Puto superbus (Putoidae) lives in mutualistic symbiotic association with bacteria. Molecular phylogenetic analyses have revealed that symbionts of P. superbus belong to the gammaproteobacterial genus Sodalis. In the adult females, symbionts occur both in the bacteriocytes constituting compact bacteriomes and in individual bacteriocytes, which are dispersed among ovarioles. The bacteriocytes also house a few small, rod-shaped Wolbachia bacteria in addition to the numerous large, elongated Sodalis-allied bacteria. The symbiotic microorganisms are transovarially transmitted from generation to generation. In adult females which have choriogenic oocytes in the ovarioles, the bacteriocytes gather around the basal part of the tropharium. Next, the entire bacteriocytes pass through the follicular epithelium surrounding the neck region of the ovariole and enter the space between oocyte and follicular epithelium (perivitelline space). In the perivitelline space, the bacteriocytes assemble extracellularly in the deep depression of the oolemma at the anterior pole of the oocyte, forming a “symbiont ball”.The scale insect  Scale insects (coccoids) constitute the superfamily Coccoidea assigned to the infraorder Coccomorpha, within the suborder Sternorrhyncha of the order Hemiptera  . As these microorganisms are responsible for the synthesis of amino acids missing in their diet, they are necessary for the proper growth and reproduction of the host insect     occurs inside the cells of other bacteria (betaproteobacteria Tremblaya) . Gruwell et al.  and near Wolsztyn (in the west of Poland) in the months of June and July in the year 2012. Specimens destined for molecular analyses were collected in Katowice in June and July in the year 2014.The adult females of Leonardi  were colThe dissected ovaries and entire abdomens were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), rinsed in the buffer with sucrose (5.8 g/100 ml), and postfixed in 1% buffered osmium tetroxidae (pH 7.4). The material was then dehydrated in a graded series of ethanol and acetone and embedded in epoxy resin Epon 812 . Semithin sections (1 μm thick) were stained with 1% methylene blue in 1% borax and examined and photographed with a Nikon Eclipse 80i light microscope. Ultrathin sections (90 nm thick) were contrasted with lead citrate and uranyl acetate and examined and photographed under a Jeol JEM 2100 electron microscope at 80 kV.P. superbus using a Sherlock AX DNA extraction kit (A&A Biotechnology), according to the manufacturer’s protocol. The DNA extracted was used for the molecular identification of symbionts colonizing the P. superbus tissue in the following experiments:PCR reaction with universal, eubacterial primers: 10F (AGTTTGATCATGGCTCAGATTG) and 1507R (TACCTTGTTACGACTTCACCCCAG) . After 16 h, the occurrence of the bacterial 16S rDNA was confirmed by a diagnostic PCR reaction with the following primers: pJET For–GCCTGAACACCATATCCATCC and pJET Rev–GCAGCTGAGAATATTGTAGGAGAT.Molecular cloning: the purified PCR product was cloned into pJET 1.2/blunt plasmid vector using a Clone JET PCR Cloning Kit (Thermo Scientific). The ligated mixtures were transformed into competent MspI restrictive enzyme. The plasmids from the selected colonies were isolated using a Plasmid Mini AX kit (A&A Biotechnology) and sequenced. The nucleotide sequences obtained were deposited into the GenBank database under the accession numbers KY558891-KY558893.Restrictive analysis: PCR products of 30 colonies were subjected to restriction analysis using an Total genomic DNA was isolated from adult females of P. superbus symbiont and selected gammaproteobacterial symbionts of hemipterans. The sequences homologous to the sequence obtained were found in the GenBank database using CLC MainWorkbench 7 software. The sequences were then edited using BioEdit Sequence Alignment Editor 5.0.9  were scarce. The selected clones (three of each type) were sequenced and the sequences obtained were compared with the data deposited in the GenBank using BLAST. The result of BLAST searches has indicated that the sequences of dominant restrictive patterns show 94–95% similarity to the bacteria belonging to the genus Sodalis , whereas the sequences of pattern no. 2, 3, and 4 were identified as Wolbachia bacteria. All sequences represented by pattern no. 1 were identical; therefore, only one of them was used for the phylogenetic analysis. The 16S rDNA sequences of Wolbachia symbionts were slightly different from one another (with a 99% similarity). Since Wolbachia bacteria are widespread within different and unrelated insect groups, the sequence of 16SrDNA of Wolbachia was not used for phylogenetic analysis. The total length of sequences subjected to the phylogenetic analysis was 1322 bp, and the base composition was as follows: 21.7% T, 22.8% C, 26.3% A, and 29.1% G. The phylogenetic tree constructed using Bayesian analysis indicated that, based on 16S rDNA relationships, the obligate symbiont of P. superbus belongs to the Gammaproteobacteria within the Proteobacteria phylum and may be designated as a Sodalis-like symbiont , however, are placed in distinct lineages and are phylogenetically distant  to 14.7%   are also scattered among ovarioles constituting the ovaries  Fig. a, b. At As stated in the “Introduction,” scale insects, unlike other hemipterans living in obligate symbiotic associations with microorganisms, are host to different species of symbionts. The results of the recent molecular analyses of symbionts hosted by different scale insect species , secondary symbiont  or may co-reside with other microorganisms as co-primary symbiont   as well as extracellularly . The situation in Cicadella viridis is of special interest because in this species, in contrast to the pseudococcinae mealybugs , the Sodalis-allied symbionts both occur in their own bacteriocytes and they also co-inhabit bacteriocytes with the betaproteobacterium Sulcia muelleri , primary symbiont in Puto albicans (Putoidae)) or the ovaries of adult females comprising vitellogenic or choriogenic oocytes , Acanthococcus aceris, Gossyparia spuria (both Eriococcidae), and mealybugs (Pseudococcidae), the secondary symbiont of Palaeococcus fuscipennis (Monophlebidae)). In the case of infection of the older oocytes, the symbionts may infest the anterior pole of the oocyte  or its posterior pole   is of special interest in that their bacteriocytes differentiate into two types: (1) bacteriocytes which constitute the bacteriome and degenerate in the adult female (named by Buchner primary bacteriocytes) and (2) bacteriocytes accompanying the ovarioles which migrate to the ovarioles and are incorporated into the embryo during embryonic development (named by Buchner secondary bacteriocytes). Buchner , to which P. superbus was formerly classified (see “Introduction”), belongs to distinct group of bacteria, namely to the class Betaproteobacteria , Icerya purchasi and Drosicha pinicola (both Monophlebidae), and Kerria lacca (Kerridae) (Duron et al. Wolbachia (Werren Both ultrastructural and molecular analyses have revealed that apart from the a Werren , it seem


We aimed to evaluate whether blood conservation strategies including intraoperative autologous donation (IAD) could reduce perioperative blood transfusion for patients undergoing cardiac valve replacement including mitral valve replacement, aortic valve replacement (AVR), and double valve replacement (DVR).A total of 726 patients were studied over a 3-year period (2011–2013) after the implementation of IAD and were compared with 919 patients during the previous 36-month period (January 2008–December 2010). The method of small-volume retrograde autologous priming, strict blood transfusion standard together with IAD constituted a progressive blood-saving strategy.P < 0.001) and postoperative respiratory failure  favored the IAD group, whereas hematocrit levels were more favorable in the non-IAD group . The use of blood product transfusion was higher in the non-IAD group . Binary multivariate logistic regression analysis showed that high age, non-IAD, DVR surgery, and absent smoking history are associated with a higher risk of intra-/postoperative blood transfusion.Baseline characteristics and preoperative information showed no statistically significant difference between IAD group and non-IAD group. Most of the postoperative morbidities are statistically the same in the 2 groups. Chest tube output (415.2 vs 1029.8 mL, Blood conservation is effective and safe in cardiac valve replacement surgeries. The use of intraoperative autologous donation can lead to improved outcomes including a significantly lower rate of intra-/postoperative blood transfusion and postoperative complications. With the rapidly increasing number of surgical operations being performed in China, the limited level of blood donations cannot satisfy all of the patients’ requirements, and some hospitals even have to postpone surgery due to the lack of blood. Moreover, further studies have shown that allogenic blood transfusions can increase the incidence of infectious diseases, lung injury, and immunosuppression,5 and also reduce short- and long-term survival.,7Blood transfusions are often required in patients who undergo heart surgery due to hemorrhage. Fifty percent of cardiac operations were reported to requiring blood transfusions in America during operations and cardiopulmonary bypass (CPB) is achievable and cost-effective, which provides a theoretical basis for the feasibility of preoperative autologous blood storage followed by intraoperative and postoperative autologous blood reinfusion. A recent study showed that more aggressive blood conservation strategies during CPB in cardiac surgery, such as intraoperative autologous donation (IAD), retrograde autologous priming (RAP), and shorter length circuit, could reduce the need for postoperative allogenic blood transfusions.At present, it is well recognized that tolerance to permissive anemia (hemoglobin levels as low as 6 g/dL) Valve replacement is an effective treatment that improves the quality of and prolongs the life of patients. Our previous study has demonstrated the advantages of IAD-based conservation strategies in mitral valve replacement (MVR) patients who were most commonly affected by rheumatoid causes. However, the role that IAD played in the whole valve placement population was still to be determined. To carry out a retrospective study, we collected data on patients who underwent an autologous transfusion from 2011 to 2013 and compared these with data from patients with no autologous transfusion from 2008 to 2010 to confirm the advantages of a blood conservation strategy based on IAD.Rheumatic valvular disease is the most common reason for heart surgery in China.2After obtaining written informed consent waived by the Hospital Review Board, we retrospectively reviewed data from a database of a single surgical center, the Department of Cardiothoracic Surgery at the First Affiliated Hospital of Zhejiang University, China. The cohort included patients (aged 18 years and above) who underwent cardiac valve replacement surgeries using CPB during the 6-year period from January 2008 to December 2013. The 3 types of surgeries studied were MVR, aortic valve replacement (AVR), and double valve replacement (DVR). Exclusion criteria included emergency procedures, renal failure, infective endocarditis, preoperational hematocrit (HCT) <30% or hemoglobin <10 g/dL, patients with incomplete clinical material, and cases noncompliant with the IAD protocol  before and after the implementation of IAD, which began at the end of 2010.The autologous donation was performed before the initial systematic heparinization of cardiopulmonary bypass. Using a needle inserted into the purse string on the right auricle, the blood was siphoned into a blood bag  for storage. Each blood bag contained 100 mL of blood preservation fluid  and was used to store 400 mL of blood. The blood bags were stored at room temperature in the operation room.The IAD volume was based on the preoperational HCT level of the patients. For most of the patients (weight 50–70 kg), the IAD volume prescribed was 600 mL for patients with HCT > 40; 400 mL for patients with HCT 35 to 40, and 300 mL for patients with HCT 30 to 35. As for patients who weighed less than 50 kg, the IAD volume was decided as 400 mL for patients with HCT > 40, 300 mL for patients with HCT 35 to 40, and 200 mL for patients with HCT 30 to 35. However, for patients who weighed more than 70 kg, the IAD volume prescribed was 800 mL for patients with HCT > 40, 600 mL for patients with HCT 35 to 40, and 400 mL for patients with HCT 30 to 35.During the IAD, the intravascular volume was replaced by an equal amount of crystalloid fluid to maintain euvolemia. The volume of IAD was stored and transfused after surgery when protamine administration had been completed.During the operation, anticoagulation with 3 mg/kg of heparin was later neutralized by protamine (ratio 1:1) at the end of the CPB. Activated clotting time (480–600 seconds) was used to monitor anticoagulation, and no antifibrinolytics were used during CPB. Cardiotomy suction into the cardiopulmonary circuit reservoir was used. The Cell Saver blood salvage system  and high performance hemoconcentrator  were used during surgery to recover any blood that was shed and to concentrate the residual blood in the bypass circuit after surgery. Identical cardiopulmonary equipments were used in both groups, including stockert s3 heart lung machine (Sorin), affinity oxygenation system (Medtronic), and QUART arterial filter (Maquet). 4:1 cold blood cardioplegia was applied for both groups for myocardial protection.In addition, except for the implementation of IAD, identical blood conservation strategies, including small-volume RAP and strict blood transfusion standards, were followed in both time periods. The volume of the circuit was approximately 1200 mL, and it was not heparin-coated. The volume of the circuit was identical during the 6-year period. The retrograde autologous priming volume, approximately 200 to 500 mL, was empirically collected for each patient in both groups to determine the efficacy, safety, and blood-saving effect of this specific volume.,14 The triggers for intraoperative transfusion of packed red blood cells (PRBCs) included hemoglobin levels <6 g/dL or HCT level <18% and, in addition, at least one of the following during CPB: partial pressure of oxygen <60 mm Hg, lactate level >2.2 mmol/L, base deficit >3 mEq/L, and serum bicarbonate level <22 mEq/L. Triggers for postoperative PRBC transfusion included hemoglobin levels <7 g/dL or HCT level <21% and, in addition, at least one of the following: elevated oxygen requirement (fraction of inspired oxygen ≥60), persistent systemic hypotension (systolic blood pressure <90 mm Hg) despite the use of vasopressors, evidence of end-organ dysfunction, or evidence of persistent bleeding (>200 mL/h for at least 3 hours). Triggers for platelet transfusion included platelet count had reached 10,000 μ/L or less, platelet count had reached 50,000 μ/L or less in addition to bleeding >1.5 mL/kg/h for 2 consecutive hours, and uncontrolled profuse microvascular bleeding after surgery. Triggers for the transfusion of fresh frozen plasma included unmanageable hemorrhage after protamine neutralization, disseminate intravascular coagulation, preoperatively confirmed coagulation factor deficiency, emergency reversion of the oral anticoagulant effect, and duration of CPB more than 2 hours.Perioperative blood transfusions were performed using a validated transfusion protocol.2 test was used for descriptive, univariate statistics, while the t test was used for normally distributed data comparisons. The Mann–Whitney U test was used for group comparison of continuous non-Gaussian distributed variables. Two-tailed P values were derived from the calculated test statistics. A multivariate logistic regression model was performed to determine the risk factors for intra-/postoperative blood transfusions.Using SPSS software for windows (version 22.0), the data were analyzed and presented as means, numbers, or ratios as needed. Gaussian distributed data were reported as mean ± standard deviation. Non-Gaussian distributed data were reported as median (interquartile range). The null hypothesis set was that patient outcomes would not differ significantly. Continuous variables were expressed as means depending on overall variable distribution, and categorical variables were expressed as standard group percentages. Pearson χ3A total of 988 (60.2%) patients underwent cardiac valve replacement surgery from January 2008 to December 2010 compared with 754 (39.8%) during the 3-year period after the implementation of IAD (from 2011 to 2013). After exclusions, 919 (55.9%) patients entered our study as the non-IAD group, and 726 (44.1%) patients were included in the IAD group.Table P = 0.017).As can be seen in Table Table P < 0.001). Older age and DVR surgery were associated with an increased need for blood transfusions. In addition, patients with history of smoking were less likely to use allogenic blood products. Further, we compared HCT levels between smokers and non-smokers and found out that smokers had significantly higher HCT levels than nonsmokers . Our multivariate logistic model is based on baseline characteristics on admission; therefore, HCT levels were not included in the analysis of risk factors.According to the baseline characteristics of the 2 groups Table , a multiUsing the former multivariate logistic regression model, the following formula was devised to calculate the probability of intra-/postoperative PRBC transfusions for cardiac valve replacement patients: IAD: 1 for patients who underwent IAD and 0 for patients who did not; Age: years; Smoking: 1 for patients with a history of smoking and 0 for patients who did not; Surgery: Surgery1 = 1 with Surgery2 = 0 for MVR, Surgery1 = 0 with Surgery2 = 1 for AVR, and Surgery1 = 0 with Surgery2 = 0 for DVR patients.4 Traditionally, avoiding anemia can improve the prognosis of patients, but further evidence has proved that perioperative allogenic blood transfusions during cardiac surgery can be related to higher morbidity, mortality, and hospitalization costs.Patients who undergo cardiac surgery use nearly 20% of the total blood bank in the United States. Our method is not complicated, but requires the cooperation of surgeons, cardiologists, anesthetists, percussionists, intensivists, and nursing staff.We carried out autologous blood transfusions to achieve a maximum reduction of allogenic blood transfusion, in accordance with 2011 American College of Surgeons and The Society of Thoracic Surgeons guidelines.P < 0.001) and significantly fewer IAD patients received intra-/postoperative transfusions of blood products . Our result also showed that postoperative chest tube output favored the IAD group , similar results had been reported in literature. The specific mechanism of it has not been established; however, we assumed that there are several explanations. First, patients in non-IAD group had more intraoperative transfusion of banked blood, which might be associated with a poorer coagulation function. Also, in the IAD group, a certain amount of self-donating blood is siphoned into a blood storage bag before the initiation of systematic heparinization and transfused back after surgery, skipping the process of cardiopulmonary bypass. Therefore, to some extent, the platelet function of this amount of blood can be preserved. On the other hand, decreased transfusions are known to result in fewer respiratory complications, which was also confirmed in our study . However, no significant differences were found regarding myocardial infarction, pneumonia, or renal failure, which was consistent with other studies.Our results showed a significant decrease in allogenic blood transfusions in the group who underwent the autologous blood transfusion  and at the time of discharge (30.4 vs 31.5), such relatively low levels would not and did not affect the survival rate of patients.Unexpectedly, postoperative and at-discharge levels of HCT of the patients in the autologous blood transfusion group were lower than those of the non-IAD group. Hajjar et al,20 We compared HCT levels and found out that smokers had significantly higher HCT levels than nonsmokers. The combination of carbon monoxide in tobacco and the effects of nicotine disrupts oxygen delivery to the tissues and stimulates the bone marrow to produce more red blood cells and thereby increase HCT and hemoglobin. Thus, in this study, high HCT levels in smokers were associated with fewer PRBC transfusions.We found that patients with a history of smoking had fewer transfusions of allogenic PRBCs, intraoperatively and postoperatively, than those with no smoking history, and fewer blood transfusions in smokers after heart procedures have been reported in other studies. Balleisen et al believed that due to the high blood viscosity and slow blood flow in elderly patients, blood is hypercoagulable. The CPB process accelerates coagulation, entailing large amounts of blood clotting factors, which leads to a decrease in blood coagulation and thus an increase in the risk of bleeding.Our results indicated that older patients have higher risk of needing allogenic blood transfusions. Age has been reported to be one of the independent risk factors for perioperative bleeding in many studies.These results should be interpreted in the light of several limitations. As a retrospective analysis, the study design had some inherent disadvantages. The implementation of the IAD-based blood conservation strategy was introduced after the patients in the non-IAD group had been treated; thus, slight changes or improvements in clinical care or discharge criteria might have occurred over time and influenced such indices as duration of hospital stay and other factors. Also, although strict and identical intra-/postoperative transfusion standards were introduced in both periods, potential incompliance with the transfusion protocol might occur at the beginning of the implementation of IAD, thus might explain the difference of postoperative HCT between the 2 groups. Another limitation is that the recorded differences in patient outcomes could originate from smaller recorded or unrecorded differences between the 2 groups. Due to the disadvantage of the retrospective analysis, several characteristics and medical indices of the patients, including central blood volume, chest tube output in terms of different time periods, and others, were omitted. Therefore, a randomized clinical trial is needed for further study.In conclusion, our study showed that IAD-based blood conservation strategies can be effective and safe in cardiac valve replacement surgery. A certain degree of intraoperative and postoperative anemia would not affect the prognosis of the patients, and patients who adopted the autologous blood transfusion had better outcomes with regard to blood transfusion, postoperative chest tube output, and respiratory dysfunction. We therefore urge that more large-scale multicenter randomized clinical trials be carried out worldwide to prove the efficacy and safety of intraoperative autologous blood transfusions in cardiac surgery.The authors thank the anesthetists, the staff of the CPB division, and laboratory department in the First Affiliated Hospital of Zhejiang University for collection and managing the data presented in this report.


Tillering is an important agronomic trait for rice population quality and grain production. Nitrogen fertilizer application increased the number of rice tillers, but not every tiller contributes equally to the overall yield. Heterogeneity in tiller yield of rice increased with increasing nitrogen levels. Typically, late emerging tillers contributed less to the grain yield. Under high nitrogen conditions, most of the nutrients were retained in the straw of late emerging tillers; these tillers have high transport efficiency in vascular bundles, but there is less time for nutrients to transport into the grain. Despite the high light intensity, less than half of the light is used for photosynthesis in late emerging tillers. The low activity of enzymes related to grain filling in late emerging tillers is another reason for low yield. PSII) resulted in insufficient photosynthetic assimilate supply in late emerging tillers, whereas highly constitutive non-photochemical energy dissipation (ΦNO) might damage the photosynthetic system. Moreover, the low activity of SuSase and spikelets per panicle revealed both inadequate sink activities and storage sites. The identification of these limiting factors in late emerging rice tillers will assist in closing the ‘yield gap’ between late emerging tillers and early emerging tillers, and contribute to further increasing rice grain yields.The increasing food demands from an expanding population necessitate global efforts to increase crop production and ensure food security. The rate of nitrogen (N) fertilizer application is strongly related to crop yield. However, although the application of N fertilizer significantly increases the number of tillers in rice, late emerging tillers usually produce lower yields compared with early emerging tillers. Understanding the physiological constraints of late emerging rice tillers is critical for further increasing rice grain yields. Two-year field experiments, consisting of four nitrogen fertilizer levels, were conducted in order to study variations in the physiological characteristics of different types of tillers. The results revealed that the contributions of late emerging tillers to population rice grain yields improved with increased N levels. However, spikelets per panicle and the grain filling of late emerging tillers were significantly lower than that of the main stems or early emerging tillers under all N levels. The nitrogen harvest index of late emerging tillers was lower than that of main stems and early emerging tillers, and differences gradually increased under higher N rates. Nutrient source deficiency was a primary factor for the low productivity of late emerging tillers. Additionally, rapid malondialdehyde accumulation and delayed emergence determined the short growth duration of late emerging tillers. Further, low actual photochemistry efficacy (Φ Branching comprises a protective mechanism for higher plants, by assisting with reducing the impacts of injury, and facilitating adaptation to the environment . Moreoveet al. 2008), but no obvious decline in straw N concentration was discovered in late emerging tillers  according to the time of emergence of individual tillers under field conditions. The specific objectives were (i) to compare the yield performance of different types of tillers under contrasting N availability levels; and (ii) to determine the responses of physiological traits in different types of tillers to variable N applications.−1, 1.43/1.67 g kg−1, 14.2/13.3 mg kg−1 and 101.9/109.8 mg kg−1, respectively.Field experiments were conducted at two adjacent fields located in Wuxue County (30°11′ N 115°59′ E), Hubei Province, in Central China, during the 2014 and 2015 rice growing seasons. The soil type was hydromorphic paddy soil, which is a silty clay loam derived from quaternary yellow sediments. Prior to experimentation, soil samples extracted from the upper 20 cm layer were collected for chemical analyses. In 2014/2015, the soil pH, organic matter, total N content, available P and available K were 6.06/5.62, 23.4/29.4 g kgindica hybrid varieties ‘Fengyuanyou-299’ and ‘C-Liangyouhuazhan’ were grown in 2014 and 2015, respectively. Both of these rice varieties are widely grown in the Yangtze River area, China. Prior to formal study, a preliminary field experiment was conducted in 2013, where two varieties ‘Liangyou-287’ and ‘Fengyuanyou-299’ were planted to observe the phenomenon of heterogeneity in rice tillers. In the preliminary experiment, the number of tillers and grain yields of different tillers were quantified . Flooding was maintained in the field during transplantation until 10 days prior to crop maturity. Weeds, diseases, birds and insects were intensively controlled during the entire growing season to avoid yield losses in both years.0 (no N fertilizer application); (ii) N82.5 (82.5 kg N ha−1); (iii) N165 . Nitrogen (urea) was applied as 50 % at the basal stage, 25 % at the tillering stage  and 25 % at the panicle initiation stage (40 DAT). Phosphorus, in the form of calcium superphosphate (75 kg P2O5 ha−1), and zinc, in the form of zinc sulphate heptahydrate (5 kg Zn ha−1), were applied as basal dose. Potassium (potassium chloride) was applied at 75 kg K2O ha−1, 70 % at the basal stage and 30 % at the panicle initiation stage.The study was laid out in a randomized complete block design with three replications. Treatments comprised of four N application rates: (i) NFor the determination of tiller order, the main stems (M) of 15 randomly selected plants from each plot were labelled with small plastic tags, while other tillers were tagged according to the emerging tiller order. At the panicle mature stage, six hills were sampled, and the grain yield and yield components of individual stem/tillers were measured. The panicles were subsequently hand-threshed, and the filled spikelets, unfilled spikelets, and grain weight were quantified using a seed analysis instrument . Except for the main stems, tillers under the same treatments were divided into early emerging tillers  and late emerging tillers  based on their emergence time. In order to study, the physiological differences among different types of tillers during their growth duration, the first and second emerging productive tillers were regarded as early emerging tillers (E), while the last two emerging productive tillers were denoted as late emerging tillers (L).At the heading stage, four tagged tillers of different types in each treatment were selected and the culm was cut at 5 cm below the panicle neck. The first internode at the top of the stem was fixed with FAA liquid  for 24 h at 4 °C, and then stored in 15 % (v/v) hydrofluoric acid for 25 days (desiliconization). The paraffin section process was based on a method described in a previous study . The ana2SO4 and H2O2 (Bao 2000). The N content of the digested samples was determined using an automated continuous flow analyser . The grain N accumulating rate (G) was calculated using the equation:G is the grain N accumulating rate (mg d−1); Ni and Nt are the initial and terminal N accumulations during grain filling, respectively (mg), and T is the grain filling time (d).To determine the N accumulation rate of the grain, eight tagged panicles of different tiller types from each plot were sampled every 10 days. The grains of the sampled panicles were oven dried at 105 °C for 30 min and then at 75 °C until they were at a constant weight. The grains were then combined, ground to pass through a 1-mm mesh screen, and then digested by H−1 μm−2); G is the grain N accumulating rate (mg d−1); VB area is the summed area of large and small vascular bundles (μm2); Loadi is the load of any treatment; Loadmax is the maximum of load for all treatments.For a further comparison of N flow efficiency among different tillers, a new concept referred to as ‘load’ was utilized to represent the level of transport efficiency. We then normalized the value of load by dividing it by maximum load for all treatments. The normalized load was given as:The N transport efficiency, from source organ to sink organ, was expressed as the nitrogen harvest index .oF) was measured in a dark-acclimated state, followed by a saturating pulse in order to measure the maximum fluorescence yield (mF).Four tagged tillers of different types in each plot were selected, and the flag leaves were used to measure the fluorescence parameters with a portable photosynthesis apparatus that was coupled with a 6400–40 leaf chamber at the grain filling stage. In the morning (2:00–3:00 am), the initial fluorescence yield (oF), steady-state chlorophyll fluorescence (sF) and maximum fluorescence (In the late morning (9:00–11:00 am), the minimum chlorophyll fluorescence yield  as;Quantum efficiency of PSII photochemistry was estimated (Genty The fraction of still open PSII reaction centres (qL) was measured as;−2 s−1, and the CO2 concentration was set at 400 µmol mol−1 during measurements.Within the leaf chamber, the leaf temperature was maintained at 30 °C, the photosynthetic photon flux density was maintained at 1200 μmol mPSII), regulated thermal dissipation , a product of lipid peroxidation, following the method of The method for preparation of enzyme extracts was modified from Protein concentrations were determined via the method described by 10 transformed to meet the assumption of homogeneity of variance and normality. In cases where the ANOVA assumptions continued to be violated following data transformation, treatment differences were assessed using the more conservative Kruskal–Wallis nonparametric test. When variances of data were homogeneous, one-way ANOVA was used to determine differences among yield, yield components, number of VB, area of VB, photosynthetic parameters and activity of SuSase per tiller type only. For all analyses, the significance level was set at P < 0.05. Two-way ANOVA was used to test the significance of main effects (N level and tiller type) and their interaction with the above-mentioned parameters. The figures were plotted using the Origin 8.0 software program .Data were statistically analysed using SPSS for Windows . All data were previously tested for normality using the Shapiro-Wilk method and homoscedasticity using a Levene test. Where appropriate, the data were logTwo-way ANOVAs showed that the number of panicles per square meter, number of spikelets per panicle and yields per hectare were significantly affected by nitrogen level (N) and tiller type (T) in 2014 and 2015 . Also, tRice tillers were divided into early emerging tillers and late emerging tillers in accordance with their emergence times. Typically, the main stems possessed the highest number of spikelets per panicle, grain filling percentage and grain weight, followed by the early emerging tillers; the poorest yields were produced by the late emerging tillers under all N treatments . In comp0 treatment, no obvious differences between the yields of main stems and late emerging tillers were observed. However, the application of N significantly increased the number of late emerging tillers, which resulted in higher yields from the late emerging tillers than those from the main stems. The application of N decreased the yield contribution of the main stems, while increasing the yield contributions of the late emerging tillers. The highest yield contribution came from early emerging tillers  under all N fertilizer levels.It is recognized that fertilization with N significantly increases rice population yields. For the same types of tillers, grain yields were increased under higher N application in both years . Under N165 and N247.5) was higher than that under low N treatments (N0 and N82.5). Under identical N treatments, G was the highest in the main stem, followed by the early and late emerging tillers. Furthermore, a slow G was observed in the main stems and early emerging tillers under N247.5 treatment, which may have been due to excess N fertilizer application that acted to delay the vegetative growth stage.N accumulation in grains increased during the filling stage, and the curve shape for different types of tillers at the same N levels was similar . The appP = 0.002); however, the differences caused by N treatment were lower than those caused by the tiller type (P < 0.001). Similar to the number of vascular bundles, the area of LVB or SVB was the highest in the main stem, followed by the early emerging tillers, with the lowest value for the late emerging tillers  or SVB  and area of LVB or SVB were affected by N levels and tiller type . Neverth tillers . The add247.5 condition.To determine whether the flow between the source and sink was unrestricted and to compare N flow efficiencies between different rice tillers, the transport load was computed . The appPSII and ΦNPQ; however, their interactive effect was not statistically significant for any of the variables measured  was lower than that of other light energy (ΦPSII or ΦNPQ)  . The resPlant senescence, recognized as an increased concentration of MDA in the flag leaf, was low at the heading stage, and the highest at maturity in all types of tillers in both years . The MDASucrose synthase  in grains is the initial rate-limiting enzyme in the sucrose-to-starch pathway . The resThe application of N fertilizer may increase the number of productive tillers ; howeverPrevious studies have reached different conclusions on yield-related parameters , which either increased  in late emerging tillers . In the present study, the MDA concentration in different types of tillers gradually became similar toward maturity, and late emerging tillers showed a high senescence rate . Kariali tillers . A high pparatus . These rThe present study demonstrated that the number and contributions of late emerging tillers to rice grain yields were improved with increased N levels. However, the number of spikelets per panicle and grain filling of late emerging tillers were significantly lower than that of the main stem or early emerging tillers at all N levels. Cumulatively, the inappropriate light distribution in PSII and the premature aging of flag leaves led to insufficient resources for late emerging tillers. The low activity of SuSase and number of spikelets per panicle were the sources of inadequacies in grain sink activities and storage sites. In future, investigation of the physiological constraints associated with the low grain yield of late emerging rice tillers would likely be an effective approach to further enhance rice yields.This work was supported by the Special Fund for Agro-scientific Research in the Public Interest of China (grant no. 201503123), and the Fundamental Research Funds for the Central Universities (grant no. 2662015PY135).Y.W., J.L. and X.L. conceived the idea, Y.W., C.G., S.W., S.H. and R.C. conducted the experiment, Y.W., J.L. and T.R. analysed the results, Y.W., X.L. and S.H. wrote the paper.None declared.


The skin wounds were exposed to three daily plasma treatments for 1 or 2 minutes and were evaluated 3, 7 and 14 days after the wounding by histological and gene expression analysis. NTP treatment significantly enhanced epithelization and wound contraction on day 7 when compared to the untreated wounds. Macrophage infiltration into the wound area was not affected by the NTP treatment. Gene expression analysis did not indicate an increased inflammatory reaction or a disruption of the wound healing process; transient enhancement of inflammatory marker upregulation was found after NTP treatment on day 7. In summary, NTP treatment had improved the healing efficacy of acute skin wounds without noticeable side effects and concomitant activation of pro-inflammatory signalling. The obtained results highlight the favourability of plasma applications for wound therapy in clinics.Non-thermal plasma (NTP) has nonspecific antibacterial effects, and can be applied as an effective tool for the treatment of chronic wounds and other skin pathologies. In this study we analysed the effect of NTP on the healing of the full-thickness acute skin wound model in rats. We utilised a single jet NTP system generating atmospheric pressure air plasma, with ion volume density 5 · 10 Non-thermal plasma technology and its use in medicine (“plasma medicine”) has become a rapidly developing interdisciplinary field that brings a new innovative approach in a wide range of biomedical applications. Primarily due to its bactericidal properties, non-thermal plasma (NTP) represents an effective tool for various procedures in human as well as in veterinary medicine, particularly in tissue disinfection and treatment of chronic wounds, such as diabetic foot ulcers, pressure and venous leg ulcers, burns and other skin pathologies with microbial etiology133), reactive nitrogen species (RNS)6NTPs are generated from a flow of neutral gas in a locally high-strength electric field, while the gas remains at atmospheric pressure and near ambient temperature. Generally, NTP composition is very complex and includes excited particles, such as electrons, ions, reactive oxygen species . On day 7, significant upregulation in expression of Nos2 was found after 1, as well as 2 min of plasma treatment (p < 0.05), while upregulation of Ptgs2 was found after only 2 min of plasma treatment (p < 0.05). Conversely, expressions of KbNF and Sod1 significantly decreased after both 1 and 2 min of plasma treatment when compared to the control wounds (p < 0.005). Inflammatory markers Il-6, Ptgs2 as well as Nos2 declined on day 14, while upregulation persisted for the other observed genes, and no significant changes were found between control and plasma treated wounds.Importantly, no significant changes between control and plasma treated wounds were detected on day 3, except of a more profound decrease in expression of Wound healing comprises a cascade of multiple biological and biochemical processes, which consist of a series of phases involving blood clotting, inflammation, cell proliferation, migration and differentiation, leading to the formation of granulation tissue, wound contraction, and final tissue restoration and remodelling. The key roles in these processes are played by growth factors, cytokines and inflammatory mediators released at the wound siteChronic non-healing wounds with a persistence of various bacteria represent an increasing health problem worldwide associated with high mortality, morbidity, and economic expenses. This emphasises the need for the development of new concepts and strategies for the elimination of bacterial load, and an improvement of chronic wound care. Based on its non-specific antimicrobial effects, NTPs appear to be a promising biomedical tool for effectively eliminating wound contamination and restarting the normal wound healing process. Indeed, several clinical trials have already approved the potential of NTP in the elimination of bacterial load, as well as for improved chronic wound healing20212) and reactive oxygen species such as ozone (O3), superoxide (O2−) and hydroxyl radicals (∙OH). In contrast to air plasma, the composition of the argon plasma significantly differs, and only low amounts of reactive oxygen and nitrogen species are produced, by mixing the argon plasma with the ambient air.In the present study, we evaluated the efficacy of our NTP system in the healing of acute skin wounds in rat models. While most of the already published studies on wound healing were carried out using argon plasma1617in vitro on cell cultures. While several reports demonstrated that argon NTP promotes cell migration and proliferation, NTP generated from air induced cell death, due to the apoptosis or necroptosis related mechanisms26in vitro, the enhancing plasma effect on wound healing was demonstrated in both argon1617The distinct effects of argon and air plasma are apparent from the experiments Initially, we observed wound contraction, which is a primary healing mechanism in rodents. The wound area was significantly more reduced in the rats treated with NTP than in that of the untreated rats on day 7, and also histological analysis confirmed an enhanced epithelisation and a reduced granulation tissue area after plasma treatment. Respectively, improved wound contraction after the plasma treatment has also been confirmed in other studies using NTP generated from argon, helium or nitrogen16789101112131415161718According to histological evaluation, reduced collagen deposition in the wound area in the early inflammatory phase of healing was lower after plasma treatment, than that in the control group. This might indicate that plasma treatment slows down the collagen remodelling in the early phase of the healing process. However, in accordance to the wound healing progression and tissue remodelling in the later phases of the healing, collagen deposition gradually increased in all groups without any significant differences, and the skin layers were nearly fully remodelled on day 14 without any signs of abnormal scarring.Il-6, Nos2 and Ptgs2 during the inflammatory phase of the healing (day 3) in both control as well as plasma treated wounds, without significant differences. However, while the level of expression of these factors declined in control wounds on day 7, an upregulation of Nos2, Il-6 and Ptgs2 persisted after 2 min of plasma treatment.The inflammatory reaction, which was investigated by analysis of macrophage infiltration in the wound area, was not affected by plasma treatment. Similarly, mRNA analysis revealed an upregulation of inflammatory markers et al., who used daily, a 2 min argon NTP treatment of skin wounds in mice, and found an increase expression of Il-6, and monocyte chemoattractant protein-1 five days after woundinget al. showed that argon plasma treatment promoted acute inflammationOur results are in line with the findings of the study by Arndt In particular, inducible nitric oxide synthase  is an enzyme responsible for the endogenous NO release, which is a highly diffusible intercellular signaling molecule that has been shown to regulate many processes in human skin physiology. The beneficial effects of NO on wound repair may be attributed to its functional influences on angiogenesis, inflammation, cell proliferation, matrix deposition, and remodellingAs air plasma treatment is capable of increasing NO accumulation in the living tissue16Mmp2 and Mmp14) or collagen (Col1a2), angiogenesis (Vefga), cell proliferation (Mki67), growth factors  or macrophage polarization . This suggests that plasma exposure with the doses which are effective to kill bacteria has no harmful effects on a wound healing scenario nor will it promote excessive scar formation.Notably, no significant changes between control and plasma treated wounds were found for the expression of genes related to wound remodelling, such as matrix metalloproteinase , and the Gram-positive bacteria (Staphylococcus aureus). The bactericidal efficacy depended on the treatment time, and reached a 99.999% reduction of Gram-positive and a 99.9999% reduction of Gram-negative bacteria after 60 s of plasma exposure .All experiments were performed in accordance with the European Communities Council Directive of 22nd September 2010 (2010/63/EU) regarding the use of animals in research and were approved by the Ethics Committee of the Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, in Prague.Forty one male Wistar rats  with body weight in the range of 280–320 g were used in the study.One day before wounding, the animals were placed under isoflurane anesthesia , and the hair on their backs was removed using an electric shaver and depilatory cream . The following day, two full-thickness excisions with 8 mm in diameter were created by punch biopsy  on the rats’ upper back midline, under sterile conditions. The plasma treated wounds were exposed for 1 min (right side) and 2 min (left side) to NTP with a distance of 1 cm from the plasma jet. To reduce pain, caprofen  was injected subcutaneously near the wound area immediately after wounding.The wounds were then covered with Vaseline Gauzes dressing , gauze Mesalt  and fixed with Tegaderm™  and a self-adhesive elastic bandage , which was enfolded around the animal body, with trimmed holes for the forelimbs. The wounds were exposed to additional plasma treatments on days 1 and 2 after wound generation, with a total of three daily plasma treatments during the experiment. The control animals underwent the same procedure as plasma treated animals, only without exposure of the wound to plasma. The animals were euthanized on days 3, 7 and 14 after the wounding, and skin samples including the wound region were harvested for further assessments.c (control wound) = 18 and np (plasma treated wound) = 23, on days 7 , and on days 10 and 14 . The surface area of the wound was determined by tracking the wound margins and calculating the wound area using ImageJ software . The percentage of wound area was calculated as  ×100.Digital photographs of the wounds were taken using a digital camera  at days 0–3, while nThe skin samples were fixed in 4% paraformaldehyde  and mounted into paraffin. A series of 5 μm thick paraffin sections were stained with hematoxylin/eosin (H&E) and Masson’s trichrome stain  for histological analysis. To identify macrophages, a mouse monoclonal antibody ED-1directed against CD68  and AlexaFluor 594-conjugated goat anti-mouse IgG  secondary antibody were used. The nuclei were visualised using 40, 6-diamidino-2-phenylindole (DAPI) fluorescent dye . Images were acquired by a laser scanning confocal microscope  and Leica microscope .Quantitative image analysis for collagen deposition was performed on sections stained with Masson’s trichrome stain. Three randomly selected sections from each animal (n = 5) were scanned on the Leica microscope using a 20x objective and used for the analysis. Three photos from each slide were taken in the centre, and the edges of the wound and the relative area of collagen was determined using HistoQuest Analysing Software .Skin samples stained with Masson’s trichrome were also used for histological assessment of the wound remodelling. The extent of epithelialisation was determined by measuring the linear epithelial gap, characterised as the length of a line segment between the margins of the ulcer. The thickness of the granulation tissue was measured at the wound centre. The wound area was determined as an area of the granulation tissue between the margins of the ulcer.The number of macrophages was quantified as a fraction (%) of CD68 positive cells in relation to the total cell number of cells determined by DAPI staining in the analysed area. Three randomly selected sections from each animal (n = 5) were scanned on the Leica microscope using a 20x objective and used for the analysis. Three photos from each slide were taken in the centre, and the edges of the wound and analysed using HistoQuest Analysing Software .kbNF, Tgfb1, Ccl3, Ccl5, Cd86, Cd163Il-2, Il-6, Il-10, Nos2, Ptgs2, ), oxidative stress , matrix metalloproteinases , tissue remodelling (Col1a2), growth factors , angiogenesis (Vegfa), proliferation (Mki67), and apoptosis (Bax) were studied using quantitative real-time reverse transcription polymerase chain reaction (qPCR) at 3, 7 or 14 days after the wounding . RNA was isolated from tissue sections using the RNeasy Fibrous Tissue Mini Kit , following the manufacturer’s recommendations. RNA amounts were quantified using a spectrophotometer . The isolated RNA was reverse transcribed into cDNA using Transcriptor Universal cDNA Master (Roche) and a thermal cycler . The qPCR reactions were performed using cDNA solution, FastStart Universal Probe Master  and TagMan® Gene Expression Assays . All amplifications were run under the same cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. All samples were run in duplicate, and a negative control was included in each array. Relative quantification of gene expression was determined using the ΔΔCt method. Results were analyzed with StepOnePlusAll data were expressed as mean ± standard error mean (SEM). The statistical significance of differences between the treated groups and the control group was analyzed using one-way ANOVA with Holm-Sidak multiple parameter test  with a level of p < 0.05 considered statistically significant.How to cite this article: Kubinova, S. et al. Non-thermal air plasma promotes the healing of acute skin wounds in rats. Sci. Rep.7, 45183; doi: 10.1038/srep45183 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This fMRI study was designed to investigate the effect of cross-modal conditioning in 28 female volunteers. Subjects underwent initial fMRI block design scanning during which three pleasant olfactory stimuli were presented and had to be rated with respect to intensity and pleasantness. This was followed by an odor identification task spread out over 3 days: the experimental group was rewarded for successful trials (correct odor identification) with emotionally salient photos, whilst the control group only received randomly displayed, emotionally neutral, pictures. In the final scanning session, the odors were again presented, and subjects rated pleasantness and intensity. Both pleasantness ratings and fMRI data showed effects of the rewarding procedure. Activation in nucleus accumbens and the orbitofrontal cortex confirmed the hypothesis that learnt association of odors with visual stimuli of emotionally positive valence not only increases pleasantness of the olfactory stimuli but is also reflected in the activation of brain structures relevant for hedonic and reward processing. To our knowledge, this is the first paper to report successful cross-modal conditioning of olfactory stimuli with visual clues. Coriandrum sativum) with its characteristic smell of the green shield bug  but a specific taste highly appreciated by connoisseurs. However, it appears to be easier to turn a pleasant odor into an unpleasant one than vice versa, probably due to the higher significance of an unpleasant experience , VDFLO117 with a floral scent (“FLO”), and DGFRUI067K with a fruity odor (“FRU”). In Table Subjects were required to evaluate both intensity and pleasantness of olfactory stimuli during fMRI procedures. They were familiarized with two rating scales in a preliminary session after passing inclusion procedures. The intensity scale ran from zero (“not noticeable”) to 10 (“very strong”), whereas the hedonic scale ranged from –5 (“very unpleasant”) to 5 (“very pleasant”), with zero representing “neutral.”t-tests with p < 0.05.Psychophysical data were analyzed using the SPSS software package , applying ANOVAs for repeated measurements, and subsequent Bonferroni adjusted In the experimental group (XG), 12 baby photographs were used as conditioning visual stimuli with positive emotional connotation.In the control group (CG), 12 photographs showing neutral items of everyday life, with no emotional association, were used as visual stimuli.Subjects were pseudo-randomly assigned to two conditions, thus creating two groups of equal sizes:The study was subdivided into three parts:Basic fMRI scans.Training/conditioning sessions: three sessions on separate days performed within 6 days.Final fMRI scans, 1 week after basic scans.Prior to the basic scanning procedure, subjects were successively shown all 12 pictures pertaining to their respective groups on a computer screen. During scanning, the three odors were presented in separate runs, with sequences randomized across subjects. According to the general scheme of a block design Figure . Pulses Subjects participated in three training sessions, and in the case of the experimental group, they were designed as conditioning sessions. All participants performed a computer based memory task requiring them to discriminate the three test odors from other odors not previously introduced. Odors were presented using brown glass bottles of 100 ml volume, containing 4 ml of each odor, from which subjects were asked to take sniffs. In XG, whenever a correct response occurred, subjects were rewarded by a commendation displayed on the screen (“this answer was correct”) which was followed by the display of a baby photograph. In case of failure, the item was repeated without reward, until successful identification of the target odor. Subjects had to complete 12 successful trials, thus being rewarded by the entire set of baby photographs. In CG, no rewarding displays occurred. The pictures showing neutral objects were interspersed at random during the memory task.During the final scanning procedure the three odors were again presented in separate block designed runs in a randomized fashion across subjects.As in the basic scan, stimuli had to be rated with respect to intensity and pleasantness. A sketch of a single fMRI session is shown in Figure TE) = 35 ms, repetition time (TR) = 2,500 ms, flip angle = 90°, and 1 average.Scanning was performed with a 1.5 T Sonata Scanner . For functional imaging, a spin echo/echo planar imaging sequence  was applied using software version syngo MR 2002B 4VA21A, with echo time  sequence at a resolution of 1 × 1 × 1 mm; this data set was used as reference images in fMRI analysis to account for the poor resolution of functional scans (3 × 3 × 3.7 mm).Analysis of fMRI data was performed with the SPM8 software package , “repetition” (2 levels), and “odor” (3 levels). Data were evaluated both in a pooled fashion, with all three odorants combined, and for the stimulants separately. Differences were tested between parts III and I (post- vs. pre-training/conditioning measurements) within both groups , and between parts III of group XG vs. CG (XG-III vs. CG-III). Standard thresholds were set to puncorrected < 0.005 and 10 voxels as a minimum cluster size.The WFU pickatlas  was used to identify activated brain regions. Regions of interest (ROI) were also established using the pickatlas. ROI_O included primary and secondary areas of olfactory processing  = 34.0], VAN was significantly weaker than the other two odors. FLO received the highest scores, and was significantly stronger than VAN and FRU. However, in terms of odor intensity at baseline, there was no significant difference between groups .Intensity ratings revealed significant differences among odors: In the analysis of ratings at baseline from the entire sample .In both the experimental and control groups, differences in odor intensity did not change after association of the odors with baby photos or emotionally neutral pictures respectively ; in addition, no significant difference was found between the three odors .Most individual pleasantness ratings were above zero, although none of the three odors was unanimously rated as pleasant. At baseline, there was no significant difference between the two groups in terms of odor pleasantness . In contrast, for CG, although olfactory stimuli were rated more pleasant in part III than prior to training, these increases were not significant . The findings for both groups are demonstrated in Figure r = 0.73; p < 0.05).A significant effect of the conditioning procedure could be established in the experimental group: pleasantness ratings of all three odors were found to be significantly increased after as compared to before the conditioning session [F1, 1 = 5.50; Both intensity and pleasantness ratings are listed in Table p < 0.01 prior to ROI analysis.Exposure to FLO, FRU, and VAN odors induced activation in brain regions associated with olfactory processing. In Figure With respect to the effect of conditioning with emotionally positive photos, pooled analysis of all three odors was first performed. Comparisons were calculated between groups for part III, and between parts III and I for the experimental group. Neither analysis with ROI_P, to check for activations in regions involved in pleasantness processing, nor with ROI_R, testing reward relevant activation, yielded significant results in the pooled approach.Selective analyses with the three separate odors were then performed in the same fashion Table . Using tThe current study revealed that the pleasantness of odors increased when their correct recognition was associated with photos of happy babies whereas no such effect occurred when the same odors were presented in a random fashion with emotionally neutral pictures.Several fMRI papers have shown effects of odors on various visual variables. Gottfried and coworkers , or in part III, and none between parts I and III for either group. This supports the idea that the significant increase of pleasantness ratings found in the experimental group in part III is independent of intensity and specifically reflected to the hedonic aspect. Moreover this also applies to the imaging results where significant differences were observed when contrasting XG vs. CG in part III, and parts III vs. I for the experimental group. Apparently the rewarding experience of the conditioning sessions brought about increased pleasantness ratings for the olfactory stimuli and associated activation in OFC and nucleus accumbens. Thus the results support the hypothesis that visual cues with a positive emotional connotation enhance the basic agreeableness of fragrances and induce activation of cortical networks related to hedonics and reward  about the experiment.TH, JW, CH, DB, and TF developed the idea and the design of the study; TF and DB ran the experiments; CH, TF, DB, and TH were involved in data analysis; TH, CH, TF, DB, JW, and VS contributed to the writeup.This research was supported by Takasago Inc., Paris, France.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VB and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Behçet's disease (BD) is a multisystemic disorder of unknown etiology mainly defined by recurrent oral aphthosis, genital ulcers, and chronic relapsing bilateral uveitis, all of which represent the “stigmata” of disease. However, many other organs including the vascular, neurological, musculoskeletal, and gastrointestinal systems can be affected. The gastrointestinal involvement in Behçet's disease (GIBD), along with the neurological and vascular ones, represents the most feared clinical manifestation of BD and shares many symptoms with inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. Consequently, the differential diagnosis is often a daunting task, albeit the presence of typical endoscopic and pathologic findings may be a valuable aid to the exact diagnosis. To date, there are no standardized medical treatments for GIBD; therefore therapy should be tailored to the single patient and based on the severity of the clinical features and their complications. This work provides a digest of all current experience and evidence about pharmacological agents suggested by the medical literature as having a potential role for managing the dreadful features of GIBD. Streptococcus sanguinis have gained increasing importance as potential infectious agents of BD [α partakes probably in somehow the disease onset and the successful use of anti-TNF-α agents has substantiated the role of this cytokine in BD [Behçet's disease (BD) is a rare relapsing systemic inflammatory disorder of unknown etiology characterized by recurrent oral ulcers, genital sores, and ocular lesions; however many other organs including the vascular, neurological, and musculoskeletal systems as well as the gastrointestinal system can be involved –3. Genetts of BD , being ats of BD . Severalts of BD , 7–9: tune in BD –12. Convne in BD . Recent ne in BD –17.Although oral aphthae and genital ulcers are the earliest and the most frequent manifestations of BD, anticipating by many years other typical BD clinical symptoms, GIBD is one of the major causes of morbidity and mortality, often leading to severe complications. GIBD occurs in 3–60% of patients, on average 4.5–6 years after the onset of oral ulcerations , varyingGastrointestinal lesions are typically irregular, round or oval, punched-out, large (>1 cm), single to a few in number, deep, and with discrete margins in a focal distribution . On the Although a wide number of conventional immunosuppressive drugs have been used to induce remission in GIBD, several failures have been reported. This article reviews the progress in the management of GIBD focusing on current treatment strategies and possible future perspectives. An electronic literature search was conducted using the PubMed database and the clinicaltrials.gov search engine. We looked for all studies published in the last years, including case reports, clinical trials, and cohort studies .α antagonists and thalidomide (THD) have proven useful in resistant and complicated cases [As underlined in the guidelines of the European League Against Rheumatism (EULAR) for the management of BD, evidence-based recommendations regarding GIBD are not provided due to the poor amount of published clinical trials . Medicaled cases , 40, 41.Nevertheless GIBD management is still largely empirical due to the lack of not yet standardized medical treatments, the heterogeneity of this disorder, and the unpredictable exacerbations of BD. To date, several conventional immunosuppressive drugs may be employed, although none of them has been proven actually effective in preventing disease relapse.CC are the first-line therapy, especially in patients with severe systemic symptoms, recurrent gastrointestinal bleeding, or when treatment with 5-aminosalicylic acid (5-ASA)/SSZ is not enough . CC are 5-ASA/SSZ is indicated in all cases of GIBD due to its safety profile and current limited therapeutic options . It is uα because of degradation of its encoding mRNA [THD is an immunomodulatory drug, used mainly in the treatment of specific tumors. It is usually considered the last-line therapy for GIBD, albeit its use is well-documented . The imming mRNA . A piloting mRNA  and threing mRNA –57 have ing mRNA .α antagonists and/or THD; a clinical and endoscopic remission was obtained in 10 out of 13 patients (about 75% of the cases) proving the favourable response with anti-TNF-α agents and/or THD in GIBD [Yasui et al. reported the benefits of THD in 7 juvenile‐onset patients with severe, recurrent GIBD who had previously failed immunosuppressant treatments and developed significant CC toxicity. THD was given at the initial dose of 2 mg/kg per day and was increased to 3 mg/kg per day if necessary. All patients showed dramatic improvement in clinical symptoms, and CC were successfully withdrawn . Yet, th in GIBD .Despite the efficacy and safety profile reported in several bodies of evidence of the literature , 62, 63,Thiopurines including 6-mercaptopurine (6-MP) and its prodrug AZA have been traditionally thought to decrease reoperation rate in patients with GIBD who already had undergone surgical interventions . The iniYet, a recent retrospective observational study was carried out to assess the efficacy of postoperative thiopurine therapy in 77 patients with GIBD; although a lower postoperative recurrence was found in patients who received thiopurines than those taking 5-ASA, the rates of reoperation, readmission, and death were not significantly different between the 5-ASA and thiopurine groups .Evidence concerning the efficacy of methotrexate combined with infliximab (IFX) in refractory entero-BD has been also reported in 9 out of 10 patients who experienced the disappearance of ileocecal ulcerations at 12 months of therapy . In addiα could represent a promising treatment option for neurologic and gastrointestinal involvement in BD [α [α also derive from the study by Monastirli et al. [Interferon (IFN) is a cytokine able to render cells resistant to infection by many viruses. It was introduced for the treatment of BD by Tsambaos in 1986, because of its antiviral and antiproliferative properties . The mosnt in BD . In thisin BD [α , and simi et al. .During the last few decades intravenous immunoglobulins (IVIg) have been increasingly administered for a wide number of autoimmune and systemic inflammatory diseases. To the best of our knowledge, IVIg have so far been evaluated in few patients with GIBD. In this regard, the efficacy of IVIg has been reported in a patient with BD-related colitis who initially had failed under CC and immunosuppressive therapy , as wellα in the pathogenesis of BD; a remarkable upregulation of TNF-α and soluble TNF receptors [γδ+ T cells producing TNF were found in the peripheral blood of patients with active disease [α IFX [Several data suggest a role of TNF-eceptors  as well  disease . Currente [α IFX  and adale [α IFX  along wie [α IFX  have beee [α IFX . A clinie [α IFX . On the e [α IFX .Yet, a Korean multicenter retrospective study aimed at investigating the response to IFX in 28 patients with GIBD showed a clinical response rate of 64.3% with a clinical remission rate of 28.6% at week 4, following IFX infusion. Furthermore, an older age at diagnosis (≥40 years), the female sex, a longer disease duration (≥5 years) as well as the concomitant immunomodulator use, and achievement of remission at week 4 were regarded as predictive factors of sustained response .The efficacy of IFX in GIBD has also been corroborated by a retrospective cohort study on 15 patients with active disease refractory to conventional medications. 80% of patients exhibited a good response to IFX and 53% of them achieved remission after 10 weeks. Moreover 64% and 50% of patients maintained the response to IFX at 12 and 24 months, respectively . More reNCT02505568).Finally, an interventional open-label single-arm study testing the efficacy of IFX by assessing the mean decrease in DAIBD score in patients with active intestinal disease refractory to conventional therapies is currently recruiting participants  testing the safety and efficacy of ADA for the treatment of GIBD are now ongoing.However, the most consistent evidence regarding ADA efficacy derives from a phase 3, multicenter, open-label uncontrolled study evaluating Japanese patients with active intestinal BD nonresponsive to standard therapies. Twenty patients received induction treatment with ADA ; a composite index, combining GI symptoms and endoscopic assessments, was used to evaluate the efficacy of treatment. A marked improvement, defined as values ≤1 for both the global GI symptoms and endoscopic assessment scores, was seen in 60% of patients at week 52. Interestingly 20% of patients achieved a complete remission, defined as global GI symptoms and endoscopic scores of 0, at weeks 24 and 52, suggesting that ADA was an effective therapy to induce and maintain clinical improvement and remission in patients with GIBD . More reLess experience has been gained focusing on the management of GIBD with ETA treatment . RecentlAlthough the employment of anti-IL-1 agents on various BD manifestations has been well-documented –97, limiHaematopoietic stem cell transplantation (HSCT) has been used in the treatment of severe autoimmune and inflammatory conditions unresponsive to conventional therapy. Although several treatment options, including biologic agents, are till now available for BD management, there is still an unmet need for more effective therapies for patients who are refractory to conventional treatments. In this regard encouraging results derive from several case reports describing HSCT in GIBD patients transplanted for accompanying haematological conditions. A complete remission of GI findings was observed after HSCT and there was no need to treat patients with any medications , 104. ThSurgery is indicated when patients with GIBD are refractory to medical treatment or serious complications, such as when gastrointestinal bleeding, perforation, fistulae, obstructions, and abdominal masses occur . Bowel pα inhibitors, which represent new tools in the therapeutic armamentarium for managing patients with GIBD, particularly those who are resistant to conventional immunosuppressant drugs. On this basis the main goal of treatment should be aimed at avoiding and preventing the feared complications of GIBD that endanger the life of these patients.BD is a complex syndrome characterized by significant heterogeneity of clinical manifestations with usually frequent relapses. GIBD is one of the most severe manifestations of this disease, causing serious complications such as perforation and gastrointestinal bleeding. GIBD shares many clinical features with inflammatory bowel diseases, and for this reason it represents a pitfall for physicians regarding differential diagnosis at the first presentation. Nevertheless, a careful evaluation of endoscopic findings may help in the diagnostic interpretation, whereas the endoscopic biopsy is necessary to confirm a histopathologic diagnosis. Treatments have been largely unsatisfactory, creating significant unmet needs, and the lack of evidence-based treatment makes the management of GIBD very challenging. Currently, several immunosuppressive drugs such as SSZ, CC, and AZA are generally well-tolerated but often associated with increased risk of disease relapses as a result of which surgery is required in many patients. In the last years, new pathogenetic hypotheses supported the use of biotechnological drugs, mostly TNF-


Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments , while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out. Optimization of the MPS workflow is important for both economic and timewise reasons7. Maximizing the number of samples that can be sequenced in one experiment is one obvious way of reducing the costs. It requires accurate quantification of each sequencing library and pooling of the libraries in equal molar ratios. If the quantifications underestimate the number of molecules in the libraries, the sequencing experiments may fail completely because the number of molecules per bead or flowcell exceeds the capacity of the sequencing platform. If the library concentrations are overestimated, the sequencing capacity will not be used in an optimal way. If the quantification of some libraries is underestimated and others are overestimated, the sequencing depth (coverage) of the samples will vary. Some samples may have high coverage with many redundant sequencing reads and loss of sequencing capacity as a result. Some samples may have low coverage and sequencing of these samples may have to be repeated because the results of the sequencing analysis are unreliable.Massively parallel sequencing (MPS) is widely used in biological research10. The target sequences are generated by fragmentation, capture protocols, or PCR amplification14, and the adapters are ligated to the fragments. Ideally, the quantification should differentiate between molecules with target sequences and other molecules, e.g. adapter and primer dimers.Each library consists of the target sequences flanked by adapter sequences needed for the downstream reactions  and subsequent data analysis 17. Finally, various quantitative PCR (qPCR) assays have been developed for MPS library quantification18. qPCR is highly sensitive and capable of quantifying specific molecules, e.g. molecules with the required adapters in the 5′ and 3′ends of the fragments.Quantification methods vary in quantitative range, sensitivity, costs, workflow simplicity, etc. Spectrophotometry can be applied for library quantification, either by measuring light penetrance as in e.g. the NanoDrop instrument , or by measuring the fluorescence as in e.g. the Qubit instrument (Thermo Fisher Scientific). Electrophoresis displays the size distribution of the DNA fragments in the library, which makes it possible to distinguish between molecules with target sequences and molecules only comprising adapter or primer dimersIn this study, eight different methods for MPS library quantification were used to quantify PCR amplicon, capture, and shotgun fragment libraries along with chemically synthesized double-stranded DNA (dsDNA) oligos. The concentrations, fragment lengths, adapter dimer abundance, and PCR inhibitor content were investigated.12. DNA was extracted from the crime case samples using Chelex 100  , EZ1 DNA Investigator , QIAquick (Qiagen) in combination with Amicon Ultra  (DNA from a bone), or phenol/chloroform (DNA from a jaw bone)19. The DNA extracted from crime case samples was archived with low TE buffer , and stored at −20 °C for 2–15 years before this study. The work was approved by the Danish ethical committee (H-4-2011-081). Samples were taken from the biobank of the Department of Forensic Medicine, University of Copenhagen . The Danish ethical committee waived the requirement for informed consent (H-4-2011-081). The DNA extracts from the crime case samples were investigated in agreement with Danish criminal law.Blood samples from 41 individuals, buccal swabs on Flinders Technology Associates cards (FTA cards) from 15 individuals, and six crime case samples from forensic case work were used  (Thermo Fisher Scientific). DNA libraries were constructed using the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) according to the manufacturer’s recommendations. The libraries were grouped as either “well amplified”, “adapter dimer rich”, or “PCR inhibited” based on analysis with the 2100 Bioanalyzer using the High Sensitivity DNA Assay   with peak power 175 W, duty factor 10%, 200 cycles per burst, and 120 sec treatment time. DNA libraries were constructed using the Ion Xpress Plus Fragment Kit (Thermo Fisher Scientific) following the manufacturer’s protocol.11.DNA extracted from blood samples from six individuals was processed into NimbleGen  capture libraries as previously describedDNA extracted from blood samples from 35 individuals was amplified using the Precision ID Ancestry panel  (Thermo Fisher Scientific). DNA libraries were constructed using the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific) following the manufacturer’s protocol with one exception: the number of PCR cycles was increased from 21 to 25 cycles in order to obtain sufficient amounts of PCR products.Two synthetic dsDNA oligos were designed . The Ion Torrent oligo was identical to the “A” and “P1” adapter sequences , the High Sensitivity Assay with the Qubit 2.0 (Thermo Fisher Scientific), the High Sensitivity Assay with the 2100 Bioanalyzer (Agilent Technologies), the High Sensitivity D1000 ScreenTape Assay with the 2200 TapeStation (Agilent Technologies), the DNA High Sensitivity Assay with the GX Touch (PerkinElmer), and the High Sensitivity NGS Fragment Analysis Kit with the Fragment Analyzer  following the manufacturers’ protocols . The GeneRead Library Quant Kit (Qiagen) or the Ion Library Quantitation Kit (IonLibQuant) (Thermo Fisher Scientific) were used following the recommendations of the manufacturers. qPCR molar concentrations were converted into concentrations in pg/µL using fragment lengths of 203 and 280 bp for Precision ID Identity Panel and Ion Xpress fragment libraries, respectively.20 with Bonferroni correction were performed using the “friedman.test.with.post.hoc” application in R 3.3.0 to test for significant differences in concentration estimates among the quantification methods. Due to the differences in library concentrations, the data were not expected to be normally distributed. Two tests were done: one including all the libraries (n = 30) and excluding the qPCR quantifications, and one excluding the NimbleGen libraries (n = 24), but including quantifications with all methods. P values for comparisons among quantifications with NanoDrop, Qubit, Bioanalyzer, GX Touch, TapeStation, and Fragment Analyzer were obtained from the first test with a level of significance at α = 0.0033 after Bonferroni correction. P values for comparisons to the real time-PCR quantifications were obtained from the second test with α = 0.0018 after Bonferroni correction.All quantifications were performed in duplicates. The averages were used for evaluation of the quantification methods. Two-sided Friedman’s tests21.Three Precision ID Ancestry Panel libraries were diluted 2×, 4×, 8×, 16×, 32×, and 64×. Each dilution was quantified three times with the TapeStation and the ABI7500 instruments using the IonLibQuant assay. The means of each of the triplicate measurements were used for further analysis. A fragment length of 207 bp was used to convert molar concentrations into weight concentrations. The relationship between the TapeStation and the ABI7500 concentration estimates were tested using linear regression analyses with the “lm” application in R 3.3.0 and the square of the Pierson correlation coefficient. For each library, the limit of quantification (LOQ) was determined as 10 times the standard deviation of the regression line’s ordinate intercept divided by the slope of the regression line as previously describedThirty-five Precision ID Ancestry Panel libraries were sequenced in pools containing 18 libraries (one library was not used in this study), which had been pooled in equimolar concentrations according to Qubit, TapeStation, or ABI7500 (IonLibQuant assay) quantifications. Each pool had a total library concentration of 50 pM. Template preparation consisting of emulsion PCR, enrichment of beads containing template, and chip loading was performed with the Ion Chef instrument and the Ion PGM Hi-Q Chef Kit according to the manual (Thermo Fisher Scientific). The loaded sequencing chips were placed onto the Ion PGM™ instrument (Thermo Fisher Scientific) together with Ion PGM Hi-Q Chef 400 Supplies Kit (Thermo Fisher Scientific) and sequenced for 500 cycles according to the manual. Sequence analysis was done using the Torrent Suite Software v.4.4.2 with the HID_SNP_GenoTyper v. 4.2 plugin. Linear regression and two-sided F test were performed using the “lm” application in R 3.3.0 to test for correlations between concentration estimates and library coverage using data from all 35 libraries and α = 0.05.All data generated or analysed during this study are included in the article and the supplementary information.−4, p = 1.11*10−16, p = 2.65*10−13, and p = 1.43*10−14 for comparison to Qubit, Bioanalyzer, GX Touch, and TapeStation, respectively), except for the comparison between the NanoDrop and the Fragment Analyzer (p = 0.128). The majority of the tested libraries had concentrations below the quantitative range of the NanoDrop . Similar results were obtained with the ABI7500 qPCR instrument with the GeneRead and the IonLibQuant kit. The ABI7500 quantification results were lower than those obtained with the other methods, and they were statistically significantly different from the estimates of the NanoDrop, the Qubit, and the Fragment Analyzer after correction for multiple testing . The variation in the relative concentration estimates of the six “PCR inhibited” libraries was large . The linear model indicated that the concentration estimates of the qPCR were on average 12.5 times lower than the TapeStation estimates. The ordinate-intercept of the linear model, corresponding to the limit of quantification, was on average 92.6 ± 34.5 and 0.053 ± 0.037 pg/µL for the TapeStation and the ABI7500 qPCR (IonLibQuant assay), respectively . However, the library coverage had no correlation with the TapeStation  or the Qubit measured concentrations .The correlations between the total number of reads for libraries and the library quantification estimates were analyzed by quantifying 35 Precision ID Ancestry Panel libraries with the Qubit, TapeStation, and ABI7500 qPCR (IonLibQuant assay) instruments prior to sequencing with the Ion Torrent platform . The reagent cost of the GeneRead assay was similar to those of most of the electrophoresis assays, and half the price of the IonLibQuant assay  of MPS is also a PCR reactiones Table  even thoThe Qubit instrument gave concentration estimates close to those of the electrophoresis-based instruments when quantifying chemically synthesized dsDNA oligos Fig. , but estThe costs and ease of workflow vary among the electrophoresis-based instruments Table . The Bio25. Commercial bead-based normalization kits are available for both Illumina and Ion Torrent libraries. Beads are added to each library, and library molecules bind to the beads until the beads are saturated. Unbound libraries are discarded, and each library will be represented by the number of bead-bound molecules. Reagents for Illumina bead-based normalization are included with the library kits, which does not cause additional expenses, whereas Ion Torrent bead-based normalization reagents must be purchased in addition to the library kits. This will cause an additional price of app. US$6 per library (Thermo Fisher Scientific). However, the Ion Library Equalizer Kit can be used for all Ion AmpliSeq, Ion Plus Fragment, and Ion Xpress Plus Fragment libraries. Whether bead-based normalization results in smaller library coverage variation than quantification and subsequent normalisation should be addressed in future studies. In a recent study, the genotype reproducibility and the concordance were higher for MPS with qPCR quantification than with bead-based normalization26. Sequencing has also been suggested as a way of accurately quantifying MPS libraries, e.g. using the MiSeq sequencer for quantifying libraries for later sequencing on the HiSeq.27. However, this is a high cost solution.Bead-based normalization of libraries has been suggested as an alternative to quantification. Previous studies indicated that bead-based normalization resulted in smaller variation in library coverage than sequencing based on library quantification with e.g. NanoDrop and Bioanalyzer, and subsequent pooling of the libraries in equal molar ratiosIn conclusion, the specific needs of the laboratory experiment determine which concentration measuring and normalization method is the most beneficial. For instance, if similar sample coverage for all samples typed in the same experiment is a requirement, or the sample throughput per experiment must be maximised, qPCR quantification of the libraries seems to be the best solution. Faster and cheaper quantification methods may be useful if the purpose is to identify libraries with too low DNA concentrations to be successfully sequenced. These samples may be excluded from the library pool, and re-typing of the samples can be initiated immediately. In some workflows, library quantification may be left out entirely if the downstream analysis of the sequencing results is sufficiently stringent to prevent misinterpretation, and if it is acceptable to delay re-typing of the sample(s).Supplementary material