A number of recent studies have demonstrated the essentiality of dietary taurine in many commercially relevant cultured fish species. Taurine is involved in many physiological functions in fish and represents an essential nutrient, which exert powerful antioxidant properties and is required as a supplement in the feed when a relevant percentage of vegetable protein sources are utilized. Our results show that dietary taurine can reduce the oxidative status of marine fish under swim stamina stress conditions. These data could be essential for the development of diets with fishmeal/soybean meal substitutions in the effort to improve the sustainability of the aquaculture.Dicentrarchus labrax) growth and oxidative stress response induced by swimming exercise. Individually tagged fish of 92.57 ± 20.33 g mean initial weight were fed two experimental diets containing the same low percentage of FM and high percentage of SBM. One diet was supplemented with 1.5% of Tau. Tau supplemented in the diet had a positive effect on fish growth, and enhanced swimming performance and antioxidant status. Two swim endurance tests were performed during the feeding trial. Metabolic oxygen consumption (MO2) was measured during exercise at incremental swimming speeds  s−1, until fatigue). Fish maximal sustainable swimming speed (Ucrit) was determined too. To investigate the antioxidant effect of dietary Tau, we also measured ROS production in fish blood by RBA (respiratory burst activity) assay and quantified the expression of genes coding for antioxidant enzymes by qPCR (quantitative polymerase chain reaction) , such as SOD (superoxide dismutase), GPX (glutathione peroxidase), and CAT  in red muscle and liver. There was a significant effect of Tau upon Ucrit during exercise. Additionally, ROS production was significantly lower in fish fed with Tau supplemented diet, supporting the role of Tau as ROS scavenger. The protective effect of Tau against oxidative stress induced by forced swimming was denoted also by a significant decrease in antioxidant enzymes gene expression in fish liver and muscle. Taken together these results demonstrate that Tau is beneficial in low FM-based diets for seabass.Taurine (Tau) is an amino sulfonic acid, which is widely distributed in animal tissues, whereas it is almost lacking in plants with the exception of certain algae, seaweeds, and few others. In the aquafeed industry, Tau is mainly used as a feed additive to promote growth in marine fish species with limited cysteine sulfinate decarboxylase activity. In particular, Tau supplementation is required in feeds in which fishmeal (FM) is substituted with high percentages of plant-derived protein sources such as soybean meals (SBM) that have much lower levels of Tau than FM. In addition to being a growth promoter, Tau exert powerful antioxidant properties being a scavenger of the reactive oxygen species (ROS). Under sustained swimming conditions, an intracellular increase in ROS production can occur in fish red muscle where the abundance of mitochondria (the main site of ROS formation) is high. Accordingly, this study aimed at investigating the effects of dietary Tau on European seabass ( Fishmeal (FM) supplies are declining and the aquaculture sector is urgently searching for alternative sources of protein. Terrestrial plant sources, such as soybean meal (SBM), are promising due to their high-protein content that satisfy nutritional requirements of carnivorous fish species. Indeed, SBM is the most common protein source used in aquafeeds due to its availability worldwide and consistent amino acid supply profile at low cost . AlthougIn mammals, the beneficial effects of Tau have been widely investigated ,4,5,6,7.Oncorhynchus mykiss), and Japanese flounder  showed high levels of CSD activity, but they were however lower than levels found in mammalian species [The role of cysteine sulfinate decarboxylase (CSD) in fish is crucial for Tau synthesis. In marine fish, the activity of this enzyme in the liver is low or absent  and diet species . Neverth species ,15.In addition to its role as a growth promoter, the nutraceutical Tau has several other properties, such as functioning as an anti-oxidizing agent, supporting osmoregulation, stabilizing and protecting cell membranes , and sca2 that occurs in the mitochondria under physiological conditions. When intracellular homeostasis is altered and the balance between pro- and anti-oxidants cannot control the ROS level, the oxidative stress develops. As a consequence of this stress, ROS attack proteins, phospholipids, or DNA, promoting cell necrosis. However, to contrast ROS, different enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) are produced by the cells to act as a first line of defense against the oxidative stress [2, CAT catalyzes the breakdown of hydrogen peroxide to water and molecular oxygen, and GPX decomposes peroxides [ROS, such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals, are all produced through sequential one-electron reduction of Oe stress . SODs reeroxides . 2 flow, which are associated with high ROS production [Fish physical exercise can alter intracellular metabolism. Sustained or burst-type swimming can increase the demand for ATP and enhance Ooduction . SwimminThe aerobic red and anaerobic white muscle are the main types of striated muscle, and according to muscle type involved, three different types of swimming can be recognized: sustained, prolonged, and burst .The burst-type swimming that is fueled by anaerobic glycolysis in white muscle can only be maintained for brief periods of less than 20 s and is terminated by exhaustion of intracellular energy supplies or by accumulation of waste products. By contrast, sustained swimming is fueled by aerobic metabolism carried out in the red muscle. In this case, fish can swim at lower speeds that they can sustain for hours, days, or even weeks. Between these two levels there is a zone of endurance swimming or prolonged exercise that can last between 2 and 200 min and, depending on the speed, is terminated by exhaustion. Hammer  assumes Anguilla anguilla).Therefore, red muscle is the most suitable tissue for studying the existing relationship between aerobic fish metabolism and ROS production during sustained activity . Indeed,Dicentrarchus labrax), by assessing both the critical swimming speed (Ucrit) and the metabolic oxygen consumption (MO2). With the aim to investigate the antioxidant effect of Tau supplementation in the diet, we also measured ROS production in fish blood by using the respiratory burst activity (RBA) assay. In parallel, the expression of a suite of genes coding for key antioxidant enzymes, such as SOD (superoxide dismutase), GPX (glutathione peroxidase), and CAT  was quantified in both red muscle and liver. Accordingly, this study aimed at investigating the effects of dietary Tau on swimming performance in European seabass  for the use of experimental animals and have been approved by a properly constituted Research Ethics Committee of the University of Insubria . Project identification Code #08/13 of November 7, 2013, signed by the President of the Ethics Committee, Prof. Elena Monti.The entire experiment was conducted at the recirculating aquaculture system (RAS) of the University of Insubria . Twenty-eight tagged sub-adult seabass with an initial mean body weight of 92.57 ± 20.33 g were divided equally into four circular experimental tanks of 600 L and let to acclimate for 7 days. During this period fish were fed daily with a commercial diet manufactured by Raanan Fish Feed Ltd.3- < 0.2 mg/L, N-NO2- <0.02 mg/L, and salinity 22 g L−1. Dissolved oxygen was maintained at 99%–100% of the saturation value by adding pure O2 to the system.The water parameters were monitored every day: temperature 20 ± 2 °C, pH 8.56, total N-NHAfter acclimation, and for all the duration of the experiment (64 days), fish were fed two isoenergetic diets (Control and 1.5% Tau) containing the same low percentage of FM and high percentage of SBM and with one of the diets being supplemented with 1.5% of Tau . To prepare the diets, all the ingredients were ground and thoroughly mixed in a blender. Then, oil and a small quality of water were incorporated to make a smooth dough, which was extruded using an electric-extruder homemade pasta-machine with a mesh plate of 2 mm size. Extruded pellets were dried overnight at 45 °C and stored at −18 °C until further utilization. The formulation and the proximate composition of each diet are shown in 3)). Furthermore, in the interval between weightings, to avoid feeding restriction that may have limited the growth potential of fish, the daily feed portion was also adjusted based on European sea bass growth curves that are available at our lab. These curves have been generated with data obtained from previous growth trials conducted with the same fish species.Fish were individually weighed and measured for their length at the beginning of the experiment (t0), after 30 days of feeding (t1), and at the end of each test  (see below). These data were used to calculate weight gain (WG), specific growth ratio  – ln )/days), feed conversion ratio (FCR = feed intake/WG), and condition factor , whereas the second one was measured at 30 days of feeding (t1) with the experimental diets in both fish groups (Control and 1.5% Tau). For RBA analysis, blood was collected from five fish/diet. Fish were anesthetized and after blood collection (1 mL from each fish) they were placed to the same communal tank. RBA, Ucrit, and MO2. Five fish/diet (not taken from the same tank) were tested for their swimming capacity for a total of 10 fish tested in five days (two fish per day were tested as just one swim-tunnel respirometer was used for the measurements). Fish were not fed the day of the swimming test. After the swimming test of each fish was finished, a blood sample was collected for RBA analysis and the fish was placed into the communal tank.After 4 days of recovery from blood collection and individual weight measurements the first swim stamina test was conducted (t2), measuring U3) was performed after 20 days of recovery from the first test (t2), following the same aforementioned protocol. Ucrit, MO2, and RBA were again measured from five fish/diet [The second swimming stamina test  with a 10 L swim-chamber respirometer (10 cm × 10 cm × 40 cm) was employed to test fish in a non-turbulent water flow with a uniform velocity profile. A computerized system with fiber optic oxygen sensing technology, and the AutoResp™ software with the automated intermittent respirometry system allowed to measure oxygen uptake by fish. A flush pump provided water exchange between the respirometer and the buffer tank where the physio-chemical characteristics of the water were checked using a thermoregulator, an air pump, and a filter. Mean water temperature was maintained at 20.0 ± 0.1 °C and salinity at 22 g L−1. A data acquisition system continuously recorded oxygen saturation in the respirometer (mean oxygen saturation ± SEM (standard error of the mean) = 98.4 ± 0.4%).An Auto−1 (15 cm s−1). At this swimming velocity, the seabass was positioned on the bottom of the chamber, gently sculling its pectoral fins and occasionally the tail flicked. The chamber was covered with a black net to encourage fish to stay in the anterior part of it.To perform the swimming stamina test, fish were individually collected from their rearing tank using a knotless net and placed directly into the swimming chamber. We minimized fish capture time, avoided collecting multiple fish and did not hold fish for more than a few seconds in the net, as these factors can increase stress affecting swimming performance. Fish were left in the swim-tunnel respirometer to acclimate for 2 h in a current at a low speed of 0.7 BL (body length) s−1, until fatigue [2, in mg kg−1 h−1) was recorded every 15 min. After the acclimation period, fish were exposed to gradual increments (every 30 min) of the swimming speed: 0.7, 1.4, 2.1, 2.8, 3.5, and then 4.2 BL s fatigue . Fish we fatigue . At each fatigue . The thrTo test recovery ability and determine individual variation in performance, the same fish were given a second swimming endurance test (t3) after a recovery period of 20 days in the communal tank. The same aforementioned protocol was followed for the second swimming test. −1);Ui is the highest velocity maintained for the entire interval (cm s−1); Uii is the velocity increment (cm sTi is the time elapsed at fatigue velocity (s), and Tii is the prescribed time interval (s). The Ucrit values for both t2 (Ucrit1) and t3 (Ucrit2) swimming tests were calculated using the following formula ,29:Ucrit−1 and converted in BL s−1. No correction for the solid blocking effect was considered, as the total cross-sectional area of the fish did not exceed 5% of the swimming chamber [The critical swimming speed values (Ucrit) for each fish were calculated in cm s chamber . −1 phorbol myristate acetate  for 1 h at 24–25 °C. Luminol-amplified chemiluminescence was measured every 3 min using a plate luminescence reader to generate kinetic curves. Each sample was run twice and read against a blank to which neither blood nor PMA was added [The induction of respiratory burst activity (RBA) in blood leucocytes was measured according to the method described by Sitjà-Bobadilla et al. . Brieflyas added . The int® 16 Tissue LEV total RNA purification Kit and the Maxwell® 16 Instrument . Concentration and purity of RNA were assessed by using the NanoDrop™ spectrophotometer Thermo Scientific™  stained with ethidium bromide. Then, 1 μg of RNA was transcribed into complementary DNA (cDNA) with GoScript™ Reverse Transcription System  using oligo d(T)16 primer. Total RNA was extracted from 120 mg of liver and red muscle (5 fish/treatment). Lysis and homogenization of tissues were performed in special disposable sterile tubes , in order to minimize the possibility of cross-contamination between samples and using the gentleMACS Dissociator . The RNA was isolated through an automated purification method using Maxwellsod, GenBank acc. no. FJ860004), catalase , and glutathione peroxidase  were quantified via One-Step Taqman® real time PCR analysis with the standard curve method. The number of transcript copies of genes superoxide dismutase (®-T easy vector system (Promega) and subsequently sequenced. Standard curves, one per each target gene, were constructed using the known copy number of a synthetic gene-specific mRNA. The entire protocol is described in detail in Terova et al. . BrieflyIn vitro transcriptions were performed using T7 RNA polymerase and the Promega RiboProbe In Vitro Transcription System kit, according to the manufacturer’s protocol. The concentration of the in vitro-transcribed RNAs was spectrophotometrically determined. Further details on the generation of standard curves are reported in One hundred nanograms of total RNA extracted from the biological samples were run in one-step qPCR, in parallel (in the same 96-well plate) to defined amounts of standard mRNAs , used to establish the standard curves. To reduce pipetting errors, master mixes were prepared to set up duplicate reactions (2 × 25 μL) for each sample. SM PCR primers and gene-specific fluorogenic probes were manufactured by Thermo Fisher Scientific   . Raw dat2, and Ucrit were subjected to two-way ANOVA  according to the GLM procedure, to determine differences due to the effect of time, diet, and their interaction (time x diet). Statistics were performed using STATISTICA 7 software. Data related to the fish growth parameters, MOSince the average initial weight of fish belonging to the two dietary groups was different, fish fed with Tau-fortified diet being heavier  than the control fish, a covariance analysis (ANCOVA) was performed to adjust subsequent fish weights and weight gains to a same initial weight.0, t1, t2, t3) were calculated with randomized block ANOVA, considering time as a block. The significance values of RBA at different times , being higher in fish fed a 1.5% Tau-supplemented diet than in the control group  from t1 through the end of the trial, showed statistical differences due to the factor diet (ol group . p < 0.01), whereas the interaction between diet and time for BW did not result as statistically significant. SGR showed statistically differences for the factor diet (p < 0.01) being higher in fish fed the Tau-supplemented diet, whereas the interaction between diet and time for SGR did not result as statistically significant. No significant differences were reported for FCR and K between dietary groups and times . Similarly, at t3 fish fed 1.5% Tau-supplemented diet recorded an Ucrit value significantly higher than the Ucrit value displayed by the control group (p < 0.01). Time determined a significant difference for the Ucrit values recorded by the control and 1.5% Tau group at t2 and t3 (p < 0.01). The interaction between diet and time for Ucrit did not result as statistically significant.At t1, after 30 days of Tau administration, a significant difference in RBA levels was observed between seabass fed with the control diet and the group fed with the 1.5% Tau addition (p < 0.05). Subsequently, at t2 and t3, fish were subjected to the swim stamina test and the RBA assay was performed immediately thereafter in the blood. The RBA of leukocytes after PMA stimulation at t2 and t3 was significantly lower (p < 0.05) in fish fed with 1.5% Tau than in the group fed with the control diet. Indeed, seabass fed with 1.5% Tau consistently showed a tendency to produce less ROS than sea bass fed the control diet at each time point. Furthermore, in the control group, swimming performance increased the RBA from t1 to t2 and a further increase was recorded at t3.At tAt the end of trial, the expression of genes involved in the oxidative stress response was evaluated by real-time PCR in fish liver and red muscle tissues. cat gene showed a significant down regulation in the liver of fish fed with the 1.5%Tau-supplemented diet in comparison to the group fed the control diet without Tau , as shown in The sod gene was not influenced by dietary Tau supplementation in either of the analyzed tissues (p > 0.05). In contrast, expression of the Atractoscion nobilis) fed with high levels of terrestrial plant-derived protein. Another study of the same research group, investigating the growth of the same fish species at the juvenile life stage, showed an improved performance due to 0.99% Tau supplementation in SBM-based diets [Dentex dentex) sizing 39.1 g, a 40% replacement of FM with SBM decreased fish growth rates and feed efficiency, which were then improved by adding 2 g/kg of Tau in the diet [Pagrus major), meagre (Argyrosomus regius), and white seabream (Diplodus sargus) juveniles also benefited from diets low in FM containing 5–20 g/kg, 10 g/kg, and 1% Tau, respectively [In aquaculture nutrition, one of the drawbacks of using plant-derived proteins to substitute FM in fish feed is related to the low level of Tau and indispensable amino acids . Indeed, Tau is almost lacking in plants with the exception of certain algae, seaweeds, and a few others, such as cactus pear. In fish, reduced growth rates and feed efficiency can be attributed to imbalances in the amino acidic profile of plant-derived proteins such as SBM. Jirsa et al.  showed aed diets . In commthe diet . The redectively ,37. D. labrax) juveniles require a dietary Tau level of 0.47%–0.51% dry matter for maximum growth performance, whereas in the study of Brotons Martinez et al. [(D. labrax) fry was increased when 0.2% and 0.3% Tau was added to the feed, with respect to fry fed 0% and 0.1% Tau-supplemented diets. This suggests that the growth stimulatory effect of Tau supplementation in the diets is correlated with ontogenetic stages of fish [Martins et al.  showed tz et al. , the SGR of fish ,12. In p of fish  for a re of fish . This coFor this reason, as reviewed by El-Sayed , juvenilTau is known for its role in protecting the organism from two major causes of cellular toxicity, oxidative stress and calcium overload . Howevercrit was found to be significantly higher in the first swimming endurance trial (t2), with even more marked differences between the two dietary groups in the successive trial (t3). Indeed, adding Tau in the diet increased the swimming resistance in fish and the higher Ucrit provided a maximum performance measure of sustained swimming not only in terms of speed but also of time elapsed at final velocity. A similar physical endurance enhanced by Tau was previously observed by Yatabe et al. [Seabass fed on 1.5% Tau showed a good swimming performance and Ue trial t. Indeed,crit observed in our experiment in fish fed with the 1.5% Tau-supplemented diet is in line with the results of Ripps and Shen [The increased Uand Shen  who repoThe link between swimming training and ROS production was studied for the first time by Mortelette et al.  in train2 and t3 after swimming performance. Seabass fed on 1.5% Tau showed a reduction in RBA in comparison to the control group. Both swimming stamina tests (t2 and t3) induced an increase in ROS production in the control group, whereas dietary Tau in fish of the other group led to an increase in ROS but at much lower levels than in the control group. This result supports a role of Tau as a scavenger of ROS as described by Jong et al. [We also investigated ROS production by measuring the respiratory burst activity (RBA) in isolated leucocytes, at time tg et al. . In partg et al. . Particu2 and t3 allowed fish to adapt their metabolism to swimming stamina test in both groups. With regard to oxygen consumption, no differences were detected between seabass dietary groups. The recovery time between tLittle is known about the relationship between ROS production and metabolic rate in ectotherms, especially in fish. Therefore, our study represents a contribution. Fish represent a good model to gain insights into the link between oxygen consumption and ROS production because variations of fish metabolic rate can be easily induced through experiments conducted under physiological conditions. In this regard, our study is in line with Mortelette et al. , who com•) parameter as a control of oxidative status, the proton leak was considered as an antioxidant mechanism by Skulachev [In addition to the hydroxyl radical (HOkulachev  through kulachev . In troukulachev . At the kulachev . The oxikulachev . ConsideA further support for Tau potent anti-oxidant properties, comes from the pattern of expression of genes coding for three antioxidant enzymes, analyzed in the present study. It is known that the antioxidant defense system includes enzymes such as SOD, GPx, and CAT that act as ROS scavengers protecting cell membrane lipids from oxidative damage . A few rcat and gpx expression in liver and muscle, respectively. The downregulation of cat and gpx, is in line with the other result found in the present study i.e., the reduction of ROS in seabass fed on 1.5% Tau in comparison to the control group. Indeed, high levels of antioxidant enzymes demonstrate a stress-induced adaptive response in attempting to neutralize the generated ROS [In our study, Tau supplementation had a protective effect against oxidative stress induced by swimming endurance in seabass, as denoted by a significant decrease in ated ROS  and one The feed additive Tau was beneficial in low FM-based diets for European seabass, as it enhanced fish growth performance and decreased ROS production under conditions of oxidative-stress-inducing activity, such as forced swimming. The protective effect of Tau against oxidative stress was denoted also by a significant decrease in antioxidant enzymes gene expression in fish liver and muscle, confirming the role of Tau as an ROS scavenger in these tissues.


NIN and SIGLEC5 identified in previous genome-wide association studies (GWASs) of samples from Chinese Han individuals. Association signals between clinical severity indicators of CP and relevant single nucleotide polymorphisms (SNPs) were also examined. A total of 3,160 study subjects, including 1,076 CP patients and 2,084 healthy controls, were recruited. A total of 32 SNPs, including 22 from NIN and 10 from SIGLEC5, were selected for genotyping. SNPs rs12883458  and rs4284742  were significantly associated with CP disease status. rs4284742 was significantly associated with all 3 clinical severity indicators, including bleeding on probing (BOP), probing depth (PD) and clinical attachment loss (CAL). According to evidence from bioinformatics analyses, both significant SNPs, rs12883458 and rs4284742, are likely surrogates of underlying variants with true effects. In summary, our findings provide direct evidence for the association of NIN and SIGLEC5 with CP susceptibility.Chronic periodontitis (CP) is a common oral disease characterized by the slow progression of alveolar attachment loss and bone destruction. Genetic components have been reported to play an important role in the onset and development of CP. In the present study, we aimed to replicate the association signals of  The prevalence of CP varies among different populations, ranging from 40% to 80%; this variance is likely due to differences in diagnostic criteria and methodology6. CP has severe impacts on quality of life and is considered one of the major causes of tooth loss in adults8. In addition, CP is also linked to other conditions, including Alzheimer’s disease9, head and neck squamous cell carcinoma10, coronary heart disease11 and pregnancy outcomes12.Chronic periodontitis (CP) is a common oral disease characterized by the slow progression of alveolar attachment loss and bone destruction15. Genetic components have been reported to play an important role in the onset and development of CP17. Early family-based studies have estimated that approximately 50% of phenotypic variations of CP could be explained by genetic factors17. A few genome-wide association studies (GWASs) have been published, and several susceptible loci, including SIGLEC518, DEFA1A318, NIN22, NPY19, WNT5A19, NCR219, EMR119, ABHD12B20, WHAMM20 and AP3B220, have been found to be associated with CP status and periodontal health. Although these previous GWASs have provided novel insights into the potential genetic etiology of CP and identified several candidate susceptible genes, further research is still needed due to the challenges inherent to GWASs, including a low portion of heritability explained by identified loci and non-consideration of biological and pathological mechanisms.Several risk factors, including age, race, obesity and smoking status, have been shown to contribute to the risk of CPNIN gene. Cytotoxic T lymphocytes, which are strongly related to the pathogenesis of CP, are functionally associated with the centrosome. In this sense, a potential link in biological mechanisms can be drawn between NIN and CP. At least two previous GWASs have reported genetic connections between NIN and CP20. Sialic acid-binding immunoglobulin-like lectins (SIGLECs) are a group of immunoglobulin families that mediate protein-carbohydrate interactions. Siglec 5 protein has been identified to be present mainly in neutrophils and plays a regulatory role in the activation of myeloid cells in the prevention of inappropriate reactivity against self tissues24. A recently published GWAS linked SIGLEC5 to aggressive periodontitis18. Because the underlying molecular mechanisms of periodontitis are still largely unclear, the effects of the NIN and SIGLEC5 genes on periodontitis have not been clarified, although significant associations with periodontitis were identified in the European population. Because the roles of the NIN and SIGLEC5 genes in periodontitis susceptibility in Han Chinese individuals have not previously been evaluated, we performed a case-control study to assess the relationship of the NIN and SIGLEC5 genes with periodontitis susceptibility in Han Chinese individuals to improve our current understanding of the roles of these genes in periodontitis predisposition. In the present study, we aimed to explore the relationship of the association signals of the NIN and SIGLEC5 genes identified in a previous GWAS with CP in a Han Chinese population, and association signals between clinical severity indicators of CP and relevant single nucleotide polymorphisms (SNPs) were also examined.Ninein is a centrosomal microtubule organization and anchoring protein encoded by the P = 0.73) and gender (P = 0.93). Informed consent was obtained from all subjects. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki (version 2002) and was approved by the Ethical Committee of Xi’an Jiaotong University.The subjects consisted of 1,076 CP patients and 2,084 healthy controls ranging from 36 to 64 years of age. All subjects were unrelated Han Chinese individuals from Shaanxi Province and were recruited from the inpatient and outpatient clinical services at the First Affiliated Hospital of Xi’an Jiaotong University between May 2014 and November 2016. The diagnosis of CP was made based on the criteria established in 1999 at the World Workshop for a Classification of Periodontal Diseases and Conditions. The healthy controls were enrolled according to the following criteria: (i) probing depth ≤3 mm; (ii) absence of any clinical signs of gingival inflammation; and (iii) no history of periodontal disease. Individuals who met the following criteria were excluded from the present study: (i) current and former smokers; (ii) pregnant or lactating women; (iii) use of antibiotics or anti-inflammatory medications in the past six months; (iv) severe medical disorders that could influence periodontal pathogenesis and/or treatment outcomes; (v) modifiers of periodontal disease, such as rheumatoid arthritis, diabetes and osteoporosis; and (vi) periodontal therapy performed within the previous six months. The clinical parameters of each subject were recorded. Bleeding on probing (BOP), probing depth (PD) and clinical attachment loss (CAL) were measured at six sites per tooth using a manual probe . All four examiners were dentists and performed examinations using a manual probe . All examiners were calibrated on a set of 40 patients probed twice before examinations were conducted in this study. We used a kappa statistic to measure agreement between different examiners, and the kappa index and intraclass correlation coefficient for PD ranged from 0.80 to 0.93 and 0.76 to 0.89, respectively, for intraexaminer calibration. The interexaminer kappa index and intraclass correlation coefficient ranged from 0.83 to 0.92 and 0.79 to 0.87, respectively, for PD  test results, is summarized in Supplemental Table Tag SNPs with a minor allele frequency (MAF) ≥ 0.05 located within the gene regions of 2 tests for each SNP. We performed a one degree of freedom test  for single marker-based association analyses. An additive mode of inheritance was assumed, and the effect of the minor allele was tested for each marker. Linkage disequilibrium (LD) blocks were constructed, and haplotype-based association analyses were performed. Stratification analyses were then performed according to the clinical severity of CP. CP patients with CAL ≥ 5 mm were defined as severe CP patients, while those with 3 mm ≤ CAL < 5 mm were defined as moderate CP patients. In addition, we also performed association analyses between significant SNPs and several clinical severity indicators of CP, including BOP, PD and CAL, using our CP samples. Linear regression models were fitted for this analysis. BOP, PD and CAL were used as phenotypes, and genotypes were used as variables. An additive mode of inheritance was assumed, and genotypes of each marker were coded as 0, 1 and 2 when 0, 1 and 2 minor alleles were present. Gene-gene interactions were also investigated by case-only analyses25. LD block construction and haplotype analyses were conducted using Haploview26. LD blocks were constructed by an algorithm proposed by Gabriel et al.27. Haplotype-based association analyses were then performed for each LD block. Haplotypes were estimated by an expectation maximization algorithm. Counts for case-control association tests were obtained by summing the fractional likelihoods of each individual for each haplotype. Single marker-based association, stratification, clinical severity association and gene-gene interaction analyses were performed by Plink28. Bonferroni corrections were applied for multiple comparisons. The P value threshold used for single marker-based analyses was 0.05/32≈0.0016.Single marker-based associations were examined by performing χhttps://www.gtexportal.org/home/)29. Expression quantitative loci (eQTL) data of significant SNPs on NIN and SIGLEC5 from 47 human tissues were extracted.We examined the potential functional consequences of significant SNPs using data from the GTEx database  and rs4284742  were significantly associated with CP disease status. A Q-Q plot for the results of genetic association analyses is shown in Supplemental Fig. NIN and SIGLEC5, respectively  of rs4284742 was present (P = 5.92 × 10−37).We performed association analyses between the significant SNPs, rs12883458 and rs4284742, and the clinical severity of CP, including BOP, PD and CAL, using CP samples. The results of this association analysis are summarized in Table NIN and SIGLEC5 in multiple human tissues . This SNP altered the amino acid sequence of the protein product encoded by SIGLEC5 and might therefore be a potential candidate for a true effective variant. In recent decades, with the fast developments of high-throughput technologies, candidate gene association studies have successfully identified many susceptibility locus for complex diseases40. Nevertheless, the data of our single study were not sufficient to confirm this hypothesis. Further studies, especially studies based on sequencing technology, are still needed in the future to systematically unravel the genetic architecture of CP.In the present study, we identified a significant association between two SNPs, rs12883458 and rs4284742, and the disease status of CP in a Chinese population. Our findings successfully replicated loci reported by several previous GWASsSIGLEC5 had differential patterns of effects in moderate and severe CP patients. The minor alleles of these SNPs were significantly related to elevated CP risk in moderate CP cases, while these same minor alleles had protective effects in the severe CP group (5 of 6 were significant). This observation has never been reported in previous studies, although stratification analyses by severity of CP have often been conducted20. One possible explanation for this observation is that SIGLEC5 might play different roles in the disease pathology in moderate and severe CP. A hypothesis is that the associated SIGLEC5 variants might affect the progression of CP from moderate to severe, rather than the risk of disease. Another explanation is that the estimated LD structures of these reported SNPs and the underlying variants with true effects were not similar between the moderate and severe CP groups in the study. In the stratification analyses, each group had fewer samples than the full sample set. Therefore, the estimation of LD structures might be significantly altered. In this sense, the patterns of association signals would be altered in turn. Future replication studies are needed to validate both assumptions. If our findings of this differential pattern cannot be replicated by other studies, then the differential pattern of association signals is likely explained by the decreased sample size in the stratification analyses. However, if this differential pattern in the effect direction of rs4284742 could be replicated by several follow-up studies, then this SNP could play a role in the underlying pathological mechanisms of CP.In the stratification analyses, we identified SNP rs4284742, and the other 5 SNPs of G, was significantly associated with elevated levels of BOP, PD and CAL. Each copy of the G allele could increase the CAL level by approximately 0.6 on average. This significant signal was not identified for rs12883458 of NIN. Our findings indicated that rs4284742 not only contributed to the risk of CP but also was related to an elevated level of CP severity.Associations between both significant SNPs and clinical severity indicators of CP identified a significant hit for rs4284742. The major allele of rs4284742, allele NIN and SIGLEC5. However, regions several kb up/downstream of these genes have been proven to be very important regulatory regions. Omitting these regions would reduce the genetic information coverage and may miss some potential significant hits. Second, we did not apply any procedures to control population stratification, which is a major confounder in genetic association studies. According to the Q-Q plot of the results of the genetic association analyses, no systematic inflation of P value significance could be observed. Nevertheless, drawing conclusions regarding population stratification from a Q-Q plot made from genotyping variants of two genes is insufficient.This study had several limitations. First, in this study, we only genotyped SNPs located within the gene regions of NIN and SIGLEC5 to be significantly associated with CP. SNPs in SIGLEC5 were also found to be significantly related to the clinical severity of CP. According to evidence from bioinformatics analyses, both significant SNPs, rs12883458 and rs4284742, are likely surrogates of underlying variants with true effects. Studies based on sequencing technology are still needed in the future to investigate the true effective markers.In the present study, we found SNPs of Supplemental Materials


This study aimed to investigate changes in types of dental arch form during adolescence and explore adolescent changes in size and form of dental arch. Hong Kong Chinese were recruited and digital dental arch models were obtained at ages 12, 15, and 18 years. Geometric morphometrics was used to investigate adolescent changes of dental arch form. There were 225 participants from whom digital models at all three age periods were available. Three types of dental arch form were identified through clustering. Significant changes (p < 0.001) in types of dental arch form were noted during age 12–18 years. During age 12–18 years, significant changes in centroid size and form of dental arch were observed (p < 0.001). No significant changes were observed during 15–18 years. Adolescent changes of dental arch form occur primarily during age 12–15 years, whereas dental arch form was relatively stable during age 15–18 years. In the dental context, adolescence spans from the age of 12 (±1 year) to 18 (±1 year) years, a period that starts from the end of mixed dentition/beginning of the permanent dentition and ends when maturation of dentition is achieved surrounding the beginning of adulthood.Adolescence is a critical period during which dramatic physical and psychological development occurs. In the biological context, it has been defined as the physical transformations that occur from the onset of puberty to termination of growthAdolescence is a crucial period in orthodontics and craniofacial orthopedics, during which the mainstream interventions of orthodontics and pre-surgery orthodontic treatment in patients with skeletal deformities are performed. Decision making in clinical practice needs to take adolescent growth potential of the craniofacial complex into consideration. For some patients, puberty growth may further compromise existing malocclusion and needs to be compensated for during orthodontic treatment. In other cases, however, growth and development may partially correct for malocclusion. In-depth knowledge on growth and development of dental arch during adolescence is therefore of distinct clinical significance.3, depth5, length6 of the dental arch, tooth inclination8, and intra- and inter-arch discrepancy10. Findings from current studies have been inconsistent, which are commonly attributed to variations in study design, sample composition, age period under investigation, and methods of assessment. However, an additional limitation inherent in these studies is their use of a sparse set of parameters to characterize dental arch. Traditional measures of dental arch, such as width, length, and depth, cannot capture the complete geometric information stored in the complex 3-dimensional (3D) models of dental arch. These studies are therefore of limited power in identifying changes in dental arch during adolescence.A large volume of literature has investigated dimensional changes in dental arch during adolescence. Most existing studies were devoted to growth changes in width11 to analyzing geometric characteristics of objects. It captures the complete geometric information contained by all landmarks12, thus increasing the power to detect changes in dental arch that are indiscernible using traditional methods, facilitating the discovery of robust changes in geometry of dental arch, and providing the opportunity to visualize adolescent changes of dental arch in 3D. In geometric morphometrics, size of an object is quantified by a composite measure termed centroid size, defined as the square root of sum of squared distance between each landmark and the centroid13. Shape, on the other hand, refers to the geometric properties of an object that remain after removal of the effect of location, scale, and rotation13. Geometric morphometrics has been used in analyzing shape of craniofacial complex14. Dental arch shape is of interest in fields as diverse as anthropology, oral and maxillofacial surgery, and orthodontics. Geometric morphometric analyses have been performed to investigate fluctuating asymmetry of dental arch among healthy population15 and dental arch shape of cleft16 and obstructive sleep apnoea17 patients. In orthodontics, dental arch characteristics of patients with orthodontic treatment need have been investigated19. In addition, Celar et al. explored the effect of different treatment modalities on shape changes of dental arch20.In contrast to traditional linear and angular measures of dental arch, geometric morphometrics is a revolutionarily new approach grounded on rigid statistical theory of shape13 of dental arch will be analyzed in this study. Based on a longitudinal sample of Southern Chinese in Hong Kong, the present study aimed to categorize dental arch form into clusters, to investigate changes in types of dental arch form during adolescence, and to explore adolescent changes in size and form of dental arch.In spite of the moderately sized literature, credibility of these studies is limited by their relatively small sample size and unrepresentative sampling methods. Furthermore, adolescent changes in shape of dental arch have not been studied longitudinally. Since both size and shape are of interest when considering adolescent changes of dental arch, form (combination of size and shape)This was a prospective longitudinal study conducted among a population-representative sample of Southern Chinese in Hong Kong. Participants were excluded from current study if they (i) had mixed dentition; (ii) were undergoing or had received orthodontic treatment; (iii) had history of systematic diseases known to alter dental arch shape; (iv) had premature loss of at least three permanent teeth in either dental arch; (v) the quality of dental digital model was unacceptable; and (vi) had missing occlusal records/inaccurate occlusal recording.Baseline of the study was performed when participants were aged 12 years. The sampling frame was all local secondary schools in Hong Kong. A random sample of 45 secondary schools from 18 districts in Hong Kong was selected as the primary sampling unit, approximating 10% of all local secondary schools. Within each school, all students in Form 1 and Form 2 classes  born between April 1 and May 31 were invited to participate in an oral health screening. Parents/primary caregivers provided written informed consent for their child to participate in the study and children provided their verbal ascent to participate. Two rounds of follow-ups were performed when participants were aged 15 years and 18 years, respectively. The study protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB reference number: UW 13–584 and UW15–278). We confirm that all methods were performed in accordance with the relevant guidelines and regulations.21 by trained operators. Impressions were evaluated and repeat impressions were obtained if anatomical coverage was deficient. Impression were wrapped in wet gauze and transported in a pressure-free environment to the dental laboratory at the end of each assessment session. Conventional plaster models were obtained from alginate impressions and then these plaster models were scanned to provide 3D digital models by the Modern Dental Laboratory .Conventional upper and lower alginate dental impressions were obtained from participants. Impression-taking was conducted in a standardized manner with specific guidelines on tray selection, mixing and loading of alginate impressions, and the anatomical extent requirements of impressionset al.6, Thilander22, Heikinheimo et al.10. Clinical center point , mesial/distal point , and buccal gingival points  were digitized for all teeth irrespective of the tooth type. In addition, incisal point  was digitized for central and lateral incisors, cusp point (tip point of tooth cusp) was digitized for canine, and occlusal point  was digitized for premolars and molars. Digitization was performed using a software written in C++ version 11.0 and complied in the platform of visual studio 2013. The landmarks were digitized by one trained investigator (TP) and were checked for accuracy by another (HMW).For both maxillary and mandibular dentition, landmark digitization was performed for all teeth from bilateral central incisors to first permanent molars. All landmarks were digitized in accordance with the definition by Bishara To quantify reliability of landmark digitization, 60 participants were randomly selected whose digital dental arch models were digitized on four occasions separated by two weeks’ interval. Landmark coordinates digitized on the first and second session were averaged and compared to the average of landmark coordinates obtained from the third and fourth session. Reliability of landmark digitization was determined through the absolute difference between landmark coordinates from the two averages, the Dahlberg’s formula, method of moments, and intraclass correlation coefficient.23, was used to categorize participants’ dental arch into 3 clusters. Analyses were performed separately for dental arch data on age 12, 15, and 18 years. Changes in types of dental arch form from age 12 through 15 to age 18 years were evaluated with Chi-square test. The level of statistical significance was set at 0.05. Analyses were performed in SPSS version 20.0.Landmark coordinates obtained from the 3D dental arch models provided raw data for geometric morphometric analyses. K-medoids clustering, a non-supervised clustering algorithm with superior robustness to outliers than the conventional k-means clusteringLandmarks digitized from the 3D dental arch models were then submitted to Generalized Procrustes Analysis (GPA), which served to remove nonshape information including location, orientation, and centroid size of the configurations. Since the present study aimed to investigate changes in form instead of shape of dental arch, the Procrustes shape coordinates obtained after GAP was then augmented by centroid size through multiplication of Procrustes shape coordinates by centroid size. GPA was performed combining landmark data for all participants at all three age periods so that the resultant coordinates are in the same shape space where valid comparisons of age-related form changes were made possible. Changes in centroid size of dental arch throughout the three age periods were determined by Friedman ANOVA. Post-hoc pairwise comparisons between age 12 and 15 years, age 15 and 18 years, and between age 12 and 18 years were performed using paired t-test. The changes of arch form from age 12 through 15 to 18 years were determined by Procrustes ANOVA, followed by pairwise comparisons among the three age periods under investigation. GPA and further statistical analyses were performed in MATLAB.At age 12, 668 children participated in the baseline survey and 627 digital dental arch models were obtained: 17 declined to provide assent to have a dental impression and 24 were excluded because they were undergoing orthotic treatment. At age 15, 435 children participated in the first round of follow-up and 376 digital dental arch models were obtained: 5 declined to provide assent to have a dental impression and 54 were excluded because they were undergoing orthotic treatment. At age 18, there were 383 participated in the second round of follow-up and 352 digital dental arch models were obtained: one declined to provide assent to have a dental impression and 31 were excluded because they were undergoing orthotic treatment. There were 281 participants who participated in all three phases of this study and among them, complete set of digital dental arch models at age 12, 15, and 18 years were available for 225 participants.The absolute difference between landmarks digitized at the first and second session and landmarks digitized at the third and fourth session ranged from 0.071 to 0.239 mm. Dahlberg’s error values ranged from 0.054 to 0.248 mm. method of moments values ranged from 0.029 to 0.197 mm. ICC values ranged from 0.691 to 0.993.The upper and lower dental arches were clustered into three distinct shape groups: narrow-type, middle-type, and wide-type at age 12, 15 and 18 years . In contrast, significant changes in types of mandibular dental arch form was observed from 12 to 15 years (p < 0.001). Between ages 15 and 18 years, there were significant changes in types of both maxillary (p < 0.001) and mandibular (p < 0.001) dental arch form. Between ages 12 and 18 years, significant changes in types of maxillary (p < 0.001) and mandibular (p < 0.001) dental arch form were observed.There were significant changes in centroid size of maxillary dental arch from age 12 through 15 to 18 years  who had normal occlusion, it was observed that approximately 30% had ‘narrow-type’ maxillary dental arch, 40% had ‘middle-type’ maxillary dental arch, and 30% had ‘wide-type’ maxillary dental arch. For mandibular dental arch, approximately 20% had ‘narrow-type’ arch, 45% had ‘middle-type’ arch, and 35% had ‘wide-type’ arch. Although cross-sectional classification of dental arch form has been performed, longitudinal changes in types of dental arch shape, particularly during adolescence, has not yet been investigated.Arch form is an important aspect to consider in orthodontic treatment planning and in deciding what type of appliances should be employed in the management of malocclusion26 level of measurements from the four measures of reliability: the absolute difference between landmark coordinates from the first two and last two digitization sessions, the Dahlberg’s formula, the method of moments, and intraclass correlation coefficient. Significant changes in types of maxillary dental arch form were observed during adolescence. At age 12, 37.2% of arch forms were ‘narrow-type’, whereas by age 18, the number decreased to 19.7%. The proportion of ‘wide-type’ arch forms increased from 22.5% at age 12 to 33.9% at age 18. No significant changes in types of maxillary dental arch forms were observed between ages 12 and 15 years. In contrast, significant changes were observed between ages 15 and 18 years.Repeatability analyses revealed that there was congruence in landmark digitization across the four sessions, as indicated by the “acceptable”Significant changes in types of mandibular dental arch form were observed during adolescence. At age 12, 27.2% of arch forms were ‘narrow-type’, whereas by age 18, the number increased to 41.3%. The proportion of ‘wide-type’ arch forms decreased from 34.4% at age 12 to 21.6% at age 18. Significant changes in mandibular dental arch forms were observed between ages 12 and 15 years and between ages 15 and 18 years.19. Papagiannis et al.19 examined sexual dimorphism, allometry, age-size and age-shape relationship, and differences between arches in a cross-sectional study based on 133 digital models (age range: 10.6–26.6). No sexual dimorphism was observed in the study. In contrast, shape was significantly associated with arch size albeit the weak strength of association. Age was likewise significantly but weakly associated with arch shape. Shape of maxillary dental arch was found associated with that of the mandibular arch. To date no studies have examined changes in arch form using geometric morphometrics analysis. Geometric morphometric analyses identified significant changes in centroid size and form of both maxillary and mandibular dental arch during adolescence. Significant changes in centroid size and form of dental arch were observed between ages 12 and 18 years for both arches, which were primarily driven by size and form changes between age 12 and 15 years.The use of geometric morphometrics to examine arch form has recently emerged in the dental literature27 evaluated width, length, and height of Chinese dental arch. However, only deciduous dentitions were examined and no age-related changes of dental arch dimensions were investigated. Therefore, their findings were not directly comparable with findings of the present study. For other Asian populations, dental arch dimensions of Vietnamese have been investigated based on a large sample of 4565 12-year-old children28. The cross-sectional nature of the study likewise impeded comparison with findings of the present study.The present study examined changes in dentoalveolar features among a large, population-representative Chinese sample that had not received any orthodontic treatment. Dental arch of Chinese has rarely been characterized. A study33. In white population, a decrease of arch length was observed during adolescence29. Bishara et al.30 reported that dental arch continued to increase in length until age 13 years in the maxilla and 8 years in the mandible. After 13 years, Lundström31 and Bishara32 found that both maxillary and mandibular arch length decreased with age until around age 30 years. Regarding dental arch width, a continuous increase was observed until 13 years in the maxilla and 12 years in the mandible10, which remained stable afterwards33. Taken together, findings from the white population suggest a transition towards relatively wider and shorter dental arch during adolescence in both maxillary and mandibular dentitions. Consistent with the decrease in dental arch length observed among the white population during adolescence, we noted significant decrease in size of dental arch among Chinese during adolescence. In addition, consistent with the transition towards wider and shorter dental arch in white population, an increasing proportion of “wide-type” dental arch was observed among Chinese adolescents for the maxillary dental arch. For the mandibular dental arch, however, increasing proportion of “narrow-type” was noted. It should be noted that dental arch width was measured as the distance between canines in earlier studies while33 while our geometric morphometric approach takes a more holistic view of dental arch shape. Further studies are needed to determine whether the observed difference is due to real ethnic differences or due to methodological variations.Worldwide, most existing studies on dental arch growth and development have been performed among the white populationCurrent studies examining changes of dental arch over time are limited in the number of participants and are potentially biased by being studies of clinical participants rather than of community-based participants. The present study benefits from the power of the revolutionary methods of k-medoid clustering and geometric morphometrics, which provides novel insight into longitudinal form changes of dental arch. In addition, this is the first time dentoalveolar features have been comprehensively evaluated in Southern Chinese.Several limitations of the present study bear noting. The response rate of the study was less than favorable. This can be attributed to the long period of follow-up (from age 12 to 18 years) and the exclusion of relatively large number of participants who had undergone or were undergoing orthodontic treatment. The relatively high attrition rate may have introduced bias that compromised the generalizability of the study findings to the general population. However, the final sample size was adequate for evaluation of developmental changes in dental arch form, which is substantiated by the statistically significant form changes observed in this study. Post-hoc sample power calculation was not performed for two reasons: first, there is currently a lack of comparable studies based on which to perform the estimation; and second, the study was primarily a proof of concept, i.e., to verify that changes in dentoalveolar features do occur during adolescence.34. Thus, the effect of developmental form changes of dental arch during adolescence must be taken into orthodontic treatment planning. Between ages 15 to 18, less frequently were changes in the dentoalveolar complex observed. This would suggest that if treatment is completed at around age 15, the dentoalveolar complex will be relatively stable post orthodontic treatment and chances of relapse due to growth and development is minimal. Nonetheless, some changes in dentoalveolar complex do occur between ages 15 to 18, which still warrants the need for retention after orthodontic treatment. Furthermore, the observed transition towards “wide-type” dental arch in the maxillary dentition and towards “narrow-type” arch in the mandibular dentition provided evidence of the need to adopt differential strategies during orthodontic treatment of maxillary and mandibular dentitions.The present study is of clinical implications. Of note, most changes in the dentoalveolar complex occurred during ages 12 to 15 years as opposed to 15 to 18 years. Age 12–15 years corresponds to a period when most orthodontic treatment is conductedIn conclusion, our findings suggest most adolescent changes of dental arch take place between ages 12 and 15 years, whereas dental arch form was relatively stable during age 15–18 years.


This study assessed the clinical value of regional ischemic preconditioning (RIP) and the role of the mitogen-activated protein kinase (MAPK) pathways in the protective mechanism of RIP in cirrhotic hepatocellular carcinoma (HCC) patients undergoing hepatectomy.Liver resection was performed with hemi-hepatic vascular inflow occlusion (HHV) under RIP (RIP group) or with HHV alone (HHV group). Clinical data, surgical outcomes, and the levels of phosphorylated MAPKs before occlusion and 30 min after reperfusion were estimated.P = 0.042), postoperative plasma transfused , and a higher level of prothrombin activity at postoperative days 3, 5, and 7 compared to HHV alone. The level of phosphorylated ERK protein was significantly increased and the levels of phosphorylated p38 and JNK proteins were significantly decreased 30 min after reperfusion compared to HHV group in the RIP group.HHV under RIP was associated with less intraoperative blood loss  contains supplementary material, which is available to authorized users. Blood loss during liver resection is prevented by vascular occlusion, which can result in ischemia/reperfusion (IR) injury. It is generally accepted that the cirrhotic liver is particularly sensitive to IR injury.3 In China and other Asian countries, more than 80% of HCC patients have underlying liver cirrhosis due to hepatitis B virus infection.5 Therefore, the need for effective methods to reduce the IR injury associated with vascular occlusion during liver resection in cirrhotic HCC patients has become increasingly important.Worldwide, liver cancer is the fifth most frequently diagnosed cancer, accounting for more than 700,000 deaths per year. In 2008, almost 55% of global liver cancer cases and deaths occurred in China.6 In patients who received hepatic resection under IP, intraoperative bleeding and postoperative complications were significantly reduced compared to no IP,7 and blood transfusions.8 hepatocyte injury, and duration of surgery9 were decreased compared to alternative methods of vascular occlusion. Other studies showed that IP (10 min ischemia/10 min reperfusion) had no clinical value for postoperative liver function and reducing postoperative complications and mortality in major liver resection,10 and there were no significant differences in operative time, blood loss, hemodynamic changes, postoperative liver function, postoperative complications, and length of hospital stay in patients undergoing elective liver resection under the Pringle maneuver12 or complex hepatectomy under total vascular exclusion (TVE) with or without IP.13Currently, ischemic preconditioning (IP) is recommended to protect against hepatic IR injury. However, most studies supporting this approach focused on patients with benign tumors or HCC without liver cirrhosis, which are not applicable to Asian countries, and outcomes of human studies on IP during hepatectomy are controversial. Some studies have confirmed the protective effect of IP. In a randomized controlled trial, patients undergoing hemihepatectomy under IP (10 min ischemia/10 min reperfusion) had significantly lower levels of markers of hepatic injury  during the immediate postoperative period than controls.14 IR injury15 and may accelerate tumor recurrence.16 Hemi-hepatic vascular inflow occlusion (HHV) is increasingly used as an alternative to the Pringle maneuver.18 HHV keeps the contralateral hepatic blood supply open, ameliorates IR injury, contributes to portal hemodynamic stability, prevents intestinal mucosal damage induced by mesenteric vascular congestion, and achieved earlier postoperative recovery of liver function compared to the Pringle maneuver in a randomized controlled trial of patients undergoing partial hepatectomy.19Most surgeons use the Pringle maneuver to reduce blood loss during liver resection. However, recent reports suggest that the Pringle maneuver is associated with portal vein thrombosis, spontaneous rupture,20Accounting for the controversy surrounding IP and the advantages of HHV, we propose the use of regional ischemic preconditioning (RIP) in cirrhotic HCC patients. This process involves occluding only 50% of the hepatic blood inflow. Data from our preliminary clinical trial indicated that HHV under RIP may be safer and achieve earlier postoperative recovery of liver function than HHV alone in HCC patients.21 Recent studies have identified mitogen-activated protein kinase (MAPK) as the major intracellular signal transduction system in IR injury.22 MAPKs are regulated by phosphorylation and have key roles in the control of cell proliferation, differentiation, and apoptosis.23 In mammalian cells, the MAPK cascade includes the ERK pathway, JNK pathway, and the p38 pathway. ERK is an important transcription factor, and the JNK and p38 pathways are involved in cellular responses to environmental stress and inflammatory cytokines. IR injury, heat shock, hypertonic conditions, and certain inflammatory cytokines such as tumor necrosis factor can activate the MAPK signaling pathway. MAPKs are involved in IR injury;25 however, the most of the previous researches were performed in cell or animal models, clinical studies in hepatic IR injury and those considering IP are limited.The protective mechanism of RIP remains to be elucidated. The pathophysiology of hepatic IR may include a number of mechanisms that the formation of pro- and anti-inflammatory mediators, expression of adhesion molecules, and the role of oxidant stress, preventing microcirculatory disturbances by nitric oxides.The objective of the current study was to conduct a prospective non-randomized controlled trial comparing HHV under RIP with HHV alone in cirrhotic HCC patients undergoing liver resection to assess the clinical value of RIP and explore the role of the MAPK pathways in the endogenous protective mechanism of RIP in these patients.The study protocol was approved by the Institutional Review Board at the Cancer Hospital, Chinese Academy of Medical Sciences. A total of 91 cirrhotic patients requiring resection for HCC were eligible for this study. Inclusion criteria were as follows: ≥ 18 years of age; primary HCC diagnosed by imaging and serum alpha-fetoprotein (AFP); compensated cirrhosis (Child-Pugh score grade A); tumor located in the right lobe of liver, close to the right hepatic vein, right portal vein, inferior vena cava or other important blood vessels, or not near major blood vessels but with a diameter > 5 cm; single or multiple lesions confined to three segments; laparotomy; indocyanine Green Retention rate at 15 min (ICG-R15) < 15%; and absence of serious cardiopulmonary complications. Exclusion criteria were as follows: other liver pathology or previous interventions .As a non-randomized cohort research, patients were assigned to a RIP group (HHV under RIP) or an HHV group  alternately with a ratio of 1:2. Case number was calculated by software PASS according to previous research. All patients consulted with a research nurse, who provided patients with comprehensive information about the advantages and disadvantages of the two occlusion techniques. All patients provided written informed consent before participation in the study.3) were obtained from subjects before occlusion and 30 min after reperfusion. Each specimen was divided into two parts. One part was immediately placed in liquid nitrogen and stored at −80 °C until Western blotting. The other part was washed three times in PBS buffer, fixed in 10% neutral formalin solution, dehydrated, embedded in paraffin, and sectioned for H&E staining and immunohistochemistry.Specimens of liver tissue  and the liver surface were repaired, and the continuous hemi-hepatic inflow occlusion was de-clamped. The natural position of the remnant liver was retained after covering the cut surface with a hemostatic sponge.26 The first porta hepatis was elaborately dissected to reveal the left and right branch of the hepatic artery and portal vein; a rubber band was preset on each vessel , phosphorylated ERK , p38 , phosphorylated p38 , JNK , and phosphorylated JNK . Sections were counterstained with Harris’ hematoxylin. Slides were scored according to staining intensity and the proportion of positive cells. Staining intensity was determined according to the staining characteristics and was scored as follows: no staining, 0 (None); light yellow, 1 (Weak); brown yellow, 2 (Moderate); brown, 3 (Strong). Five fields were randomly selected at a high magnification and 100 cells were counted in each field in which the proportion of positive cells was determined and scored as follows: 1 = 0 to 10%; 2 = 11–25%; 3 = 26–50%; 4 = > 50%. The total score was the multiplication of both scores: 0, negative (−), 1 to 4, weakly positive [+]; 5–8, moderately positive [++]; 9–12, strongly positive [+++]. All the sections were scored independently by two experienced pathologists.Immunohistochemistry was performed as previously described.31 using liver homogenates and the above-mentioned antibodies. All the antibodies (including GAPDH (Proteintech)) were used at 1:1000 dilution. Densitometry was performed using ImageQuant LAS 4000 mini . Data was analyzed with Image J software.Western blot analysis was performed with the use of conventional protocols as described previouslyt test or Wilcoxon rank sum test. Categorical data were compared using the χ2 test or Fisher’s exact probability test. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS software version 16.0 . Continuous data are expressed as mean ± standard deviation and were compared using an independent Student’s A total of 91 patients were eligible for inclusion in this study. Of these, 15 patients did not meet the inclusion criteria, and 4 patients refused to participate; therefore, 72 patients were included in the analyses. A total of 23 patients chose to be included in the RIP group and 49 patients chose to be included in the HHV group. Demographic and clinical characteristics of the study subjects are shown in Table Intraoperative and postoperative outcomes are shown in Table P = 0.042) and postoperative plasma transfused , in the RIP group compared to the HHV group. There were no deaths or biliary fistulas in either group at postoperative 30 days. There were no significant differences in postoperative length of hospital stay, postoperative infection, pleural effusion, and cardiac and pulmonary dysfunction between the two groups.There was less intraoperative blood loss  and ALT returned to normal levels within 1 week in significantly more patients in the RIP group than the HHV group . There were no differences in postoperative AST, ALT, ALB, or TBIL levels between the two groups in each detection point (P > 0.05).Outcomes of liver function tests after liver resection are shown in Table P < 0.001)    Fig. a, b. The05) Fig. c.Fig. 4PP < 0.001; Table Immunohistochemistry staining showed the similar results. In all tissues, phosphorylated ERK and phosphorylated p38 proteins were localized to the sinusoidal endothelial cells, and phosphorylated JNK protein was localized in the hepatocytes. Before occlusion, there were no significant differences in the levels of phosphorylated JNK, phosphorylated p38, and phosphorylated ERK proteins between the two groups (data not shown). Thirty minutes after reperfusion, the levels of phosphorylated JNK and phosphorylated p38 proteins were significantly lower in the RIP group than the HHV group, while the level of phosphorylated ERK protein was significantly higher in the RIP group P < 0.00; Table 1This prospective non-randomized controlled trial studied the effect of HHV under RIP on cirrhotic HCC patients undergoing liver resection. HHV under RIP was associated with less intraoperative blood loss and less postoperative plasma transfused compared to HHV alone.32 This may be one of the main reasons for blood loss.These findings suggest that RIP may reduce intraoperative blood loss in cirrhotic HCC patients undergoing liver resection. However, the results should be interpreted with caution as their clinical significance remains to be elucidated. We attribute the reduced intraoperative blood loss in the RIP group compared to the HHV group to the higher intraoperative PTA observed in the RIP group. However, this supposition requires confirmation through dynamic studies that monitor intraoperative PTA in HHV under RIP and HHV alone. In our cohort study, there was no significant difference between the two groups in body mass index (BMI) and resection volumes, respectively, so we speculated that they may be not the main cause for intraoperative bleeding. However, four more TACE-treated patients enrolled in the HHV group compare to RIP group because of unclear reason. Previous study have showed that patients underwent preoperative TACE had more intraoperative blood loss because of perihepatic organs adhesion.20 and a randomized controlled trial of 61 patients who received hepatic resection under the Pringle maneuver with or without IP.7 In these studies, IP significantly reduced intraoperative bleeding20 and postoperative complications7 and promoted early recovery of liver function20 compared to no IP.We determined the need for postoperative plasma transfusions according to intraoperative exudation in the cirrhotic liver, volume of postoperative blood-tinged fluid, and in particular postoperative PTA (representing coagulation function), in order to determine the amounts of coagulation factors and plasma proteins to replace. There was no significant difference between the two groups in PTA on postoperative day 1, but PTA was significantly lower on postoperative days 3, 5, and 7 in the HHV group compared to the RIP group, which showed that the recovery of PTA in the HHV group was slower than that of the RIP group. This suggests that RIP may have some protective effect on coagulation function, causing patients in the RIP group to have less intraoperative exudation and less postoperative plasma transfused. More importantly, it should be noted that our current study included only patients with HCC and liver cirrhosis. Over 50% of tumors were located near the right or middle hepatic vein, right portal vein, the third porta of the liver, or the inferior vena cava. These were considered high-risk patients who were likely to experience postoperative liver failure. To the authors’ knowledge, the current study is the first to investigate the effects of RIP in cirrhotic HCC patients undergoing liver resection. The outcome of the current study is in accordance with our preliminary study of 54 HCC patients who underwent hepatectomy under HHV with or without RIP20 This study showed the same maximum tolerance time. Total ischemic time in this study group ranging from 10 to 70 min was determined by difficulty of surgical procedure instead of a fixed time period. There was no significant difference of mean ischemia time between the two groups. But, the changes of molecular and histopathology profile were truly observed in all the patients even the least ischemic time was only 10 min. Although the degree of changes of molecular and histopathology profile theoretically correlated with ischemic time, the detail of the correlation was not the target we focused, this could be an exploratory goals in future. We propose that HHV under RIP can prolong the ischemic tolerance time of the liver and therefore has potential to increase the duration of surgery. This is especially important in patients with severe disease, such as those with cirrhosis or tumors located close to intrahepatic structures. Furthermore, we found that HHV under RIP was associated with faster postoperative recovery of transaminase levels and therefore liver function, and less intraoperative liver tissue damage compared to HHV alone.For cirrhotic patients, continuous portal triad clamping (Pringle maneuver) exceeding 30 min may cause irreversible injury. Previously, we have shown that the maximum ischemic tolerance time of the liver in HCC patients is 70 min.33 that used the Pringle maneuver under IP.The current study showed that the operative time, length of hospital stay, and frequency of postoperative complications were not different between the RIP and HHV groups, indicating that the safety of HHV was not compromised by RIP. These data are in accordance with a prospective randomized controlled study34 MAPK is a highly conserved family of protein kinases that is regulated by phosphorylation and includes JNK, p38, and ERK. JNK regulates cell viability, apoptosis, and cell proliferation. In the current study, 30 min after reperfusion, the levels of phosphorylated JNK protein and phosphorylated p38 protein were significantly lower in liver tissue from the RIP group compared to the HHV group. These findings are in accordance with a previous study that showed JNK activity was decreased in rat liver after IP, and hepatic injury was alleviated. This suggests that JNK activation may be associated with IR injury.35 The role of p38 in tissue damage is controversial. In rats,36 p38 activation was associated with increased levels of F-actin in liver cells, changes in the cytoskeleton, and liver failure. ERK is associated with cell proliferation, transformation, and differentiation.37 After reperfusion, the level of phosphorylated ERK protein in liver tissue of the RIP group was significantly higher than the HHV group. Studies on ischemia in the myocardium propose that a “RISK path”  that includes ERK1/2, PI3K/AKT, PKC, and PKG has a protective effect against IP.38 Zhang et al.39 reported IP could reduce ischemia/reperfusion-induced cell apoptosis by activating the ERK signaling pathway and inhibiting Rho-kinase activity. Importantly, MAPK pathways are part of a phosphorelay system.40 During IP, direct feedback between the ERK and p38 and JNK and p38 pathways is important.43 p38 and JNK pathways are activated within a few minutes after reperfusion, inducing cell apoptosis and necrosis.44 Application of a p38/JNK activator elevates transaminase levels and increases liver necrosis, while a p38/JNK inhibitor may reduce hepatic IR injury.45IP can mobilize endogenous protective mechanisms and enhance liver tissue tolerance of ischemia. MAPK cascade pathways are involved in cellular responses to extracellular stimuli.33 In the current study, patients were not stratified by age due to a small sample size. Third, recovery of transaminase and molecular profiles seems different according to the use of ischemic preconditioning. However, there was no difference in short-term clinical outcomes between the two groups. The current results suggest that preconditioning may have protective effect for the liver from ischemic injury. However, it remains difficult to determine the necessity of this maneuver in actual clinical settings.This study is associated with several limitations. First, patients were not randomly assigned to RIP or HHV; this may result in patient selection and confounding bias. However, demographic and clinical characteristics of the two groups were not significantly different. Second, evidence suggests that the protective effect of IP is age-specific. IP is particularly effective in young patients (< 60 years of age) requiring a prolonged period of inflow occlusion, and is possibly related to preservation of ATP content in liver tissue.Our study indicates that HHV under RIP may reduce intraoperative IR injury, improve coagulation. and increase the rate of recovery of postoperative liver function compared with HHV alone in HCC patients with cirrhosis requiring resection. The protective mechanism of RIP may be associated with changes in the protein phosphorylation level of MAPK pathways.Supplementary Fig. 1Representative cases of immunohistochemistry staining for pJNK, pp38, and pERK in RIP group and HHV group are shown. Magnification: ×100. (PNG 819 kb)High resolution image (TIF 13752 kb)


Artemisia tridentata) ecosystem in western Wyoming. This is important given that sagebrush ecosystems are poorly understood, despite their prevalence in the western US. In addition, large-scale nitrogen additions have begun on sagebrush landscapes in Wyoming in order to mitigate population declines in mule deer (Odocoileus hemionus). The study objectives were (1) to evaluate the effectiveness of a nitrogen fertilization pulse in increasing sagebrush biomass and forage quality, and (2) to assess effects of nitrogen addition on soil biogeochemistry and vegetation community structure. We fertilized 15 plots across 5 locations in western Wyoming using a single pulse of urea (5.5g N m-2). In addition, we immobilized available nitrogen through surface hay treatments (250g hay/m2). Nitrogen additions failed to increase growth of sagebrush, alter nitrogen content of sagebrush leaders, or alter greenhouse gas efflux from soils. The plant community also remained unchanged; total cover, species richness, and community composition were all unaffected by our treatment application. Over the two years of this study, we did not find indications of nitrogen limitation of ecosystem processes, despite a wet growing season in 2014. Thus, we have found a general lack of response to nitrogen in sagebrush ecosystems and no treatment effect of a single pulse of N to sagebrush biomass or forage quality.Nitrogen additions are known to elicit variable responses in semi-arid ecosystems, with responses increasing with precipitation. The response of semi-arid ecosystems to nitrogen are important to understand due to their large spatial extent worldwide and the global trend of increasingly available nitrogen. In this study, we evaluated the impact of a single nitrogen addition pulse on a semi-arid big sagebrush ( Nitrogen (N) additions are known to elicit varying responses in semi-arid ecosystems . EcosystIn a meta-analysis of research studies on the effects of N fertilization studies in water-limited drylands worldwide, Yahdjian et al.  found thArtemisia tridentata spp.) ecosystems are the dominant dryland system [x compounds [In the western US, big sagebrush  over the 15 years since the onset of natural gas development [The Pinedale Anticline of western Wyoming is one of the most disturbed regions of big sagebrush. Land managers are particularly concerned with large population declines in mule deer  to evaluate the efficacy of the addition of a N pulse for increasing big sagebrush biomass and forage quality, and (2) to assess effects of N addition on soil biogeochemistry, plant biomass, and plant community composition. We hypothesized that the N fertilization pulse would increase N availability in the soil, however it would not increase growth, biomass, or forage quality of big sagebrush due to primary limitation by water. In addition, we expected changes in plant community composition and a high potential for increased GHG emission.Artemisia tridentata wyomingensis) and mountain big sagebrush (Artemisia tridentata vaseyana). Other common plant species present include Sandberg bluegrass (Poa secunda), western wheatgrass (Pascopyrum smithii), chaffweed (Antennaria microphylla), squirreltail (Elymus elymoides), and longleaf phlox (Phlox longifolia).We conducted this study between 2012 and 2015 in semi-arid big sagebrush plant communities in western Wyoming, near the town of Pinedale (42°51′58″ N 109°51′53″ W). The area surrounding Pinedale is consistent with sagebrush steppe in other parts of Wyoming; plant communities are co-dominated by Wyoming big sagebrush . We obtained permission from three local land-owners to use their privately-owned lands because of constraints associated with imposing manipulations on public areas adjacent to the Pinedale Anticline. We identified site locations with similar soil types, textures, and landscape positions, with all sites positioned on a slight slope. Each site had previously been moderately grazed for over a century; this grazing history is typical of the region, big sagebrush plant communities throughout the western U.S., and—most importantly–consistent with the history of the Pinedale Anticline, for which we are trying to characterize potential effects of N fertilization.-2) and surface hay (well-aged for at least one year) was applied  for the above respective treatments in September 2012. The addition of hay to the surface immobilizes soil inorganic N, through incorporation of N into microbial biomass during decomposition of the hay [To address our hypotheses, we applied three treatments  in a generalized randomized complete block design with split-plots. Treatments were randomly applied to subplots and replicated in three main plots; this split-plot setup was replicated at five sites (blocks). The hierarchy of locations is site > main-plot > subplot. The five replicate sites were located on three privately-owned ranches. Within each site, we created three plots (10m x 10m). Each main-plot was enclosed by cattle-proof fencing and contained three subplots (2.5m x 8.5m) each randomly assigned one of the above treatments. Subplots were fertilized with N in the form of urea (4.5g N m the hay –41. All 3-), and ammonium (NH4+), were estimated in situ using Plant Root Simulator (PRS) probes. The PRS probes are ion exchange resin membranes held within a rigid plastic frame  and provide an index of nitrogen availability to plants. PRS probes provide an index of soil N availability that integrates across the time of their deployment (i.e. soil N availability from Oct-May for this study). PRS probes are placed in pairs, one anion probe (collecting NH4+) and one cation probe (collecting NO3-). Multiple pairs were placed within a single treatment. All probes were washed with DI water following the manufacturer’s protocol and shipped to Western Ag Innovations, Inc.,  for analysis.Inorganic nitrogen availability, in the form of nitrate (NOIn order to estimate available soil nitrogen (N) during the first year of the experiment (Sept 2012 –May 2013), we placed PRS probes within each treatment block at all fifteen plots. We installed probes over winter to catch the spring snowmelt, the largest pulse of nitrogen for the year , as plotDuring the first year they were placed on the edge  of the canopy of a big sagebrush individual. During the third year of the study (Oct 2014 –May 2015), probes were placed at two locations to measure soil N available under both the big sagebrush canopy and within the interspace between individual shrubs. For example, if an individual sagebrush had a canopy that extended 20cm from its main stem  then the Year 1 probes would be placed at 20cm , while the Year 3 probes would be placed at 10cm  and 30cm  from the stem. Probe placement was stratified by shrub location in an effort to represent a key component of soil N variability in drylands, as N availability is known to be elevated under shrub canopies . This wa4+ and NO3-. Thus, PRS probes provide an index of N availability that can be compared among treatments within a sampling campaign, but cannot be compared between sampling campaigns. As such, our analyses are limited to comparisons within a campaign .The sets of probes from each sampling campaign  were retrieved from the field and analyzed after their 7–8 month deployment. A limitation of PRS probes is that temperature and soil moisture affect the ability of the anion and cation resin to adsorb NHWe estimated big sagebrush annual growth by measuring leader growth  and the ephemeral branch growth  in 2014. Leaders and ephemeral branches were collected at the end of the growing season in late October 2014 and dried in the laboratory for five days at 55°C and then at 35°C until mass remained constant over a 5-day period. We then weighed and measured the length of each individual leader and branch. The dried leaders were ground in a ball mill and submitted to the Stable Isotope Facility at the University of Wyoming for total carbon and nitrogen analysis , as a pr2) within each subplot. The canopy cover of each plant species was estimated using a modified Daubenmire [We sampled the plant communities at each site over two days in August 2014. We surveyed the plant community using 20 randomly placed Daubenmire quadrats  pipe (10 cm high x 20 cm diameter) were permanently buried in the ground to a depth of 6 ± 2cm, which then acted as bases for the chamber lid. Chamber lids were round, 20cm inside diameter and 8cm deep, made of opaque PVC and vented following Hutchinson & Mosier . The cha5.3 , using the following libraries: knitr, tidyverse, lubridate, plotrix, emmeans, ecodist, labdsv, nlme, Rmisc, vegan, soiltexture, and here.We conducted all data analysis and figure creation using R version 3.lme function, ‘nlme’ package v 3.1–139 [emmeans function, ‘emmeans’ package v1.3.4 [We used a linear mixed effects model ( 3.1–139 ) to exame v1.3.4 ).3-, NH4+), sagebrush growth , and forage quality .The mixed effects model was used to evaluate the effects of our N and hay treatments on inorganic soil N availability  were calculated for each treatment and plot using the rate of change for each of the gases analyzed over the 30-minute period, calculated as the slope values of a linear regression. These fluxes were then similarly analyzed using the mixed effects model previously described.Soil trace gas fluxes .We examined the effects of N and hay treatments on the plant community by evaluating differences in total plant cover and species richness using the same mixed effects model and associated processes. To explore the effects of treatment applications on plant community composition, we used a permutational multivariate analysis of variance (PERMANOVA). Total cover and abundance by species was assessed by summing the cover class midpoint for each species by site to obtain the total percent cover for each species and then standardized these values to relative abundance (–4 for R ). We theWe also explored the effects of total soil N on leader length through calculating a one-way analysis of covariance (ANCOVA) with site and plot as nested, random factors. This is similar to the original model described above except treatment is removed, as it is confounded with soil N availability. We then obtained fitted values at the population and hierarchical level .4+ between treatments, however NO3- was significantly more available in the N fertilized treatments (p<0.001) than either the control or N removal (p = 0.035). By the end of second year of the experiment, no significant differences remained between treatments for total inorganic N, NO3-, or NH4+. During year one, NO3- represented 99.0% ± 0.4 of the total available inorganic N across all treatments/locations, while in year two it was 92.0% ± 1.7.The addition of N significantly increased the availability of total inorganic N in soils p<0.001;  during tThe addition of N and the application of hay had no treatment effect on the growth of big sagebrush leaders; they were unchanged in length, mass, and density. While the treatment term was significant in predicting leader length (p = 0.006), neither the nitrogen additions (p = 0.103) nor hay treatment (p = 0.079) were significantly different than the control when examining contrasts. Ephemeral branches  were unchanged by the addition of N fertilizer, though the addition of hay reduced their mean length by approximately 8% or 1.1cm  inappropriate to a certain extent. Precipitation between the two years, 2012 and 2014, varied by approximately 200mm depending on the ranch examined and could account for a portion of the variability seen in the control plots between years. Due to differing conditions between years, we have focused on comparing PRS probe measurements of total inorganic N within single years.-2 (or 45 kg N ha-1), which is 150–900% of annual N mineralization [2O and NOx) accounted for an additional 10–20 kg N ha-1 yr-1 loss from the soil, which could contribute to the lack of increase observed in inorganic soil N among treatments in year two [2O compared to these studies one year after fertilization, it may be that fluxes were greater immediately after fertilization or during the spring when water was more available, therefore not captured in our sampling period.The lack of significant increase in soil N availability in the second year in fertilized plots is likely due to ‘normal’ N losses through N mineralization, gaseous loses , and leaching. Additions of N were conducted at 4.5 g N mlization . Trace gyear two . While wIncreased N availability had minimal effect on the length of big sagebrush leaders over the two years of the study. Additional N did not result in an increase in forage quality; C:N ratio and mass of N were the same between the control and N fertilized plots. While the addition of hay minimally decreased the length of ephemeral branches, the addition of N had no effect. It is possible that the decrease in soil N caused by the hay treatments decreased growth by increasing N limitations. These limitations on the growth of inflorescences by N may have been more apparent during 2014, when water was more available.Our prediction that there would be no differences in plant growth between treatments was generally supported. Conversely, we also found that leader length was weakly positively correlated with inorganic soil N availability at each of our sites. On the face of it, these two statements are contradictory. While we have shown that our treatments significantly affected the availability of total N in soil across all sites, there was no associated treatment effect on plant growth. Leader growth correlated with N availability at the site level. It is possible that other factors may drive or mediate plant growth at these sites. Site-specific factors such as soil texture , precipitation, and leaching can affect the conversion of urea fertilizer to N, the retention of that in soil, and/or to the uptake and allocation of that N by plants to increased leader growth.Interestingly, differences in leader length between ranches was strong. While this could be due to many factors, the ranch with highest precipitation in each year from 2012–2015 also had the longest sagebrush leaders. However, we did not measure precipitation at any of our sites or ranches, making it impossible to assert this with any confidence.P. smithii (western wheatgrass) is known to be nitrophilic and respond to additions of N [Contrary to our predictions, the addition of a single pulse of N did not alter plant community composition. ons of N ,51, howe2, CH4, and N2O) from soil. Fluxes of CO2 and N2O fell into similar ranges for dry, unwetted sagebrush reported by Norton et al. [2, CH4, and N2O may be limited primarily by water availability, not N availability.There was no effect of N additions on GHG efflux (i.e., COn et al. . The lacOur findings are not consistent with other studies, which have generally found a positive response of plant productivity to N fertilization ,8 indicaOur results indicate that a one-time application or single pulse of N does not affect soil N availability in the long term within the sagebrush communities of the Pinedale Anticline. We suggest that annual fertilization would be necessary to maintain high levels of soil N availability within big sagebrush systems, as the levels of soil N were the same between the fertilized and control plots by the second year. Yet increases in N availability over the long term may have negative consequences, including higher volatile N losses, and increases in invasive plants . It shouThis study was conducted to investigate the role of N limitation in dryland sagebrush ecosystems and to consider the effect of a pulse of N addition to increase forage and forage quality for mule deer. We evaluated responses of soil N availability, annual growth rate of big sagebrush, and forage N concentration in response to N additions. In addition, we investigated the response of trace gas production and plant community structure to increased N. This study demonstrates that a single addition of N, in the form of urea fertilizer, does increase N availability in soil for one year after fertilization. However, this increased soil N availability is only loosely associated with increased growth of big sagebrush and not likely to impact forage availability and quality for mule deer. The results of this study suggest that the addition of N in a single pulse is likely a poor choice for mitigation of mule deer population declines on the Pinedale Anticline due to the minimal gains in forage, if any, and associated high cost.