The present study tested the combined effects of using emoticons and colours on labels amongst a nationally representative sample of the UK population (n = 955). In a 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) ×2  between-subjects experiment, participants rated the level of desirability, healthiness, tastiness, and calorific content of a snack bar they had been randomised to view. At the end they were further randomised to view one of nine possible combinations of colour and emoticon labels and asked to choose between a chocolate and a cereal bar. Regardless of label, participants rated the chocolate as tastier and more desirable when compared to the cereal bar, and the cereal bar as healthier than the chocolate bar. A series of interactions revealed that a frowning emoticon on a white background decreased perceptions of healthiness and tastiness of the cereal bar, but not the chocolate bar. In the explicit choice task selection was unaffected by label. Overall nutritional labels had limited effects on perceptions and no effects on choice of snack foods. Emoticon labels yielded stronger effects on perceptions of taste and healthiness of snacks than colour labels. Frowning emoticons may be more potent than smiling emoticons at influencing the perceived healthiness and tastiness of foods carrying health halos.Recent studies report that using green labels to denote healthier foods, and red to denote less healthy foods increases consumption of green- and decreases consumption of red-labelled foods. Other symbols ( One of the most pressing public health threats is the global obesity epidemic. Obesity, or body adiposity, is one of the four behavioural risk factors most strongly related to the steady rise of non-communicable diseases, including cardiovascular diseases, cancers, chronic pulmonary diseases, and diabetes . To tackSeveral studies to date have looked at the effects of nutritional labelling, defined as information being given about at least one nutrient in either relative or absolute terms, where the information is visible at the point of purchase and consumption choice see . ReviewsOne frequently used labelling format has been colour labels, in particular traffic light colours , to provide nutritional information of foods. A recent lab study by However, other studies have found no evidence that colour labels increase perceptions of healthiness and greater consumption of healthier products. Recent studies in supermarket settings in the UK and Australia found that the introduction of front-of-pack traffic light labels did not lead to healthier food purchasing . More reA recent paper by descriptive norm) leads to decreased engagement with the negative behaviour. However, such interventions often produce unintended “boomerang” effects, whereby people who already abstain from the undesirable behaviour actually inadvertently start engaging more with the negative behaviours in order to match the social norms surrounding that behaviour  to the descriptive normative message buffered successfully against this “boomerang” effect.This suggestion was tested in a recent study by In the domain of food a recent systematic review by There are several gaps in Moreover, the finding that a simple smiling or frowning emoticon can influence people's engagement with a certain type of behaviour may have far-reaching consequences for the design of very simple, engaging, easy to understand, and cost-effective population-based interventions that may have the added benefit of reaching the most deprived social groups. Importantly, combining smiling or frowning emoticons with different colours could make them even more potent signals of food healthiness. With these considerations in mind we examined the combined effects of emoticon expressions and colour nutritional labels, based on those used in ageM = 50.31, ageSD = 14.45) with sampling quotas set for SES occupational status  × 3 (colour label: red vs. green vs. white) × 2  between-subjects design. This gave 18 possible combinations of snack bar and label, one of which was randomly presented to each participant for the primary endpoint measures  now?” Responses were recorded on Likert-type scales anchored from 1 = Not at all to 9 = Very much.Our primary endpoint measures were single-items adapted from Compared to other bars like this, how tasty is this chocolate bar ?” Responses were given on Likert-type scales anchored from 1 = Less tasty to 9 = More tasty.Ratings of tastiness were measured by: “Compared to other bars like this, how healthy is this chocolate bar ?” was an item used to assess healthiness ratings. Response options were anchored from 1 = Less healthy to 9 = More healthy.“Compared to other bars like this, how many calories do you think this chocolate bar  contains?” Answers were given on a scale from 1 = Fewer calories to 9 = More calories.Estimates of the calorific content were obtained by a single item: “After measuring the primary endpoint, participants were further randomised to see one of the nine combinations of chocolate bar and cereal bar shown in “How hungry do you feel right now?”, answered on a 7-point rating scale anchored at −3 = Very hungry, 0 = Neither hungry nor full, +3 = Very full] and BMI were measured to examine whether there were any differences between participants randomised to different conditions that might affect the ratings of the snack foods.Hunger [The study was conducted online. The experiment was conducted in accordance with APA standards for the ethical treatment of human participants, and gained the prior approval by the Psychology Research Ethics Committee of the University of Cambridge. Electronic informed consent was obtained from all participants. In the first phase of the online study participants rated their current hunger levels. Then they were randomly assigned by the survey software platform Qualtrics to one of the 18 versions of the experiment. Once they completed the ratings on the primary endpoint, participants were asked to indicate their demographics, as well as weight and height, for BMI calculations. At the very end of the experiment, participants were further randomised into one of nine possible combinations of coloured emoticon labels and were asked to choose between a chocolate and a cereal bar (secondary endpoint).Fs < 1.1), and BMI  as dependent variables revealed no significant differences between the experimental conditions, indicating that randomisation of participants was successful. Furthermore, inclusion of hunger and BMI as covariates in the analyses did not affect the observed results for any of the dependent measures.Two separate 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) × 2  between participants' ANOVAs with hunger  = 47.51, p < .001, η2 = 0.047, whereby participants reported greater overall desire to eat the chocolate bar  than the cereal bar . For means and standard deviations see A 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) × 2  between participants' ANOVA on desire to eat the given bar at that moment revealed a significant main effect of snack bar type, F = 2.71, p = .067, η2 = 0.005. An examination of simple main effects revealed that whilst overall the current desire for consumption was greater for the chocolate bar than the cereal bar across all three colour labels, this difference was most pronounced when the colour label was white, F = 33.98, p < .001, η2 = 0.035, and least pronounced when the colour label was green, F = 7.75, p = .005, η2 = 0.008, with red labels falling in the middle, F = 10.99, p = .001, η2 = 0.012  = 8.06, p = .005, η2 = 0.008, whereby participants rated the chocolate bar  as tastier overall than the cereal bar . For a breakdown of means and standard deviations see A 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) × 2  between participants' ANOVA on tastiness ratings revealed a significant main effect of snack bar type, F = 2.86, p = .022, η2 = 0.012. Breaking down the three-way interaction revealed that there was a significant two-way interaction between food option and colour only when coupled with a frowning emoticon, F = 4.75, p = .009, η2 = 0.026, but not when coupled with a smiling emoticon or with no emoticon, Fs < 1.86, ps ≥ 0.156. An examination of simple main effects revealed that the largest difference in tastiness ratings between the chocolate bar  and the cereal bar  was obtained when there was a frowning emoticon expression on a white colour label, F = 10.59, p = .001, η2 = 0.011.The main effect was qualified by a significant three-way interaction, F = 3.21, p = .040, η2 = 0.023, but not when the label was red or green, Fs < 2.28, ps ≥ 0.103. An examination of simple main effects showed that emoticon expression had a significant effect on ratings of tastiness for the cereal bar only when the colour label was white, F = 4.91, p = .008, η2 = 0.010, whereby tastiness ratings of the cereal bar were higher in the presence of a white smiling emoticon (M = 5.34) compared to a white frowning emoticon (M = 4.44) or no emoticon white label (M = 4.53). No other main effects or interactions were significant.Furthermore, breaking down the three-way interaction differently revealed that there was a two-way interaction between food option and emoticon face expression when the label was white, F = 33.38, p < .001, η2 = 0.033, whereby participants rated the cereal bar  healthier than the chocolate bar . There was also a main effect of emoticon expression, F = 3.20, p = .041, η2 = 0.006, whereby participants rated snack bars labelled with no emoticons as healthiest , snack bars with smiling emoticons in the middle , and snack bars with frowning emoticons as least healthy . For a breakdown of means and standard deviations see A 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) × 2  between participants' ANOVA on healthiness ratings revealed a main effect of snack bar type, F = 2.80, p = .025, η2 = 0.011  = 6.74, p = .001, η2 = 0.014, and not when the colour label was green or red, Fs < .98, ps ≥ 0.376. Breaking down the interaction differently, white colour labels led to higher healthiness ratings when compared to red and green colour labels only when they were accompanied by a smiling emoticon, F = 5.11, p = .006, η2 = 0.011, but not when they were accompanied by a frowning or no emoticon, Fs < 1.65, ps ≥ 0.193.The main effects were qualified by a significant two-way interaction between colour label and emoticon expression, .011 see . An examF = 2.99, p = .051, η2 = 0.006  = 5.52, p = .004, η2 = 0.012, but did not lower healthiness ratings for the chocolate bar, F = .52, ps ≥ 0.595. No other main effects or interactions were significant.Furthermore, the effects were also qualified by a significant two-way interaction between emoticon expression and snack bar type, .006 see . An examFs < .54. For means and standard deviations see A 3 (emoticon expression: smiling vs. frowning vs. no emoticon) × 3 (colour label: green vs. red vs. white) × 2  between participants' ANOVA on ratings of calories yielded no significant main effects or interactions, all A series of Chi-square tests were carried out to analyse the explicit choice of snack bar in the nine options that participants were randomised to see. The Chi-Square tests revealed that amongst all nine options participants chose the chocolate bar irrespective of the label see .ps > .634.We also carried out a binomial probability analysis whereby we tested the likelihood of choosing one of the snack bars whilst comparing the options where both snacks were equally encouraged  with those where chocolate choice was encouraged , and those where cereal bar choice was encouraged . The analysis yielded no significant results, all In an experiment manipulating the colour and injunctive norm (emoticon expression) on nutritional labels, colour and emoticon expressions had mixed effects on perceptions and choice of snack foods. As predicted we did not find an additive effect on perceptions of labelled snack foods, in that emoticons, but not colour, affected perceptions of snacks. In particular, frowning emoticons on white background were most potent at modifying the ratings of tastiness and healthiness of snacks with a health halo . In line with recent systematic review evidence nutritional labels had no effect on choice of snacks. The efficacy of nutritional labelling therefore warrants further empirical investigation.These findings extend the extant literature in several important ways. First, we failed to replicate Our results also show that in the absence of colour there is an effect of emoticon labels, but there is no equivalent effect of colour in the absence of emoticons. One could perhaps argue that the non-significant effects of colour labelling are due to the fact that colour confuses people when emoticons are present. However, in our experimental design we also employed colour labels without emoticons as a control. Therefore, the absence of an effect of colour cannot be attributed to the presence of emoticons, since colour did not have an effect in the control conditions.Finally, we show that injunctive norms, in the form of labels with simple emoticons on a white background may be more impactful at modifying consumers' healthiness perceptions than coloured labels. In particular, our results suggest that frowning emoticons may be more potent than smiling emoticons at signalling the healthiness and tastiness of food options, especially for foods that may have a health halo, such as cereal bars. These findings fit with prior theoretical and empirical work in psychology suggesting that bad/negative is stronger than good/positive . These fOf particular relevance to our findings, research by It is interesting to note that emoticon expressions had a significant effect on changing people's perception of the tastiness and healthiness of the cereal bar, but not the chocolate bar. These results are in line with prior research that has demonstrated that nutritional labelling in unhealthy fast food does not impact healthier purchasing , possiblOur findings of the effectiveness of injunctive norms in the form of emoticon expressions diverge from the findings by A notable strength of our study is that we used a nationally representative sample of the UK population to examine the effects of colour and emoticon labelling on snack foods. Furthermore, we used an adequately powered sample to detect the effects of interest, so we could be more confident in drawing conclusions from our findings.One of the limitations of our study is that we only measured self-reported preferences of the two snack options in an online study. Future studies should examine the effects of colour and emoticon labelling on more objective measures of behaviour, such as actual purchasing and consumption. Another limitation of our study is that for the primary endpoint we asked participants to rate their perceptions of a single product. Previous research has found that nutritional labels are more frequently used when comparing two different products, or when deciding about a product that is encountered for the first time . FurtherIt is also important to note that we only examined the effects of labelling on snacks, therefore these effects may not be applicable to other types of food. Future research could usefully extend the present paradigm and examine the effects of emoticon labels on different types of foodstuffs, including main meals and sugar-sweetened beverages.It is also plausible that the effects of the frowning emoticons only arise when the emoticons and the prior expectation of healthiness of the food option are discrepant. Thus, in our study a smiling emoticon label on a cereal bar matches participants' prior expectations, but a frowning emoticon does not. Future research should examine whether emoticon labels are especially potent for foods with discrepant perceived and actual healthiness. Such a study could usefully extend the present design to include food options that are perceived to be less healthy but are actually healthier, in order to ascertain whether the diagnostic value of the frowning emoticons arises from their greater potency as negative symbols, or from being coupled with unexpected/discrepant food options.Our results point to the need for further examination of the impact of colour labelling, especially in light of the growing popularity of traffic light labels. Policy decisions regarding traffic light and other similar colour labels should wait until the effects of such labels are systematically examined and the magnitude and direction of these effects are quantified.Injunctive norms in the form of emoticon expressions may prove a beneficial addition to current nutritional labelling policies. Due to the universal nature of smiling and frowning e.g., , and theIn keeping with recent systematic review evidence  overall


By independently manipulating the rate and frequency of N input, with nine rates (from 0 to 50 g N m−2 year−1) crossed with two frequencies (twice year−1 or monthly) in a temperate steppe of northern China across 2008–2013, we found that N addition increased community ANPP, and had positive, negative, or neutral effects for individual species. There were similar ANPP responses at the community- or species-level when a particular annual amount of N was added either twice year−1 or monthly. The community ANPP was less sensitive to soil ammonium at lower frequency of N addition. ANPP responses to N addition were positively correlated with annual precipitation. Our results suggest that, over a five-year period, there will be similar ANPP responses to a given annual N input that occurs either frequently in small amounts, as from N deposition, or that occur infrequently in larger amounts, as from application of N fertilizers.Nitrogen (N) is a key limiting resource for aboveground net primary productivity (ANPP) in diverse terrestrial ecosystems. The relative roles of the rate and frequency (additions yr Few studies have considered the long-term effects of independent manipulations of both the annual rate and frequency of N addition on ANPP at both community- and species-levels.ANPP at the community-level generally shows a positive, but decelerating relationship with increasing N addition rates1415192022232527284NO3 (from 0 to 50 g N m−2 yr−1) crossed with two frequencies of N addition (twice or monthly yr−1) in a temperate steppe. To our knowledge, this is the first study that jointly considers the role of both the rate and frequency of N addition on ANPP over a relative long-term period (five years). We aimed to determine the response of ANPP, at both community- and species-levels, to different rates and frequencies of N addition. In the same experimental setup, both leaf chlorophyll content11Temperate steppe is a floristically diverse and productive grassland type of both ecological and economic importance in Eurasia311114P < 0.001). There was no significant effect of N addition frequency, and no interaction between N addition frequency and rate, on community ANPP (P < 0.001). Community ANPP increased with increasing N addition rates from 2010 to 2013, but not during the first year (2009) of the study  , similarly for both frequencies of N addition .Community ANPP significantly increased with increasing N addition rates . Nitrogen addition frequency and year interacted to affect the relative ANPP of L. chinensis . Nitrogen addition increased the relative ANPP of L. chinensis .The ANPP of all species showed no detectable difference between the two frequencies of N addition ; Fig. 2.ons yr−1 ; Fig. 2.addition . Neitheraddition . Over tihinensis . In part4+–N and NO3−–N (P < 0.05). In contrast, decreasing the frequency of N addition significantly increased soil NH4+–N (P < 0.05), but had no significant effect on the other four soil variablesIncreasing the rate of N addition decreased the top 10 cm soil water content, soil temperature, and soil pH, and significantly increased soil NHP < 0.05), soil temperature (P < 0.05), and soil pH (P < 0.001), and had a positive relationship with log10(soil NH4+–N) (P < 0.001) and log10(soil NO3−–N) (P < 0.001). The slope of community ANPP responding to log10(soil NH4+–N) was greater under 12 than under 2 N additions yr−1 . There were no apparent difference between 2 and 12 N additions yr−1 for the slopes of community ANPP response to the other four soil factors  across multiple years. This suggests that plant ANPP will respond similarly to a given annual rate of N inputs, regardless of whether N is added in a few large fertilization events or in many small deposition events in temperate grassland where air temperature peaks with rainfallPennisetum clandestinum turfgrass community was greater at low than high frequency of N addition in Australia where air temperature peaks with rainfall. In contrast, community ANPP, within one growing season treatment, was 34% greater at high than low frequency of N addition in a Kansas prairie, US where air temperature peaks with rainfallAt the community-level, previous studies might provide reliable estimates for the short-term impacts of N deposition on community ANPP, despite the fact that they have not applied N in small and frequent pulses that most closely resemble N deposition. Within two-year treatments, Barton and colleaguesCalamagrostis purpurascens, Deschampsia caespitosa and Trisetum spicatumSchismus arabicusCarex rupestris, Kobresia myosuroides and C. scopulorum at Niwot Ridge, ColoradoB. tectorum at Sanpete County, UtahPanicum antidotale at Cambridge26Ch. glaucum is an annual species and the other six response species are perennial speciesS. grandis and L. chinensis, resulting the similar ANPP of Ch. glaucum between the different frequencies of N addition. Thus, both species life forms and species interactions likely influenced species’ responses to N addition.At the species-level, both Bilbrough and Caldwell4+–N under less frequent N addition treatments can result in less recruitment, slower plant growth at both the juvenile and adult stages and more rapid species loss42434114In addition to the increase in N availability, acidification of the soil is one of the main effects of N enrichment3839Annual precipitation is often the primary driver for ANPP in semi-arid regions464849In summary, our study revealed that ANPP responses to increasing rates of N addition at both the community- and species-levels were independent of the frequency of N addition over a five-year time horizon in temperate grassland where air temperature peaks with rainfall events. We found no evidence for the hypothesis that lower N addition frequency would lead to higher ANPP, but the community ANPP was less sensitive to soil ammonium at low frequency of N addition. We also found that the enhancement of productivity by N addition is largely dependent on annual precipitation, highlighting the important role of water availability in mediating ecosystem responses to N deposition in temperate steppe. From a perspective of grassland management and hay-making, combining infrequent N addition with irrigation might help improve productivity in Eurasian grassland, and minimize the costs of N application. Further study over a longer time horizon will be needed to determine whether accumulation of soil ammonium and changes in species dominance will eventually lead to significant differences in community ANPP under different N frequency treatments. More new long-term experiments that independently manipulate the rate and frequency of N addition are needed to examine the generality of our results in other ecosystems, for example Mediterranean regions where rainfall does not peak with temperature. Conservation practitioners, land managers, and policy-makers may need to take into account the trade-off between the increases in productivity and losses of biodiversity under N fertilization or deposition.Stipa grandis P. Smirn and Leymus chinensis (Trin.) Tzvel, which together accounted for >60% of the total peak aboveground biomass , which is located in the Xilin River Basin, Inner Mongolia Autonomous Region, China. The 50 ha field has been fenced since 1999 to exclude large animal grazing. The topography of the experimental area is flat, with an elevation range of 1255–1260 m. The mean annual temperature was 0.9 °C, with mean monthly temperatures ranging from –21.4 °C in January to 19.7 °C in July for the period of 1980–2013. Mean annual precipitation was 351.4 mm, with approximately 72.8% falling during the growing season from May to August. The soil is classified as Haplic Calcisols and Calcic-Orthic Aridisol by the FAO and the US soil classification system, respectively. The plant community was dominated by two species,  biomass . There w–2 yr–1) crossed with two frequencies of N addition (2 times vs. 12 times N addition yr−1). There is also an unamended control treatment  to detect the influence of water and sand additions (see below paragraph) by comparing responses between the control and 0 N addition plots. Each plot is 8 × 8 m. Hence, there were 19 experimental treatments in total, all present in each of ten blocks.The experiment was established during September 2008, following a randomized complete block design4NO3 (>99%) addition started on 1 September 2008 and continued on the first day of each month thereafter for the high frequency treatment (12 N additions yr−1), and started on 1 November 2008 and continued on the first day of June and November thereafter for the low frequency treatment (2 N additions yr−1). That is, when the monthly N addition in August per year, it is with total annual equal N loadings between 2 and 12 N additions yr−1. During the growing season from May to October, fertilizer was weighed and mixed with purified water , and sprinkled evenly using a sprayer to each plot to simulate wet N deposition. It was estimated that less than 1 mm of water was added to each plot annually, except the control plots, which had no water added. In winter (from November to April), NH4NO3 was mixed with sand  and broadcast uniformly by hand. Sand was sieved through less than 1 mm in size, hydrochloric acid dipped, washed in purified water, and then heated at 120 °C for 24 hours in an oven. To avoid otherwise potentially confounding effects, plots received the same amount of water and sand, regardless of whether they received the high or low frequency of N addition treatment.Purified NHANPP was estimated annually from peak aboveground biomass. Peak aboveground biomass is an acceptable approximation for ANPP in this region as aboveground plant tissues die during the winter season4+–N; mg kg−1) and nitrate  concentration and soil water content (%). Subsamples were air-dried for analysis of soil pH. For soil NH4+–N and NO3−–N measurements, 10 g fresh soil subsamples were extracted with 50 ml 2.0 M KCl solution and then analyzed using a flow injection auto analyzer . The soil NH4+–N and NO3−–N concentration is expressed on a dry weight basis. The soil pH was measured in water suspension (soil:water = 1:2.5) by a pH meter (FE20–FiveEasy). Soil temperature (°C) was automatically recorded within each plot at a 1.5 hour interval by iButton digital temperature loggers (DS1922L) at a depth of 10 cm in the soil beginning in April 2012. We used the average temperature of growing seasons (from May to 9 August) for further analyses in both 2012 and 2013.Concurrent with aboveground plant sampling in 2012 and 2013, three soil cores (0–10 cm depth and 50 cm apart) were collected using a 3 cm diameter soil auger adjacent to each aboveground plant sampling plot. The soil samples were thoroughly mixed and sieved through a 2 mm mesh to obtain one composite sample for laboratory analysis of soil ammonium  were employed to test the effects of N addition frequency, N–rate (N as a continuous factor), year, and their interactions on the top 10 cm soil water content, temperature, pH, NH4+–N, and NO3−–N concentration across 2012–2013. The response ratio of ANPP, soil water content, soil temperature, soil pH, soil NH4+–N, and NO3−–N concentration was calculated as the ratio of these values in treatments divided by the mean in controls, ANPP for example, ANPPtreatment/ANPPcontrol. The response ratio of soil NH4+–N and NO3−–N were log10 transformed to meet the assumptions of normality and homogeneity. ANCOVA was employed to distinguish the slopes between the two N addition frequencies when ANPP responded to each environmental factor  at the community-level. All statistical analyses were performed with the SPSS software package .Repeated-measures analysis of covariance (ANCOVA) were used to test effects of N addition frequency, N–rate (N as a continuous factor), year, and their interactions on the ANPP of the community and of species and species relative ANPP, using the data in 2008 (pre-treatment measurement) as a covariate to control for initial differences in ANPP across plots . The species relative ANPP was calculated as, How to cite this article: Zhang, Y. et al. Productivity depends more on the rate than the frequency of N addition in a temperate grassland. Sci. Rep.5, 12558; doi: 10.1038/srep12558 (2015).


Creatine monohydrate has become a very popular nutritional supplement for its ergogenic effects. The safety of creatine monohydrate has previously been confirmed. However with each novel form of creatine that emerges, its safety must be verified. Therefore, the purpose of this study was to examine the safety of a novel form of creatine, creatine nitrate (CN), over a 28 day period.58 young males and females  participated in this study across two laboratories. Subjects were equally and randomly assigned to consume either 1 g (n = 18) or 2 g (n = 20) of CN or remained unsupplemented (n = 20). Blood draws for full safety panels were conducted by a trained phlebotomist prior to and at the conclusion of the supplementation period.Pooled data from both laboratories revealed significant group x time interactions for absolute lymphocytes and absolute monocytes (p < 0.05). Analysis of the 1 g treatment revealed lab x time differences for red blood cell distribution width, platelets, absolute monocytes, creatinine, blood urea nitrogen (BUN):creatinine, sodium, protein, and alanine aminotransferase (ALT) (p < 0.05). Analysis of the 2 g treatment revealed lab x time differences for BUN:creatinine and ALT (p < 0.05). BUN and BUN:creatinine increased beyond the clinical reference range for the 2 g treatment of Lab 2, but BUN did not reach statistical significance.Overall, CN appears to be safe in both 1 g and 2 g servings daily for up to a 28 day period. While those with previously elevated BUN levels may see additional increases resulting in post-supplementation values slightly beyond normal physiological range, these results have minor clinical significance and are not cause for concern. Otherwise, all hematological safety markers remained within normal range, suggesting that CN supplementation has no adverse effects in daily doses up to 2 g over 28 days and may be an alternative to creatine monohydrate supplementation.The online version of this article (doi:10.1186/s12970-014-0060-9) contains supplementary material, which is available to authorized users. In regards to ergogenic supplementation, creatine’s effects have been researched in humans since the early 1990’s. Earnest and colleagues examined supplemental creatine’s effects on both body composition and muscular performance -5.Creatine supplementation’s ergogenic effects are largely explained by increasing intramuscular phosphocreatine (PCr) stores . The incThis increase in fatigue resistance created by creatine supplementation allows for increased training volume. As demonstrated by several laboratories, creatine supplementation is able to increase repetitions to failure ,12. TherSimilarly, dietary nitrate has been receiving more attention for its effects on energy efficiency and work capacity. Nitrate supplementation, typically in the form of foods or juices high in inorganic nitrate such as beet root juice, has primarily been demonstrated to decrease oxygen cost despite maintaining work load -21. ThisBecause of the ergogenic effects of creatine and phosphocreatine and the ATP sparing effects of nitrate , the twoIn a randomized design, a total of 58 subjects were recruited for this study across two laboratories. Subjects were randomly divided into control (CRL), 1 serving (G1), or 2 serving (G2) groups. Wherein, the CRL group did not supplement and G1 and G2 consumed 1 serving (1 g) and 2 servings (2 g) of CN , respectively, every day for 28 days in an unblinded manner. In addition to the CN, the supplement contained 1 g of carbohydrate, 500 mg of vitamin C, 500 IU of Vitamin E, 18 mg of Calcium, and 800 mg of a proprietary blend consisting of taurine, coconut water powder, and L-glutamine per serving. Supplement containers were weighed prior to and following the supplementation period and supplement consumption logs were completed by each participant to ensure compliance. Blood draws were conducted prior to and at the conclusion of the supplementation period. MusclePharm Sports Science Institute (Lab 1) received approval from the MusclePharm Sports Science Institute IRB, and the University of Tampa Human Performance and Nutrition Laboratory (Lab 2) received approval from the University of Tampa IRB. Each subject was provided written informed consent prior to participation in the study.Forty-two subjects  were recruited by Lab 1, while Lab 2 recruited the remaining 16 . Subjects were required to be at least recreationally active (≥ 3 days/week of moderate to vigorous intensity exercise), free of any disease or disorder which may produce confounding results, non-smokers, and have abstained from creatine or nitrate supplementation for the month immediately prior to beginning CN supplementation, as assessed by pre-participation health history, exercise, and supplementation questionnaires.All measurements were taken prior to and at the conclusion of the 28-day supplementation period. Following a 10-hour fast, all subjects submitted a blood sample for analysis in the morning to prevent diurnal variations. All blood draws were performed via venipuncture by a trained phlebotomist. Samples were analyzed for complete metabolic panels and complete blood counts by an external laboratory . All collected samples were analyzed for the following markers: white blood cell count (WBC), red blood cell count (RBC), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (percent and absolute), neutrophils (percent and absolute), lymphocytes (percent and absolute), monocytes (percent and absolute), eosinophils (percent and absolute), basophils (percent and absolute), serum glucose, blood urea nitrogen (BUN), creatinine, estimated glomerular filtration rate (eGFR), BUN:creatinine, sodium, potassium, chloride, carbon dioxide, calcium, protein, albumin, globulin, albumin:globulin, bilirubin, alkaline phosphatase, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). Intra-test Coefficient of Variation (CV) for Lab 2 blood measurements were all under 3%. Inter-test reliability results from 12 men and women measures up to one week apart from Lab 1 resulted in no significant differences from day-to-day (p > 0.05) and an average inter-test %CV of 6.9%.Pooled data was analyzed using a 3x2 repeated measures ANCOVA model for all group, time, and group by time interactions with the pre value as the covariate. A bonferroni post-hoc analysis was used to locate differences. Shapiro-Wilk tests were used to determine normality of the data. The Minimal Difference (MD) needed to be considered real was determined using the method previously described by Weir . Data arSignificant group by time interactions were present for absolute lymphocytes (p < 0.05). Wherein, G1 increased to a lesser extent than CRL, G2 decreased significantly relative to CRL, and G2 significantly decreased from pre to post. Absolute lymphocytes had a normal distribution at baseline (p = 0.55), yet the distribution was negatively skewed (p < 0.05) after the supplementation period. Significant group by time interactions were present for absolute monocytes (p < 0.05). G1 increased relative to CRL and G2 increased to a lesser extent than G1. Absolute monocytes was positively skewed at both time points (p < 0.05). All data is presented in Additional file Significant group by time interactions were present for RDW (p < 0.05). Lab 2 observed greater increases in RDW relative to Lab 1 and significantly increased from pre to post. Significant group by time interactions were observed for platelets (p < 0.05). Lab 1 observed a significant decrease in platelets relative to CRL, and Lab 2 observed a significant increase in platelets compared to decrements in both CRL and Lab 1. Platelets were normally distributed at baseline (p = 0.17), and negatively skewed (p < 0.05) at post. Significant group by time interactions existed for absolute monocytes (p < 0.05). Wherein, Lab 2 observed significant increases compared to Lab 1 and CRL. Significant group by time interactions were present for creatinine (p < 0.05). Lab 2 observed significant decrements relative to CRL. A significant group by time interaction was present for BUN:creatinine (p < 0.05). Wherein, Lab 2 observed significant increases pre to post. A significant group by time interaction was observed for sodium (p < 0.05). Lab 2 observed a significant increase over CRL. A significant interaction was observed for serum protein (p <0.05). Lab 2 observed a significant increase relative to CRL. Significant group by time interactions were observed for ALT (p < 0.05). Lab 2 observed greater decrements relative to CRL and Lab 1. ALT was positively skewed at baseline (p < 0.05), but it was normally distributed (p = 0.52) at post. RDW, platelets, absolute monocytes, creatinine, BUN:creatinine, sodium, and serum protein were either normally distributed, negatively skewed, or positively skewed at baseline without a change in skewness at post. With the exception of BUN:creatinine, all of these markers remained within clinical reference ranges, indicating clinical insignificance. All data is presented in Additional file Significant group by time interactions were present for BUN:creatinine (p < 0.05). Lab 2 observed significant increases relative to CRL and Lab 1 as well as within group differences pre to post. BUN:creatinine was positively skewed at baseline (p < 0.05), and at post, it became normally distributed (p = 0.16). Significant group by time interactions were observed for ALT (p < 0.05). Wherein, Lab 1 and Lab 2 observed greater decrements than CRL. ALT was positively skewed at baseline (p < 0.05), yet it was normally distributed at post p = 0.93). All data is presented in Additional file . All datThe results of the present study confirm our hypothesis that CN supplementation will not cause abnormal changes in hematological safety markers. When analyzing the pooled data from both laboratories, significant interactions were observed only for absolute lymphocytes and monocytes, yet these remained within the accepted physiological range and not clinically significant. Also, percent lymphocytes and monocytes were unchanged. While remaining within range, we observed unusual effects between groups. For lymphocytes, the CRL group had a greater change than G1, and for monocytes, G1 increased to a greater extent than G2. These results suggest a natural variation for these markers. Alternatively, data was collected during the winter months, and subjects may have experienced these unusual outcomes as an effect of weather. Although significant interactions were observed concerning Lab 2 in the one serving treatment, it must be mentioned that many of these differences were not observed in the two serving treatment, and the CRL sample was represented solely from Lab 1. Thus, some of the differences are not likely due to CN supplementation, and variations between subjects may be due to variations in climate between Denver, CO and Tampa, FL . HoweverAnalysis of clinical significance at the individual level was conducted using the MD statistic that calculates the  error needed to be exceeded in order for an individual measurement to be considered real as described by Weir . If a suThe present findings generally agree with previous literature. The safety of creatine has been confirmed on several occasions , includiWhile creatine monohydrate is recognized as safe, the nitrate component of CN may still be troublesome to athletes or practitioners. Concerns over nitrate and other nitrosamines began as early as 1970. Lijinsky and Epstein  publisheThis is the first study to examine the safety of CN. From the present results, we can conclude that CN in doses of up to 2 g are safe for human consumption for a duration of 28 days. All measured variables remained within the normal range across groups, with the exception of BUN which was not statistically significant when the groups were compared. Therefore, CN supplementation may be contraindicated for those already high in BUN. Additionally while the differences observed for absolute monocytes and lymphocytes appear to be due to variability and remained within range, CN may be unadvisable for daily consumption for those with a weakened immune system. In the present study, CN was supplemented for only 28 days, and future research may be interested in examining CN for a longer trial period to confirm its safety. Moreover, future studies are required to determine the efficacy of CN, as the combined effects of nitrates and creatine on both longitudinal and acute changes in performance and body composition are currently unknown.


P < 0.05). Regression analysis of patients with DED revealed the PSQI and HADS scores were significantly correlated with the severity of DED (P < 0.05). Our results demonstrate that sleep quality in patients with DED is significantly worse than in patients with other irritating ocular surface diseases and it is correlated with the severity of DED.The aim of the present study was to evaluate sleep and mood disorders in patients with irritating ocular diseases. The study design was a cross-sectional/case-control study conducted in six eye clinics. Out of 715 outpatients diagnosed with irritating ocular surface diseases and initially enrolled, 301 patients with dry eye disease (DED) and 202 age-matched control participants with other ocular surface diseases were analyzed. The mean Pittsburgh Sleep Quality Index (PSQI) and Hospital Anxiety and Depression Scale (HADS) scores were 6.4 ± 3.2 and 11.1 ± 5.7 for severe DED (n = 146), 5.5 ± 3.3 and 9.8 ± 4.0 for mild DED (n = 155), 5.5 ± 3.1 and 9.5 ± 6.6 for chronic conjunctivitis (n = 124), and 5.0 ± 3.3 and 8.9 ± 5.3 for allergic conjunctivitis (n = 78). There were significant differences among these diagnostic groups for PSQI ( Dry eye disease (DED), chronic conjunctivitis, and allergic conjunctivitis are very common irritating eye diseases with symptoms including pain, burning, foreign body sensation, discomfort, discharge, and blurred vision, as well as dryness in DED and itching in allergic conjunctivitis. The lesions in ocular surface diseases are generally easy to recognize, however, DED has an unknown etiology and the reported signs and symptoms often have discrepancies12To the best of our knowledge, no study has reported whether any ocular surface disease induces sleep disorders, although every ocular surface disease may produce symptoms that may induce distress, potentially leading to sleep disorders. Ocular pain and discomfort are generally mediated by the trigeminal nerve and the discomfort spreads widely and radiates around the eyes. We hypothesized that if depression is caused by ocular surface discomfort, patients with any ocular surface disease might accordingly develop depression and sleep disorders.In this study, we conducted a comprehensive ophthalmological examination along with a questionnaire-based survey, which included questions about sleep, depression, anxiety, morningness/eveningness, and photophobia, in patients with ocular irritating diseases to evaluate their sleep and mood status. We compared psychiatric indices among patients with DED and allied irritating ocular surface diseases.Participants were recruited to the study from eye clinic patients at six institutions in Japan between January 2014 and December 2014. The Institutional Review Board and Ethics Committee of the Keio University School of Medicine approved this study and the methods were carried out in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants.Patients were invited to fill out questionnaires, which included the Pittsburgh Sleep Quality Index (PSQI)810All outpatients were examined by board-certified ophthalmologists specialized in corneal disorders and certified orthoptists, and their diagnosis of ocular surface disease was classified as DED, allergic conjunctivitis, or chronic conjunctivitis. A diagnosis of DED was made according to the Japanese Dry Eye Society15Allergic conjunctivitis was diagnosed with symptoms and signs including itching, irritation, injection, lacrimation, and eye discharge corresponding to conjunctival edema, papillae, follicles, or injection. Patients in the allergic conjunctivitis group were seen at a returning visit and/or they had a typical history of seasonal or perennial allergic conjunctivitis clearly responsive to histamine blocker and/or steroid eyedrops. Chronic conjunctivitis was diagnosed with eye discharge, irritation, pain, or any other discomfort without corneoconjunctival epithelial disorders. Patients in the chronic conjunctivitis group were seen at regular visits or they had symptoms lasting for more than a month.R , 0.1%/0.3% TearbalanceR , DiquasR , MucostaR , and 0.02%/0.1% FlumetholonR . We prescribed various histamine blockers or steroid eyedrops for allergic conjunctivitis; antibiotics, steroids, and/or nonsteroidal anti-inflammatory eyedrops for chronic conjunctivitis; and hyaluronate for trichiasis (included in the chronic conjunctivitis group) according to the symptoms of the patient.The prescribed eye drops for the treatment of DED were hyaluronate, mucin secretagogue, and steroid, i.e., 0.1%/0.3% Hyalein < −12 dB in either eye), bilateral cataracts, visual impairment (<20/30 in both eyes), acute disease presenting for less than one month, or were < 20 years old. Age was further matched among the diagnostic groups since many patients with allergic conjunctivitis were < 30 years old.Patients were excluded if they also had severe glaucoma  in DED, a regression analysis of patients with DED was carried out, with PSQI and HADS scores as dependent variables and demographic (gender and age) and ophthalmic parameters as independent variables. All analyses were performed using StatFlex  and SPSS version 21 , with P < 0.05, unpaired t test). The female gender was predominant in DED and allergic conjunctivitis. There were no differences between diagnostic groups for the other parameters of age, myopic errors, and phakic status. Eyedrop prescriptions for ocular surface diseases were consistent with diagnosis.A total of 715 outpatients initially participated in this study and finally 301 patients with DED and 202 age-matched control participants with other ocular surface diseases were analyzed following inclusion and exclusion criteria. Patient demographics and ocular parameters are summarized in P < 0.05, Kruskal-Wallis test), sleep duration (P < 0.05), sleep efficacy (P < 0.05), and HADS (P = 0.07)  . The pro = 0.07) . GeneralP < 0.001, Pearson correlation coefficient), the severity of DED (P < 0.05), photophobia (P < 0.001), and morningness/eveningness (P < 0.001). The HADS score in patients with DED was significantly correlated with the severity of DED (P < 0.05), photophobia (P < 0.001), and morningness/eveningness , which is why we showed significant results under the classifications of severe and mild DED. There is also the potential for the overlapping of DED and other diseases, and a small number of patients with DED may have been included in the allergy and chronic conjunctivitis groups. This study should be further confirmed using a comprehensive DED classification, such as the one proposed by the 2007 Dry Eye WorkshopIn conclusion, the present results indicate patients with DED suffered more seriously from sleep and mood problems compared to patients with other irritating ocular surface diseases. We speculate that sleep and mood disorders in these patients could derive from a disease-specific ocular surface abnormality and optical disability. Psychiatric treatment focusing on sleep and mood disorders might therefore be beneficial for patients with DED.How to cite this article: Ayaki, M. et al. Sleep and mood disorders in dry eye disease and allied irritating ocular diseases. Sci. Rep.6, 22480; doi: 10.1038/srep22480 (2016).


With growing interest in the connection between fat and bone, there has been increased investigation of the relationship with marrow fat in particular. Clinical research has been facilitated by the development of non-invasive methods to measure bone marrow fat content and composition. Studies in different populations using different measurement techniques have established that higher marrow fat is associated with lower bone density and prevalent vertebral fracture. The degree of unsaturation in marrow fat may also affect bone health. Although other fat depots tend to be strongly correlated, marrow fat has a distinct pattern, suggesting separate mechanisms of control. Longitudinal studies are limited, but are crucial to understand the direct and indirect roles of marrow fat as an influence on skeletal health. With greater appreciation of the links between bone and energy metabolism, there has been growing interest in understanding the relationship between marrow fat and bone. It is well established that levels of marrow fat are higher in older adults with osteoporosis, defined by either low bone density or vertebral fracture. However, the reasons for and implications of this association are not clear. This review focuses on clinical studies of marrow fat and its relationship to bone. Marrow fat is distinct from white adipose tissue and from brown adipose tissue , 2. The Higher levels of marrow fat are known to occur in humans in conditions of starvation , 8, alcoIn vitro and animal models have provided new insight into the connection between marrow fat and bone in particular, and these findings have been extensively reviewed  are widely used to assess fat in the bone marrow: magnetic resonance spectroscopy (MRS) and T1-weighted MRI. Dual energy QCT can also be used to quantify marrow fat. However, dual energy scans require relatively high levels of radiation. Single energy QCT does not give a reliable measure of marrow fat.Ifat/(Ifat + Iwater)] × 100%. With higher resolution MRI (3 T), fat content has also been reported using three of these lipid peaks (IUL + IRL + ISL)/(IUL + IRL + ISL + Iwater)] × 100%.In MRS measurements, marrow fat is expressed as a percentage rather than an absolute value. For evaluation of vertebral marrow fat, the measurement is acquired on one or more of the lumbar vertebral bodies L1–L4). A single voxel is placed in the center of the vertebral body ] × 100%  . Women wDXA and QCT measurements of bone are both vulnerable to artificially low readings in the presence of higher BMF , 32. Blap = 0.02) (p < 0.001) in those with and without a baseline vertebral fracture, respectively, and remained statistically different after adjustment for spine BMD by DXA. In AGES–Reykjavik, higher vertebral BMF was associated with prevalent vertebral fracture , in models adjusted for age, gender, and trabecular spine vBMD  and 3 T MRS show promise in distinguishing marrow and blood vessels in pore space . Future n = 15), low (n = 15), and osteoporotic (n = 20) spine BMD. Consistent with other reports, marrow fat content was 29% in young control women, and 56, 63, and 65% in older women with normal, low, and osteoporotic BMD, respectively. The highest proportion of unsaturated lipids in marrow fat was found in the younger controls (0.127) and the lowest proportion in older women with osteoporotic spine BMD (0.091). Unsaturation levels in older women with normal and osteopenic BMD were 0.114 and 0.097, respectively. The unsaturation level and marrow fat content were negatively correlated . In a study of postmenopausal women (n = 69), Patsch et al. reported an association between history of fracture and lower unsaturation levels, even after adjustment for spine vBMD  , in those from the tibia (n = 80), and in those from the proximal femur (n = 32)  MR . Similarn = 210) found a negative correlation between BMI and hip BMAT  (n = 455) of men and women aged 18–88 years identified a positive correlation between total body fat and pelvic BMAT    and a la < 0.01) .r = 0.34, p = 0.02) have been reported in obese premenopausal women  but not hip BMAT  (n = 455), there was a modest but statistically significant negative correlation between VAT and pelvic BMAT   . However > 0.05) , 29, 49.< 0.001) .n = 13) and without (n = 13) type 2 diabetes (T2D), those with T2D had higher BMF , but the difference was not statistically significant . A study in 156 older men reported higher BMF in those with T2D   .Weight loss in older adults is known to increase bone loss and fracture risk, even among those who are overweight or obese , 58. In 3 (about 8%). However, total body BMD increased by 0.01 g/cm2 (about 1%); the lack of bone loss in spite of substantial weight loss may be due to the younger age of participants, use of whole body BMD rather than hip BMD, and/or the short follow-up time. In a 12-week weight loss study (n = 10), participants lost 5.2 kg on average, but did not experience a statistically significant change in pelvic or total BMF, measured by MRI (p = 0.84) in women without diabetes (n = 5) and an apparent decrease in BMF  in those with T2D (n = 6) (In a short-term (13-week) study of diet-induced weight loss in 55 women and 17 men, age 19–46 years, participants lost an average of 9.2 kg (about 9% of body weight) . Pelvic d by MRI . In a st (n = 6) . Bariatr (n = 6)  and the These findings suggest that changes in bone and in marrow fat are not necessarily linked and that their inter-relationship is dependent on other factors including diabetes status. Further investigation of the dynamic relationships among weight loss, insulin resistance, bone loss, and changes in marrow fat promises to give us a better understanding of the interplay of marrow fat and bone.Osteoporosis is an important health issue, contributing substantially to morbidity and mortality among older adults. Health care costs for osteoporosis-related fractures in the United States were estimated as $19 billion in 2005 and are expected to increase 50% by 2025 with the growth of the older population . Animal The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Resective epilepsy surgery is an established and effective method to reduce seizure burden in drug-resistant epilepsy. It was the objective of this study to assess intraoperative blood loss, transfusion requirements and the degree of hypothermia of pediatric epilepsy surgery in our center.Patients were identified by our epilepsy surgery database, and data were collected via retrospective chart review over the past 25 years. Patients up to the age of 6 years were included, and patients with insufficient data were excluded.Forty-five patients with an age of 3.2 ± 1.6 (mean ± SD) years and a body weight of 17  kg  were analysed. Duration of surgery was 3 h 49 min ± 53 min, which was accompanied by an intraoperative blood loss of 150  ml. This corresponded to 11.7  % of estimated total blood volume, ranging from 0 to 75%. A minimal haemoglobin count of 8.8 ± 1.4 g/dl was measured, which was substituted with erythrocyte concentrate  in 23 patients. Body core temperature dropped from 36.0 ± 0.7°C at baseline to a minimum of 35.7 ± 0.7°C, and increased significantly (p < 0.001) thereafter to 37.1 ± 0.7°C until the end of surgery. A significant (p = 0.0003) correlation between duration of surgery and blood loss (Pearson r = 0.52) was observed. However, age, minimal body temperature or number of antiepileptic drugs seemed to have no impact on blood loss.Resective epilepsy surgery is a safe procedure even in the pediatric population, however it is associated with significant blood loss especially during long surgical procedures. Epilepsy surgery has become a frequent procedure in certain neurosurgical centers . Two basIrrespective of the technique chosen, epilepsy surgery has been shown to be a safe and effective treatment for medically intractable epilepsy . This apAlthough there are several studies on safety and outcome of epilepsy surgery in general ,7-10, onIntraoperatively, especially children are at risk for blood loss and hypothermia, due to their low absolute blood volume and relatively high body surface area, respectively. Since hypothermia has been shown to impede coagulation, it may further aggravate blood loss .Therefore, we performed a retrospective study to assess blood loss and body temperature in pediatric epilepsy surgery.This study was conducted in compliance with the Helsinki declaration. The responsible ethics committee  granted an exemption from requiring ethics approval for this retrospective chart analysis.Patients were identified from the epilepsy surgery program of our neurosurgical department from the year 1989 until 2011. All had received presurgical evaluation at the Department of Epileptology, which included video EEG monitoring, MRI imaging, and neuropsychological testing if possible. We included pediatric patients with resective epilepsy surgery procedures up to the age of six years. Patients undergoing disconnective surgery and children with missing anesthesia protocols or anesthesia protocols with insufficient data were excluded.In a retrospective chart review, we extracted data for sex, age, weight, body height, preoperative Hb and coagulation status, duration of surgery, duration of anesthesia, duration of ventilation, hours of ICU stay, blood loss, blood transfusion, administered liquids, minimal hemoglobin count, body temperature, and numbers of antiepileptic drugs. We estimated 80 ml/kg body weight total blood volume for calculation of percentage of blood loss ,20. BlooData are displayed as mean ± standard deviation in case of normal distribution, or as median  otherwise. Normal distribution was tested using the Shapiro-Wilk-test, and relationship between variables was analysed applying Pearson’s product moment correlation. Statistical significance was assumed at a p < 0.05.Fifty-five children with resective epilepsy surgery could be identified. Missing anesthesia protocols or charts led to the exclusion of 10 patients, leaving 45 children for final analysis. They consisted of 21 girls and 24 boys with an age of 3.2 ± 1.6 years, a body weight of 17  kg and a height of 104  cm  and partial prothrombin time (PTT) were 12.6 ± 1.6 g/dl, 99.8 ± 10.7%, 27.6 ± 1.9 s, respectively.The children received midazolam for premedication. Anesthesia was induced with intravenous thiopental at a dosage of 7.0 ± 2.4 mg thiopental per kg body weight in 26 children. 10 children received induction with Propofol. Inhalational induction with sevoflurane was performed in 9 children, in which venous access would have been difficult to achieve in the awake state. Anesthesia was maintained with isoflurane except in 4 more recent cases (2010–2011) where propofol was applied with a target-controlled infusion (TCI) pump for total intravenous anesthesia (TIVA). TIVA was guided with Bispectral Index - monitoring. Fentanyl or remifentanil were used as opioids, and vecuronium or cis-atracurium for muscle relaxation. Core temperature was monitored in all children via rectal probes All 45 patients received arterial cannulae, and 4 received a central-venous access.Mean duration of surgery and anesthesia was 3:49 ± 0:53 h and 5:26 h ± 1:06 h, respectively. Duration of ventilation (including intensive care unit) was 6:15  h, which was accompanied by a high variability in length of ICU stay ranging between 7:30 h and 27:00 h , see Table An absolute blood loss of 150  ml was observed (see Table No vasopressors had to be used, and no resuscitation or deaths occurred in any of the cases.While there is some literature regarding neuroanesthesia and even literature dealing with pediatric epilepsy surgery, complications of standard procedures in epilepsy surgery remain largely unknown ,15. To oWe found that blood loss is one major factor compromising the safety of surgery, which is in accordance with Pietrini et al. who reviewed neurosurgical procedures on pediatric epilepsy patients. Whereas Pietrini et al. focused on hemispherectomies , we wereAntiepileptic medication could impede coagulation, thus putting the patient at an increased risk of intraoperative bleeding. For instance, an association between antiepileptic medication and coagulation disorders has been reported for valproate, carbamazepine, and gabapentin -24. In cBlood transfusion strategy is a subject which is constantly under discussion . Since rAs in red-cell transfusion, FFP transfusion practices remain controversial. According to recommendations, FFP should be given to correct microvascular bleeding in the presence of coagulation test disorders or to correct microvascular bleeding during massive blood transfusion . HoweverThere is evidence that hypothermia is frequent especially in small children which can have adverse effects such as surgical site infections and coagulation disorders . In adulIn our series, thiopental and, more recently, propofol were used for intravenous induction of anesthesia. We did not observe an effect of antiepileptic drugs on the dosage of thiopental for induction, which has been described to be between 5 and 8 mg/kg body weight, depending on age . PropofoIntraoperative fluid management can be challenging especially in the pediatric setting, especially when there is significant blood loss that has to be restored. hence, sufficient invasive or non-invasive monitoring is necessary, and arterial cannulation is recommended in intracranial resections. In fact, all of our patients received arterial cannulae.In contrast, the use of central venous catheters is controversial in the pediatric setting . Only 4 We conclude that resective epilepsy surgery is associated with a significant blood loss, especially during surgery with long operating times. This has to be taken into consideration by the anesthetist and the neurosurgeon especially in pediatric patients where fluid and blood management is more critical than in the adult population. Intraoperative hypothermia is of concern in children, however forced air warming seems to be effective in restoring normothermia in pediatric epilepsy surgery.The authors declare that they have no competing interests.MT and MS designed the study and drafted the manuscript, MT, MvL, CW, and JCSB were responsible for data acquisition. All authors revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/14/71/prepub


How water and N availability influence ECE in arid and semiarid grasslands is still uncertain. A manipulative experiment with additions of rainfall, snow and N was conducted to test their effects on ECE in a semiarid temperate steppe of northern China for three consecutive years with contrasting natural precipitation. ECE increased with annual precipitation but approached peak values at different precipitation amount. Water addition, especially summer water addition, had significantly positive effects on ECE in years when the natural precipitation was normal or below normal, but showed trivial effect on GEP when the natural precipitation was above normal as effects on ER and NEP offset one another. Nitrogen addition exerted non-significant or negative effects on ECE when precipitation was low but switched to a positive effect when precipitation was high, indicating N effect triggered by water availability. Our results indicate that both water and N availability control ECE and the effects of future precipitation changes and increasing N deposition will depend on how they can change collaboratively in this semiarid steppe ecosystem.Both water and nitrogen (N) availability have significant effects on ecosystem CO Owing to the inconsistent changes in both magnitude and direction of gross ecosystem photosynthesis (GEP) and ecosystem respiration (ER), some previous studies have shown positive changes in net ecosystem productivity (NEP) to increasing precipitation235The East Asian summer rainfall has already extended its distribution northward to the northern China, and its increasing amount will range from 93 to 136 mm by the end of this centuryCarbon fluxes in the future will face the precipitation changes including not only those in summer rainfall amount and rainfall pattern, but also those in snow amount, and melting time. Snow plays an important role controlling ECE in winter and the following growing seasons9132 assimilation81920Global N deposition is expected to increase under the background of the climate change caused by intensive anthropogenic activities16et al.As both water and N are important limiting factors in the semiarid temperate steppe, changes in precipitation and N deposition at the same time may potentially induce complex interactions on ecosystem structure and functioning. Burke The semiarid temperate steppe in northern China is an important component of the Eurasian steppe. The steppe is not only sensitive to climate changePrecipitation showed remarkably different patterns and amount across the three study years . The annP = 0.04), 9.2% in 2012 (P < 0.001), while the increase was only marginally significant in 2013   , Table 1 = 0.10) .P < 0.001) in the spring snow addition treatment while no significant effect in the other two years. Nitrogen addition decreased soil temperature significantly in the summer water addition treatment by 0.2 °C in 2012 (P = 0.004), 0.3 °C in 2013 (P < 0.001) while had insignificant effect in 2011  and summer water addition treatment , but showed no significant effect in 2011 regardless of spring snow addition (P = 0.33) or summer water addition (P = 0.15) (P = 0.007) and marginally significant effect on soil temperature in 2013 . Nitrogen addition also had significant interactive effects with summer water addition on soil temperature in 2012  and 2013  and on soil moisture in 2011  . Nitrogen addition had significantly negative effect on soil temperature in 2013  , Table 1 = 0.02) , Table 1P = 0.004) and GEP  while only marginally decreased NEP  (P = 0.07) in 2012 while there was no significant effect for the all three fluxes in 2013  , Table 1P < 0.001 for all cases)  (P < 0.001) while increased ER  and had insignificant effect on GEP in 2012  in years when the natural precipitation was normal (2013) or below normal (2011), but the effect was only significant for NEP and ER when the natural precipitation was above normal (2012) , Table 1l cases) , and in l cases) . However in 2012 .P < 0.001 for all cases) respectively in 2012   respectively in 2012   under both spring snow addition and summer water addition treatments varied with natural precipitation in the three years. Nitrogen addition enhanced NEP, ER and GEP in association with spring snow addition by 29.0%, 19.3% and 25.1% ( in 2012  and by 7ctively)  while thctively) . Nitroge in 2012  and by 1l cases)  while thl cases) .P = 0.05) and GEP , and in 2012 on ER , while insignificant for all three variables in 2013 (P = 0.005) and GEP , in 2012 for ER , and in 2013 for ER  while only marginally significant in 2013 for GEP   .r2 = 0.30, P = 0.001), 394.3 mm  and 447.3 mm , respectively, in plots without N addition while at the total precipitation of 793.0 mm , 441.3 mm  and 536.6 mm  in plots with N addition  in plots pooled either with or without N addition. The NEP, ER, GEP reached their highest values at the total precipitation of 554.3 mm  and N addition experiment in a semiarid typical steppe in the northern China, we found that water addition, especially summer water addition, showed complex effects on ECE depending on ECE components when the natural precipitation was above normal. It had significantly positive effects on ECE  in years when the natural precipitation was normal or below normal, but the effect was trivial and even negative for GEP when the natural precipitation was above normal because high precipitation had a significantly increasing effect on ER but a decreasing effect on NEP. On the contrary, N addition showed strong positive effects on ECE in years when the natural precipitation was normal or above normal while the effect was minimum when the natural precipitation was below normal. Ecosystem COStipa grandis and Leymus chinensis. The soil at the site belongs to Chernozem according to the Chinese soil taxonomic system, or is classified as Ustoll based on the US Soil TaxonomyThe experiment site, located in the Inner Mongolia Grassland Ecosystem Research Station, Institute of Botany, the Chinese Academy of Sciences , is covered with a typical steppe. The dominant species include 2 (5 m × 5 m) and at least 1 m walkway between two adjacent plots. Treatments were labeled as following: control (N0W0), spring snow addition (N0W1), summer water addition (N0W2), N addition (N1W0), spring snow with N addition (N1W1), summer water with N addition (N1W2) −1 to each summer water addition plot and it was conducted from June 15 every year since 2010, and 10 mm water was applied weekly. With the spring snow addition, summer water addition and natural precipitation across the three study years, we designed an annual precipitation gradient with which changes in ecosystem C exchange (ECE) could be examined.The experiment used a randomized block design with two levels of N addition  interacted with two levels of water addition  with 5 replicates per treatment resulting in thirty plots in total, each with an area of 25 m1W2) see . Nitroge2 concentration data for NEP calculation. When the inside of the chamber was vented completely after NEP measurements, a shade cloth covered the chamber to determine the respiration using similar measurements as ER. NEP and ER were calculated according to Equation et al.et al.44Measurements of ECE, including net ecosystem productivity (NEP), ecosystem respiration (ER) and gross ecosystem photosynthesis (GEP), were performed between 8:30 and 10:30 am on the third or fourth sunny day after the summer water addition. We made three measurements every month in 2011 and once every week in 2012 and 2013 during the growing seasons from May to October. ECE were determined by an infrared gas analyzer  with a transparent chamber (0.5 × 0.5 × 0.5 m) which both attached to an air pump (LI-COR Inc.). When conducting measurements the chamber was placed on a square stainless steel frame (0.5 × 0.5 m) which was inserted 5 cm into the soil at the center of each plot in 2010. In addition, the chamber had two small fans in upper opposite corners to mix air and a temperature probe to determine the air temperature inside chamber during measurements. Each measurement lasted about 80 seconds at 1 Hz frequency to attain COFc is the CO2 flux representing NEP and ER (μmol m−2 s−1); V is the volume of chamber (m3); Pav, Wav and Tav are the average pressure (kPa), water mole fraction (mmol mol−1) and average temperature (°C) during the measurement period; R is the ideal gas constant (8.314 J mol−1K−1); S is the surface area covered by chamber (m2); and dc/dt is the slope of least squares linear regression of CO2 concentration on time. The sum of NEP and ER was used to calculate GEP as Equation where Soil temperature was determined at the depth of 10 cm with a thermocouple probe (LI 8100-201), which was continuously monitored at a 1 minute interval to calculate the means. At the same time, soil moisture was measured at the depth of 10 cm with a TDR-200 probe  and was determined by the average three measurements.Mixed model analysis was applied to examine the major and interactive effects of either spring snow addition or summer water addition, and N addition during the growing seasons on soil temperature, soil moisture content, NEP, ER, and GEP. Linear or nonlinear regression analysis was employed to explore the relationships of ECE with soil temperature in the growing seasons, soil moisture in the growing seasons, and annual precipitation across the three years. All statistical analyses were performed using SPSS 16.0 for windows .How to cite this article: Zhang, X. et al. Water and nitrogen availability co-control ecosystem CO2 exchange in a semiarid temperate steppe. Sci. Rep.5, 15549; doi: 10.1038/srep15549 (2015).


To achieve these goals, native ureters were obtained from pigs and were decellularized. The success of decellularization and the ECM composition were characterized by (immuno)histological staining methods and a DNA-assay. In vitro: scaffolds were crosslinked either with carbodiimide (CDI), genipin (GP), glutaraldehyde, left chemically untreated or were lyophilized. Scaffolds in each group were reseeded with Caco2, LS48, 3T3 cells, or native rat smooth muscle cells (SMC). After 2 weeks, the number of ingrown cells was quantified. In vivo: crosslinked scaffolds were implanted subcutaneously into rats and the type of infiltrating cells were determined after 1, 9, and 30 days. After decellularization, scaffold morphology and composition of ECM were maintained, all cellular components were removed, DNA destroyed and strongly reduced. In vitro: GP and CDI scaffolds revealed a higher number of ingrown 3T3 and SMC cells as compared to untreated scaffolds. In vivo: at day 30, implants were predominantly infiltrated by fibroblasts and M2 anti-inflammatory macrophages. A maximum of MMP3 was observed in the CDI group at day 30. TIMP1 was below the detection limit. In this study, we demonstrated the potential of decellularization to create biocompatible porcine ureteric grafts, whereas a CDI-crosslink may facilitate the remodeling process. The use of decellularized ureteric grafts may represent a novel therapeutic method in reconstruction of ureteric defects.The surgical reconstruction of ureteric defects is often associated with post-operative complications and requires additional medical care. Decellularized ureters originating from porcine donors could represent an alternative therapy. Our aim was to investigate the possibility of manufacturing decellularized ureters, the characteristics of the extracellular matrix (ECM) and the biocompatibility of these grafts  The surgical removal of tissues or organs is an aggressive therapeutic strategy to treat innate or acquired pathologies of the urinary tract. Surgical removal will present the patient and surgeon with challenges in the reconstruction of the excretory function of the urinary tract. In the case of extensive ureteral resection, surgical reconstruction by means of end-to-end anastomoses remains a major issue. Various surgical techniques were employed such as the psoas hitch  to investigate whether decellular ureters might provoke inflammatory responses or rejection reactions. Another goal of the given study was to evaluate a method to manufacture scaffolds suitable for storage by means of lyophilization.The goals of the given study were as follows: (A) to characterize tissue-engineered decellular porcine ureteral scaffolds and (B) to determine the influence of crosslinking agents on the morphological matrix properties and cell ingrowth Porcine ureters can be decellularized successfully, thus preserving ECM composition.in vitro and do not provoke rejection reactions in vivo.Tissue-engineered decellular ureteral scaffolds can be reseeded with different cells in vivo.Inflammatory reactions and degradation rate might be influenced by different crosslinking agents We addressed the following hypotheses:Deutsche Landrasse, 25–65 kg). The methods used in the present study were similar in some aspects to those of earlier published work  and stored in PBS at 4°C for a maximum of 4 weeks. SDS solution and distilled water were changed daily.All experiments were performed with ureters from pigs (n = 9). Native ureters with the same origin served as positive control (n = 9). For qualitative fragment length analysis, 5–12 μg of total DNA was electrophoretically separated in a 1.5% agarose gel . After the run, the gel was documented by light exposure in the FastGeneGelPic LED Bos .The isolation and quantification of DNA in the decellular tissue scaffolds was performed using the protocol of Qiagen  and as previously described . Dried decellular ureteral scaffolds were cut into small cross-sectional pieces of 100 mg and were homogenized using liquid nitrogen. In addition, scaffold powder was dissolved in distilled water (4 ml) and added to test tubes containing 4 ml chloroform and 2 ml methanol (5%). Furthermore, 0.5 ml of a methylene blue solution (1%) was added and mixed intensively for 1 min. After incubation time of 10 min, the absorption of the chloroform-containing phase was measured spectrophotometrically at the absorption maximum of 660 nm. For the calculation of residual SDS concentrations within scaffold pieces before and after washing (n = 12), a calibration curve was prepared.The determination of residual SDS was performed using an anionic surfactants test kit  and scaffolds were lyophilized applying a constant vacuum  in a range from −40 to 0°C.Scaffold-crosslinking with different agents was performed as previously described  containing different cell lines  or native rat SMC. Scaffolds and cells were incubated (each 1.5–2.0 × 106 cells/tube) on a rotator  at room temperature for 4 h. Afterwards, scaffolds were placed in well plates and incubated under constant conditions  for 2 weeks (n = 7–9). After incubation, scaffold pieces were fixed in 4% paraformaldehyde solution and embedded in paraffin for further histological investigations.Sterilized decellular scaffold pieces of 1 cm, which were either crosslinked, chemically untreated or lyophilized were placed in tubes  were implanted into one of the subcutaneous back pockets of rats (1 cm length). The wound was sewn with two stitches. Finally, the concentration of isoflurane was reduced to allow rats to recover from anesthesia. All surgical interventions were performed under sterile conditions. Postoperatively, rats were given carprofen  for 3 days. After a follow-up of one, nine, or 30 days, the animals were again narcotized, euthanized, and the scaffolds were explanted for further histological investigation. Therefore, the scaffolds were immersed in 4% paraformaldehyde solution and embedded in paraffin. All procedures were approved by the committee of Animal Care and Use of the relevant local governmental body (TVV15/10) in accordance with the Animal Welfare Act. Every effort was made to minimize the number of animals used.110 Sprague-Dawley rats were grouped according to scaffold crosslinking: GA, GP, CDI, BP, untreated group, and sham group , and routinely stained with Hematoxylin–Eosin and Azan  ANOVA on ranks with post hoc Tukey test. P-values of P < 0.05 were considered statistically significant.The Shapiro–Wilk test was used to determine normal distribution of the data, except immunohistochemistry data. The statistical evaluation of the immunohistochemistry including CD68 + or CD163 + cells was performed using Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks with After decellularization, the ECM composition is similar to native ureteral tissue. Azan staining of natural Figure A and decP < 0.001; each n = 9]. The DNA content in decellular scaffolds was significantly lower  compared to that in native ureter samples . An additional DNA digestion further reduced the amount of the DNA to 97.32 ± 0.7% (173.28 ± 36.6 ng/mg tissue) compared to native ureter samples . The percentage of remaining DNA after both procedures was decreased by about 91.2% compared with native ureteral tissue (P < 0.001). Qualitative analysis by gel electrophoresis showed intact DNA bands in the native samples with a size larger than 3,000 base pairs. Decellularization caused a gross but incomplete removal of this band, accompanied by a visible DNA-smear. When treating the decellular scaffolds with DNase, the smear was grossly removed  = 391.89, P < 0.001], each n = 12, Figure A significant effect of the washing duration on the SDS-concentration was observed, resulting in a 99.93% decrease, compared to residual SDS before washing. The concentration of SDS within the scaffolds after washing in distilled water was 0.006 ± 0.01 mg/g dry tissue vs. 8.449 ± 0.43 mg/g dry tissue in scaffolds before washing  = 6.84, P = 0.002]; CDI: P < 0.05, GP: P = 0.001; Figure  = 5.32, P < 0.008]; P < 0.01; Figure After incubation for 2 weeks, the ureteral scaffolds were increasingly infiltrated by tested cell lines Figures  and 5. L3,2 = 6.84, The cell infiltration in untreated Figures A,C,E andP < 0.01). At day 1 post implantation, granulocytes were present in each group. Their number decreased and diminished toward day 30 post implantation in most groups, excepting the untreated and BP group  = 9.80, P < 0.001]; P < 0.001, Figure  = 4.40, P = 0.008]; P < 0.05). At day 30 post-operative, there were no significant effects of crosslinking on the number of CD68 and CD163 positive cells   and SMC), depending on crosslinking. Highest infiltration was offered by the crosslinkers CDI and GP compared to other crosslinkers and chemically untreated scaffolds. These findings reflect not only the positive effects of crosslinking but also the effectiveness of washing out residual cytotoxic SDS concentrations (remove of 99.93% SDS). In contrast to crosslinking with GP or CDI, the infiltration of cells into GA crosslinked scaffolds was comparable to untreated scaffolds in vitro as a sign of non-optimal growth conditions. After GP crosslink, an increase of ingrown cells was described compared to GA crosslink , carboxyl (–COOH) and hydroxyl (–OH) groups of collagen may be responsible for the immunological reactions  have been reported , which corresponds with other disadvantages described in the literature , and activates collagenase1 .We reported on morphology, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Myocardial biopsies were obtained from RA and LA appendages. Mitochondrial respiration was assessed in situ in permeabilized myocardium. MiR array and real‐time quantitative polymerase chain reaction were performed to evaluate miR expression. Mitochondrial respiratory rates were similar in RA versus LA. Expression of miR‐100, ‐10b, ‐133a, ‐133b, ‐146a, ‐155, ‐199a‐5p, ‐208b, and ‐30b were different between the atria in both SR and AF patients. In contrast, differential expression was observed between RA versus LA for miR‐93 in patients with SR only, and for miR‐1, ‐125b, ‐142‐5p, ‐208a, and ‐92b within AF patients only. These results indicate that mitochondrial respiratory capacity is similar in the RA and LA of patients with SR and AF. Differences in miR expressional profiles are observed between the RA versus LA in both SR and AF, and several interatrial differences in miR expression diverge between SR and AF. These findings may contribute to the understanding of how AF pathophysiology may affect the two atria differently.Several of the cellular alterations involved in atrial fibrillation (AF) may be linked to mitochondrial function and altered microRNA (miR) expression. A majority of studies on human myocardium involve right atrial (RA) tissue only. There are indications that AF may affect the two atria differentially. This study aimed to compare interatrial differences in mitochondrial respiration and miR expression in the RA versus left atrium (LA) within patients with sinus rhythm (SR) and AF. Thirty‐seven patients with AF  may affect the two atria differentially. We assess interatrial differences in mitochondrial respiration and microRNA expression within patients with AF versus SR. The results of our study indicate that mitochondrial respiratory capacity is similar in the right versus the left atrium of patients with SR and AF. Differences in microRNA expression are observed between the right versus left atrium in both SR and AF, and several interatrial differences in miR expression diverge between SR and AF. Atrial fibrillation (AF) is prevalent in the increasingly aging population, and is associated with serious complications  regulates gene expression through inhibition or degradation of mRNA and has been implicated as an important influential element in mitochondrial respiration  or a history of AF (n = 21) scheduled for elective isolated coronary artery bypass grafting (CABG) surgery, mitral valve replacement (MVR) surgery, aortic valve replacement (AVR) surgery, or combined CABG/AVR surgery at St. Olav's Hospital, Trondheim University Hospital, Norway were included. Patients with either paroxysmal AF (pAF) (n = 11) or chronic AF (cAF) (n = 10) were included in the AF group. Blinding of patient characteristics was ensured until completion of data collection and processing. The study conformed to the principles outlined in the Declaration of Helsinki, and was approved by the Regional Committee for Medical Research Ethics of Norway. Written informed consent was obtained from patients prior to inclusion.A total of 37 patients with either SR . CPB was performed at mild hypothermia of 32–34°C. Standard St. Thomas  crystalloid or blood cardioplegia was administered for myocardial protection. Cardiac rhythm was monitored pre‐ and peroperatively by electrocardiography.Biopsies from the RA appendage were obtained during RA cannulation, and biopsies from the LA appendage during CPB. Each biopsy was immediately divided into two; one piece was promptly submerged in an ice‐cold preservation solution for assessment of mitochondrial function, and the other snap‐frozen in liquid nitrogen and kept on −80°C for miR analyses.in situ according to methods thoroughly described by other authors , 6.56 mmol/L MgCl2 (1 mmol/L free Mg2+), 20 mmol/L taurine, 0.5 mmol/L dithiothreitol (DTT), and 20 mmol/L imidazole 50 mmol/L potassium‐methanesulfonate (CH3KO3S), 5.7 mmol/L Na2ATP, 15 mmol/L phosphocreatine (PCr) (pH 7.1 at 22°C)), and subsequent 10 min rinsing in respiration solution , 1.38 mmol/L MgCl2 (1 mmol/L free Mg2+), 20 mmol/L taurine, 0.5 mmol/L dithiothreitol (DTT), and 20 mmol/L imidazole (pH 7.1 at 22°C), 90 mmol/L potassium‐methanesulfonate (CH3KO3S), 10 mmol/L sodium‐methanesulfonate (CH3SO3Na), 3 mmol/L K2HPO4, 10 mmol/L glutamate, 4 mmol/L malate, and 2 mg/mL bovine serum albumin). Mitochondrial respiratory assessment of the permeabilized atrial myocardium was performed by use of a Clark‐type microcathode oxygen electrode  covered by a fluorinated ethylene propylene (FEP) membrane, with tissue submerged in 3 mL respiration solution maintained at 22°C. Assessment of respiration in the presence of respiratory complex I substrates glutamate and malate, without adenosine diphosphate (ADP) , was followed by assessment of respiration in the presence of subsaturating 0.1 mmol/L ADP (VADP). A volume of 20 mmol/L creatine was supplemented prior to measurement of Vcreatine, and subsequently the addition of a saturating 2 mmol/L ADP allowed assessment of maximal respiration rate (Vmax) with glutamate and malate as substrates for complex I through IV of the electron transport chain. Vsuccinate was measured in the presence of complex II substrate succinate (10 mmol/L) and subsequent complex I inhibition by amytal (1 mmol/L) preceded assessment of complex II . Ascorbate (0.5 mmol/L) and N,N,N’,N’‐tetramethyl‐p‐phenylenediamine  was added to induce complex IV activity (VascorbateTMPD) and before completion irreversible inhibition of complex IV by azide (4 mmol/L) allowed assessment of Vazide. Respiratory rates are given as micromoles O2 per minute per gram dry weight myocardial tissue (μmol O2/min/g dw). Acceptor control ratio (ACR), as calculated by the ratio Vmax/V0, estimates degree of coupling between oxidation and phosphorylation. The ratio of VADP/Vmax quantifies mitochondrial sensitivity to ADP. Percent increase in respiratory rate after addition of creatine is given (↑RR Cr). The ratio Vamytal/Vmax estimates excess respiration of the cytochrome oxidase complex. Estimation of the apparent constant of Michaelis for ADP was calculated in the absence (appKm(ADP−Cr)) and presence of creatine (appKm(ADP+Cr)) . Capture probes targeting all human miRs registered in miRBASE 16.0 were applied. Hybridization was performed with a Tecan HS4800™ hybridization station . Agilent G2565BA Microarray Scanner System  was used to scan the array slides, followed by image analysis with ImaGene® 9 . Background correction of quantified signals was performed prior to normalization by applying the global Lowess  regression algorithm. miR RT‐qPCR was performed with miRCURY LNA™ Universal RT miRNA PCR pick and mix custom panel, and LightCycler® 480 Real‐Time PCR System  was used for amplification. PCR was performed for miR‐1, ‐100, ‐106a, ‐106b, ‐10b, ‐125b, ‐133a, ‐133b, 138‐1*, ‐142‐5p, ‐144, ‐146a, ‐155, ‐15b, ‐17, ‐187, ‐18a, ‐18b, ‐191, ‐193a‐3p, ‐199a‐5p, ‐19a, ‐19b, ‐208a, ‐208b, ‐21, ‐23a, ‐25, ‐26a, ‐26b, ‐29b, ‐30a, ‐30b, ‐328, ‐363, ‐451, ‐486‐5p, ‐590‐5p, ‐600, ‐92a, ‐92b, ‐93. Roche LC software was applied for analyses of amplification curves both in determining crossing points (Cp) and melting curves. Algorithms similar to the LinReg software were used to quantify amplification efficiency. Normalization was based on the average of 5 normalization assays detected in all samples. Results of miR expression after RT‐qPCR are presented as normalized crossing point  of the specific miR after subtracting the average Cp of the normalization miRs).RNA isolation, miR array and real‐time quantitative polymerase chain reaction (qRT‐PCR) were performed by Exiqon Services, Vedbaek, Denmark. RNA quality was ascertained by use of Agilent 2100 bioanalyzer  prior to labeling for miR array by use of Exiqon's miRCURY LNA2 and Fisher's exact test were used for categorical data. A two‐tailed P‐value <0.05 was considered significant. Hochberg–Bonferroni‐correction was applied to statistical analyses of miR expression. GraphPad Prism 5  was used for graphics.SPSS 21.0 for Mac  was used for statistical analyses. Paired students t‐test was applied for comparison of RA versus LA within each patient. Pearson's ChiNo significant differences were observed in paired comparison of mitochondrial respiration rates in samples from the RA versus LA within either SR or AF Fig. , nor werMiR array revealed 75 miRs that were differentially regulated between the LA and RA (data not shown). Array was used as a preliminary explorative study to identify miRs for investigation with qRT‐PCR. qRT‐PCR revealed increased LA expression of miR‐10b, ‐133a, ‐133b, and ‐30b, and decreased expression of miR‐100, ‐146a, ‐155, ‐199a‐5p, and ‐208b as compared to RA in both the SR group and the AF group .The reduced expression of miR‐93 in the LA versus RA of patients with normal SR may also be related to a difference of anatomical and physiological regions. It has been demonstrated that miR‐93 has proangiogenic effects, promotes perfusion recovery after ischemia, and is associated with improved cell survival after hypoxia and serum starvation in endothelial and skeletal muscle cells (Hazarika et al. Interatrial differences found in AF patients only include increased LA expression of miR‐1 and ‐208a and decreased expression of miR‐125b, ‐142‐5p, and ‐92b as compared to the RA. Whereas electrophysiological mapping has indicated that myocardial areas in both the RA and LA may be responsible for activating atrial premature complexes in patients with AF (Natale et al. Investigations of miR expression in human myocardium are still at an early stage, and this study provides information of myocardial miR expression that is strictly descriptive in nature. Moreover, these data do not provide information as to whether any direct link exists between miR expression and mitochondrial function. Many unknown factors still need clarification, and even associative observations are valuable in the process of obtaining an understanding of miR in cardiovascular pathophysiology. Although direct comparison of the results are precluded due to differences in patient characteristics, we identified two other studies reporting comparative assessment of miR expression in the LA versus RA, one investigating patients with AF and valvular heart disease (Cooley et al. The atrial appendage is selected for investigation as it constitutes the most accessible location for sampling human myocardial tissue. Whether AF‐related alterations on the cellular level prevail throughout the atrium has not yet been assessed in humans, thus it has not been ascertained that tissue from the atrial appendage is representative to the remaining atrial myocardium of patients. However, it has been demonstrated that structural AF‐related alterations in the atrial appendages of goats corresponded to that of the remaining atrial myocardium (Ausma et al. Due to the frequent coexistence of cardiac disease in AF, distinguishing AF‐related alterations from those of concomitant heart disease presents a challenge in investigations of AF. Although the clinical characteristics of the patients groups in our study were comparable in most aspects, there was a difference in prevalence of mitral valve disease. Although investigations of “lone” AF have been demonstrated in animal models (Morillo et al. Our investigation provides a description of associative alterations in miR expression and mitochondrial function. Further investigations are required to explore causal relations between mitochondrial respiration, miR, and AF.Mitochondrial respiration is similar between the RA and LA in patients with normal SR as well as in AF. Interatrial differences in miR expression are observed within both patients with SR and patients with AF, and the interatrial differences in miR expression profiles diverge between patients with normal SR and patients with AF.The authors are particularly grateful to the Department of Cardiothoracic surgery, St. Olavs Hospital, Trondheim University Hospital, Norway, for providing tissue samples.None declared.