duplicate analyses allowed us to independently assess the degree to which isotopic variability resulted from pretreatment versus analysis.To control for at least one potential source of variability, isotopic analysis, we reanalyzed as many samples as possible on one instrument. Eighteen of the twenty laboratories that performed collagen extractions (90%), and eleven of the sixteen laboratories that extracted hydroxyapatite (69%), returned aliquots of prepared material for reanalysis. Three aliquots of collagen and hydroxyapatite (when available) were selected from each laboratory's returned samples for reanalysis. Collagen samples were reanalyzed at the UC-Davis Stable Isotope Facility using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer . Elemental concentration was standardized by reference to Glutamic Acid, and stable isotope composition was standardized by reference to bovine liver, nylon, and USGS-41 Glutamic Acid. Hydroxyapatite samples were re-analyzed in the Stable Isotope Geochemistry Stable Isotope Laboratory at the Rosenstiel School of Marine and Atmospheric Sciences at the University of Miami using a Kiel-IV Carbonate Device  coupled to a Thermo-Finnigan Deltahttp://genome.tugraz.at/genesisclient/genesisclient_description.shtml). Significance for all analyses was set at α = 0.05.In addition to the use of a battery of well-established statistical analyses , we also used heat maps to visually identify which pretreatment protocols clustered together . Heat maps visually display data patterns by assigning a gradation of color to numerical values. The heat maps depict the difference in the values obtained by each pair of labs . Both axes were clustered using average linkage hierarchical clustering, with Euclidean distance as the distance metric. Heat maps were generated using the Genesis software package developed by Alexander Sturn and Rene Snajder . The intent of this metric is to establish a means by which to evaluate isotopic results obtained from different laboratories, or when comparing newly obtained results to previously published values in the literature. Our hope is that these values will be treated as an experimentally generated threshold value that one could quickly use when evaluating whether newly generated isotopic data are significantly more enriched or depleted than another laboratory's results or previously published isotopic data. This metric is far more meaningful than a simple t-test, for example, as it explicitly takes into account inter-laboratory variability.both laboratories in each pairwise comparison). Using this value, a researcher can evaluate with ∼95% confidence the likelihood that a newly obtained isotope value is different from another value as a consequence of bona fide biogenic differences rather than laboratory pretreatment and analysis.The development of MMDs assumed that the values obtained in the course of the present study are representative of the possible isotope values that might be obtained from any laboratory currently performing such analysis. Minimum Meaningful Differences were calculated by adding the average pairwise inter-laboratory difference for each isotope system plus four times the average of the standard deviations obtained by each laboratory participating in the present study , we present these for comparability with other studies. Across all laboratories, the respective values for these metrics were: collagen yield = 16.4±7.9% , weight %C = 41.7±5.3%, weight %N = 15.2±1.9%, and atomic C:N ratio = 3.2±0.1. These data robustly confirm the excellent quality of preservation of the collagen in the selected specimen.13Ccol values averaged −17.0±0.3‰ and had an overall range of 1.8‰ : One from Laboratory B, two from Laboratory H, and four from Laboratory L   , bottom.l values .13Ccol, the average pairwise inter-laboratory difference was 0.2‰ . Significant differences remained even after outliers were removed . The average pairwise inter-laboratory difference for δ15Ncol values was 0.4‰ . Again, significant differences remained even after outliers were removed .Analysis of inter-laboratory variation indicates significant differences among laboratories for the two isotope systems of interest. For δ13Ccol  and 0.2‰ δ15Ncol . No significant differences in δ13Ccol or δ15Ncol were observed as a result of humic acid removal reagents; no treatment, NaOH, and KOH produced indistinguishable δ13Ccol  and δ15Ncol  values. It is possible that the lack of appreciable differences in isotope values between laboratories that did and did not remove humic acids could be a consequence of the sample's low initial humic content.It is worthwhile noting that neither the choice of demineralizing agent (HCl versus EDTA) nor the decision of whether/how to remove humic acids  engendered significant differences in the resulting isotopic signatures. The offset seen between samples demineralized using HCl versus EDTA was only 0.2‰ for δ13Ccol decreasing from -17.0±0.3‰ to −17.1±0.1‰  from 9.0±0.4‰ to 9.1±0.2‰  were no longer aberrant after reanalysis. This strongly suggests that although collagen pretreatment methods are responsible for some of the observed isotopic differences among laboratories, differences in instrumentation or data calibration also drive a large amount of the observed variation in isotope values among laboratories: 69%  for δ13Ccol and 48%  for δ15NcolReanalysis of the collagen samples affected the number and distribution of outliers. For δ13Ccol , top, th  value, which takes into account both the average inter-laboratory difference and the typically observed intra-laboratory variability (in the form of the standard deviation of each laboratory's replicate measurements), was determined to be a modest 0.6‰ for δ13Ccol . This me δ15Ncol  was slig13Cap values averaged −11.7±0.6‰ and had an overall range of 3.5‰ , a finding that persisted following the removal of the four outlying values . The average pairwise inter-laboratory difference for δ18Oap values was 2.0‰ .Analysis of inter-laboratory variation indicates significant differences among laboratories for the two isotope systems of interest (note red cells in 2O2). Indeed, the isotope values of samples processed using hydrogen peroxide produced significantly enriched δ13Cap and δ18Oap values compared to those samples processed with bleach, with values of −11.8±0.7‰ versus −11.5±0.2‰ for δ13Cap  is greater than the average pairwise difference in δ18Oap values between any two participating laboratories (2.0‰), it would appear that the choice of reagent used for organic removal is a prime driver in inter-laboratory variation in δ18Oap13Cap, as the difference in means between oxidation methods (0.3‰) is less than the average pairwise inter-laboratory difference (0.6‰).One presumed driver of inter-laboratory variation in isotope values is the chosen method of organic removal, namely bleach  versus hydrogen peroxide  and 0.15‰ δ18Oap . The differences between samples treated with buffered and un-buffered acetic acid were similarly small: 0.06‰ for δ13Cap  and 0.2‰ for δ18Oap . This result is somewhat unexpected, as previous studies 13Cap and δ18Oap values.Differences in the labile carbonate removal technique (both concentration of acetic acid and the use of buffered versus un-buffered acetic) did not have a significant effect on δ18Oap, almost halving the standard deviation of the measured samples from 1.7‰ to 0.9‰ . While the variance for δ13Cap was also reduced during reanalysis, the observed difference (0.6‰ versus 0.5‰) was modest by comparison, and not significant .Subsequent reanalysis of a subset of the hydroxyapatite samples on the same instrument revealed at least two interesting phenomena. First, reanalysis produced significantly enriched isotope results for carbon isotope values, which increased modestly from −11.7±0.6‰ to −11.2±0.5‰ . Indeed, there was only one outlier among the two isotope systems, a solitary samples from Laboratory O which had a δ13Cap value with a z-score of 2.4 (p<0.01). This finding suggests that Laboratory R's aberrant isotope values in the first round of analysis were the result of analytical instrumentation, working standards, or data calibration rather than a preparation step per se, would seem to drive a large portion of the observed variation in isotope values for hydroxyapatite: 44%  for δ13Cap and 54%  for δ18Oap. These results echo the recent findings of Carter and Fry Two other phenomena of note were evident after reanalysis. First, the samples from Laboratory R, which had outlying δ13Cap values between samples oxidized using bleach versus hydrogen peroxide , a significant difference in δ18Oap values remained . Therefore, although instrumentation drove some of the isotopic variation among laboratories, differences in preparation  were also responsible for observed differences in isotopic values obtained from different laboratories, a finding that is in agreement with previous studies Second, while reanalysis on the same instrument reduced the difference in δ13Cap and δ18Oap were 0.6‰ and 2.0‰ respectively. The Minimum Meaningful Difference (MMD) value for δ13Cap was determined to be 1.2‰ , as is more fully elucidated below.As noted above, the average inter-laboratory pairwise differences for δ be 1.2‰ . This var δ18Oap  was much13Ccol and 0.4‰ for δ15Ncol. These values are of such a small magnitude as to not be cause for great concern. As to causality, neither of the most obvious differences in pretreatment between participating laboratories  had a significant effect on the resulting isotope values.The present study began with the goals of: 1) quantifying inter-laboratory variability in stable isotope analysis of bone collagen and hydroxyapatite, and 2) tracing the likely causes of this observed variability. For bone collagen, we found statistically significant inter-laboratory variation for both carbon and nitrogen isotope values among laboratories. However, the average pairwise difference between any two participating laboratories was only 0.2‰ for δ13Ccol and 0.9‰ for δ15Ncol have a high likelihood of being of biological origin rather than an artifact of pretreatment or analysis. In sum, it would appear that the results of stable isotope analysis of bone collagen from one laboratory can be compared (if cautiously) with results obtained elsewhere. Overall, inter-laboratory variability in collagen isotopes would not appear to be of paramount concern.Subsequent reanalysis of a subset of samples on the same instrument indicates that the prime driver of inter-laboratory variation in collagen stable isotope analysis is differences in analytical instrumentation and/or standardization rather than pretreatment . Finally, the Minimum Meaningful Difference (MMD) value establishes a threshold by which results obtained from two laboratories might be evaluated. Differences exceeding 0.6‰ for δ13Cap and δ18Oap was significant, and while the average pairwise difference between any two participating laboratories was only 0.6‰ for δ13Cap, for δ18Oap, that value rose to 2.0‰, a difference which could easily change interpretations of past residency or paleomobility. It is unlikely that anything more than a small portion of this variability is the result of differential diagenesis 2O2) appear to be a prime driver of δ18Oap variability, but not for δ13Cap. However, differences in the method for removing labile carbonate (acid concentration and buffering agent) do not have a significant effect on either isotope system, counter previous suggestions For bone hydroxyapatite, the results of the present study are somewhat less reassuring. Inter-laboratory variability for both δbona fide rather than an artifact of different pretreatment or analytical methods only if they exceed 1.2‰ for δ13Cap and 3.1‰ for δ18Oap. The magnitude of these MDD values, particularly for δ18Oap, might call into question the attribution of biological significance oftentimes given to different δ13Cap and δ18Oap values obtained in different laboratories. In sum, it would appear that inter-laboratory variability could be a significant concern for hydroxyapatite. Analytical results from different laboratories might not be directly comparable, particularly in the case of δ18Oap.As with collagen, the subsequent reanalysis of a subset of samples on the same instrument indicates that differences in analytical instrumentation and/or standardization (rather than pretreatment per se) were a prime driver of inter-laboratory variation in hydroxyapatite stable isotope values . Efforts to unify data correction among laboratories would likely decrease this variability Three final points merit consideration. First, it should be noted that while the present study addresses the wisdom of (over)claiming the significance of dissimilar results from different laboratories, even small differences (if replicable) obtained in one laboratory can still be considered reliable. Second, the bone hydroxyapatite results presented here may not be directly applicable to comparisons of enamel hydroxyapatite, a tissue thought to be far more resistant to diagenesis Figure S1Sample hydroxyapatite extraction protocol form.(EPS)Click here for additional data file.Figure S2Sample hydroxyapatite result form.(EPS)Click here for additional data file.Figure S3Sample collagen extraction protocol form.(EPS)Click here for additional data file.Figure S4Sample collagen result form.(EPS)Click here for additional data file.Table S1Summary of the collagen pretreatment methods for each participating laboratory.(XLSX)Click here for additional data file.Table S2Summary of the hydroxyapatite pretreatment methods for each participating laboratory.(XLSX)Click here for additional data file.