Durum wheat is susceptible to terminal drought which can greatly decrease grain yield. Breeding to improve crop yield is hampered by inadequate knowledge of how the physiological and metabolic changes caused by drought are related to gene expression. To gain better insight into mechanisms defining resistance to water stress we studied the physiological and transcriptome responses of three durum breeding lines varying for yield stability under drought. Parents of a mapping population (Lahn x Cham1) and a recombinant inbred line (RIL2219) showed lowered flag leaf relative water content, water potential and photosynthesis when subjected to controlled water stress time transient experiments over a six-day period. RIL2219 lost less water and showed constitutively higher stomatal conductance, photosynthesis, transpiration, abscisic acid content and enhanced osmotic adjustment at equivalent leaf water compared to parents, thus defining a physiological strategy for high yield stability under water stress. Parallel analysis of the flag leaf transcriptome under stress uncovered global trends of early changes in regulatory pathways, reconfiguration of primary and secondary metabolism and lowered expression of transcripts in photosynthesis in all three lines. Differences in the number of genes, magnitude and profile of their expression response were also established amongst the lines with a high number belonging to regulatory pathways. In addition, we documented a large number of genes showing constitutive differences in leaf transcript expression between the genotypes at control non-stress conditions. Principal Coordinates Analysis uncovered a high level of structure in the transcriptome response to water stress in each wheat line suggesting genome-wide co-ordination of transcription. Utilising a systems-based approach of analysing the integrated wheat’s response to water stress, in terms of biological robustness theory, the findings suggest that each durum line transcriptome responded to water stress in a genome-specific manner which contributes to an overall different strategy of resistance to water stress. Durum wheat is a major staple crop in the Mediterranean basin as a source of semolina for the production of pasta, couscous, burghul and other local products. It is cultivated primarily under rain-fed conditions across a wide range of environments varying in the profile and quantity of rainfall with drought as a major abiotic stress per se is also advocated considering the importance of genetic backgrounds and species-specific differences in adaptations to stress and for the immediate development of drought resistant crop cultivars, as demonstrated in bread versus durum wheat by To address these challenges, various national and international efforts have been proposed to enhance wheat production by integrating technologies and approaches in plant science to underpin breeding efforts Durum wheat grown in the Mediterranean is susceptible to post-anthesis terminal drought, a phase of growth which directly affects yield. Thus wheat breeders have targeted this type of drought by selecting for cultivars with a fast growth rate that rapidly accumulate biomass before flowering as a water stress avoidance strategy Triticum turgidum L. ssp. durum (Desf.) Husn.] parents plus one recombinant inbred line (RIL) from the Cham1×Lahn mapping population developed at ICARDA were selected for this study because of their combined drought resistance and yield potential established in multiple year × site field trials in the Mediterranean region. Lahn was characterised by high yield potential under favourable rain environments, Cham1 exhibited relatively higher yield under drought, and RIL2219 was established as being more drought resistant than the best parent in addition to showing good yield stability (unpublished observations). Data collected in multiple field trials (30 over four years) showed RIL2219 closer to Cham1 in its overall phenology so that it reached heading 1.2 and 6 days earlier, on average, than Cham1 and Lahn respectively; it’s grain fill duration was 1.69 and 2.69 days later than Cham1 and Lahn respectively; and it was similar to cham1 for days to maturity with both reaching it 5 days earlier than Lahn (unpublished observations).Two durum wheat ABA (20–25 ng) was added as internal standard.Flag leaf transpiration rate, stomatal conductance to water, and CO2 and total RNA was extracted using TRIZOL Reagent  following the manufacturer’s recommendations. Total RNA was DNase treated with TURBO DNase enzyme (Ambion) and cleaned using RNeasy columns (Qiagen) following the manufacturer’s instructions. High quality RNA was submitted to transcriptome profiling at Bristol University (http://www.bristol.ac.uk/biology/research/transcriptomics/). Expression analysis was carried out on Affymetrix GeneChip Wheat genome arrays using Affymetrix GeneChip Eukaryotic One cycle target labelling reagents and protocols (701028 Rev 4). Five µg of total RNA was first reverse transcribed using a T7-Oligo(dT) promotor primer in a first strand cDNA synthesis reaction, followed by RNase H mediated 2nd strand cDNA synthesis. The double stranded cDNA was purified and served as a template in a subsequent in vitro transcription (IVT) reaction carried out in the presence of T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix to produce complimentary biotinylated RNA (cRNA). The biotinylated cRNA targets were cleaned up, fragmented and hybridised to Wheat Genome arrays for 16 hours in a GeneChip Affymetrix Hybridisation oven 640. Hybridised probe arrays were then washed and stained with a streptavidin-conjugated fluorescent staining solution followed by antibody amplification of the fluorescent signal in a GeneChip fluidics 450 station using the appropriate fluidics scripts for the array format. Following washing and staining, arrays were scanned using the GeneChip scanner 3000. Cell intensity data was automatically computed from the image data using GeneChip Operating Software (GCOS) under default settings. The quality and integrity of both total RNA and labelled and fragmented cRNA was assessed on an Agilent bioanalyser RNA Nano assay.Individual flag leaves, one leaf per biological sample, were sampled for RNA extraction and transcript expression studies. For all three wheat lines, 3 biological replicates per line were taken at days 1, 2, 3, 4, 5  and 6 (RIL2219 only) of the stress plus day 0 and 1 from the well watered controls to give a total of 66 samples. Leaves were frozen in liquid Nwww.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2410. Microarray data initial analysis was carried out using GenespringGX 8.0 . The set of array raw data files (.cel files) were pre-processed using the gcRMA algorithm 2 scale with respect to leaf RWC at proposed levels of 90, 82, 74, 66, 58 and 50; these levels of RWC were chosen to reflect a range of water deficit, whilst maintaining 6 observed occasions. RWC was then modelled on time (days of stress), fitting a logistic curve to the RWC data collected from each wheat line, using nonlinear regression, RWC(time) = a+b/[1+ exp((time - c)/d)], for estimable parameters a, b, c, and d. For each transcript by replicate  by wheat line combination, the change in transcript expression data over the time course was interpolated using Lagrange polynomials 2 transformation which considered the main effect of, and interaction between, the line and RWC factors, and also accounted for the block effect. The transcripts were filtered into groups given significant F-test  results, after correcting p-values for multiple testing and false-discovery y = C – C[exp(exp(−k(RWC − m))], were used for rate of CO2 assimilation, transpiration rate and stomatal conductance, with parameters C being the asymptote, m the inflection point and k the exponential rate separate, or common, for each wheat line. ABA content showed an asymmetric rise to a peak, therefore a rational functions model was used, log(ABA) = a+[(b+cRWC)/(1+ dRWC+fRWC2)], with estimable parameters a, b, c, d and f, taking ABA on the log (to base e) scale to account for greater variability in this measure.Microarray data are available in the ArrayExpress database  background correction and constant normalisation was applied to the probe intensities The wheat Affymetrix chip was designed with 11 probe sets per gene and the majority of the analysis in this study focused on the average expression value of the 11 probes in a probe set. In our analysis, we did not filter out any probes or identify unigenes, thus we present results for each probe set signal intensity that represent changes that could be due to unigenes, splice variants, homeoalleles or several members of a gene family. Probes were assigned to orthologous genes and metabolic bins, or pathways, using the MapMan programme following the standard protocol Cessation of watering and loss of water from the soil for 6 days progressively decreased flag leaf RWC and water potential for all wheat lines : the iniLine only  were modulated in value in response to water stress, genotype, or in combination, in all lines during the stress transient. In the first three days of the stress transient after watering was stopped, probes showed moderate changes in expression and this increased sharply as the stress progressed and leaf RWC declined with some delay shown by RIL 2219 . To analr factor . A detaiStress  . Across Catharanthus roseus-like RLK1, wheat LRK-10 like, proline extension like, wall associated kinase, S-locus glycoprotein like) and others included members belonging to 14-3-3 proteins, sugar and nutrient physiology, light, lipids, MAP kinases, lysine motif and phosphinositides were particularly altered during the stress , protein folding, protein glycosylation, post-translational modification and protein targeting were also altered during the stress transient  bin 29 we stress  bin 30. e stress  bin 21.Probes belonging to each of the major categories of hormone metabolism were modulated in expression as a result of water stress with the majority showing over-expression  bin 17. The majority of probes annotated to photosynthesis were down-regulated during the stress transient with transcripts annotated to LHCII, PS1 and ATP synthase changing early at 82% leaf RWC  bin 1. MWater stress increased the expression of probes annotated to sucrose degradation , some sucrose invertases  and starch degradation . This trend was accompanied by large decreases in the expression of probes annotated to alpha-glucan phosphorylase 2, cell wall invertases such as beta-fructofuranosidase , starch synthesis   bin2 andWater stress caused down-regulation of transcripts of nitrate reductase (NIA2) and increases in those for glutamine synthetase , glutamate dehydrogenase (GDH2) and glutamate synthase (GLT1)  bin12. CProbes annotated to cell cycle, cell division, cell organisation, development and vesicle transport were modulated by water stress  bin 33. Another group of probes, assigned to miscellaneous enzyme families involved in various metabolic processes, were modulated in response to the stress and included several members of cytochrome P450 which showed relatively large changes in expression  bin 26. Line × Stress comprised 7699 probes documenting an interaction between the two independent variables wheat line (two parents and RIL 2219) and stress (transient); probes that showed statistically significant responses to the stress and also measured differences between the lines in the way they responded to the water deficit. The global nature of the stress response, in all three lines, is demonstrated by the many pathways affected and by the large number of affected probes annotated to regulatory pathways presented in Stress  with the PCo coordinates PCo1, PCo2 and PCo3  were impline, line+stress and line x stress where such differential hybridisation may be an important consideration when comparing expression differences between the lines. The results showed that for the line only ANOVA group, 15 probes out of 396 probes  showed some evidence of differential hybridisation between the lines but with no stress response . Nevertheless, a handful were identified as promising in the ANOVA group Line x stress and one in particular, probe # 602715 belonging to the probe set ta.28513.1.s1_s_at and annotated as an Auxin Response Factor family, deserves close verification since it was also identified by PCo analysis as a major candidate for within-line stability under stress Click here for additional data file.File S1ANOVA results of probe expression values of three wheat lines in response to water stress. Whole dataset of 19062 probes interpolated values, pre-ANOVA, for individual replicates at each leaf %RWC level and for the three lines Lahn, Cham1 and RIL2219 in spreadsheet ‘Interpolated data at RWC levels’. ANOVA, log (base2), split the whole dataset into 5 independent statistical groups represented by individual spreadsheets: probes showing differences in expression between the lines but not during the stress in spreadsheet ‘line only 932 probes’; probes showing differences in expression as a result of the stress transient but not between lines in spreadsheet ‘stress only 2579 probes’; probes with differences in expression between the lines at the onset of the stress with the profile changing in the same way during the stress transient in spreadsheet ‘line+stress 7644 probes’; probes with differences in expression due to line and stress combined, showing an interaction in spreadsheet ‘line x stress 7699 probes’; and probes with no differences due to stress or line after initial filtering in spreadsheet ‘no effect 208 probes’. Statistical results are given as least significant difference (LSD) at 5% and 1% levels of significance for each of the ANOVA groups.(XLSX)Click here for additional data file.File S2MapMan analysis of ANOVA group line only 932 probes. Probes showing statistically different expression values between the three lines Lahn, Cham1 and RIL2219 but with no effect of the water stress. Data were calculated as expression value beyond the relevant LSD (5%) given in (XLSX)Click here for additional data file.File S3MapMan analysis of ANOVA group stress only 2579 probes. Probes showing no differences in expression between the three lines Lahn, Cham1 and RIL2219 but changing in response to the stress. Data were calculated as expression value beyond the relevant LSD (5%) given in (XLSX)Click here for additional data file.File S4MapMan analysis of ANOVA group line+stress 7644 probes. Probes showing consistent differences in expression between the three lines Lahn, Cham1 and RIL2219 in response to the stress transient –i.e. they show a parallel profile of change in transcript response across the stress transient. Data were calculated as expression value beyond the relevant LSD (5%) of data in (XLSX)Click here for additional data file.File S5MapMan analysis of ANOVA group line x stress 7699 probes. Probes showing differences in expression between the three lines Lahn, Cham1 and RIL2219, and in response to the stress; they show an interaction. Data were calculated as expression value beyond the relevant LSD (5%) given in (XLSX)Click here for additional data file.File S6MapMan analysis of ANOVA ‘no effect 208 probes’group. Probes, post initial filtering, showing no difference in expression between the three lines Lahn, Cham1 and RIL2219, or in response to the stress from data in (XLSX)Click here for additional data file.File S7MapMan annotation of PCo results. Mapman analysis of probes responsible for maximum separation observed, as defined by F-tests for the correlation between probes and principal coordinates (PCos), following PCo applied to the RWC-interpolated data for the 19062 probes derived from 54 samples (three replicates of each of six RWC levels by three wheat lines). Individual spreadsheets show annotation for the probes relevant for the first three principal coordinates retained from the analysis . Probes are presented by Affymetrix id and MapMan annotation to bin name, pathway and description of gene model.(XLSX)Click here for additional data file.File S8The contribution of allelic differences in gene sequences to probe hybridisation for probes annotated to transcription factors. ANOVA for each perfect match probe and the significance of the ratio changes across lines was calculated in proportion to the corresponding mean probe-set abundance. The name of the probe and P value are given for the corresponding probe set taken from line only, line+stress and Line x stress ANOVA groups datasets from (XLSX)Click here for additional data file.


Cardiovascular disease is the leading cause of mortality worldwide. Stem cell transplantation has become a new treatment option for cardiovascular disease because the stem cells are able to migrate to damaged cardiac tissue, repair the myocardial infarction area and ultimately reduce the role of the infarct-related mortality. Cardiac magnetic resonance imaging (MRI) is a new robust non-invasive imaging technique that can detect anatomical information and myocardial dysfunction, study the mechanism of stem cells therapy with superb spatial/temporal resolution, relatively safe contrast material and lack of radiation. This review describes the advantages and disadvantages of cardiac MRI applied in stem cells transplantation and discusses how to translate this technique into clinical therapy.Sources of Data/Study Selection: Data from cross-sectional and prospective studies published between the years 2001-2013 on the topic were included. Data searches included both human and animal studies.Data Extraction: The data was extracted from online resources of statistic reports, Pub med, THE MEDLINE, Google Scholar, Medical and Radiological journals.Conclusion: MRI is an appealing technique for cell trafficking depicting engraftment, differentiation and survival.  Currently, stem cell research has achieved many breakthroughs in transplantation therapy and its feasibility and effectiveness has been demonstrated in animal experiments as well as in small-scale clinical trials. Cardiovascular disease is a worldwide leading cause of mortality. In recent years interventional and surgical treatment of coronary heart disease using coronary artery bypass grafting (CABG) has made great progress. However, it’s mortality rate is still as high as 13%, while the five-year prevalence of heart failure remains as high as 50%.2For myocardial infarction stem cell transplantation therapy, many issues remain unresolved: How are the optimal number of cells determined when performing cell transplantation therapy? How is the survival time of transplanted cells best monitored? Do the transplanted cells undergo differentiation ? Can transplanted cells produce electrochemical coupling of normal myocardial and cardiac cells and their functions change? What is the mechanism of cells transplantation in the treatment of myocardial perfusion and cardiac function after a short enhancement ? These are examples of the current key issues that cardiac stem cell therapy research must address.3,4 Imaging methods allow us to trace and track stem cells in vivo and better evaluate the efficacy of cell therapy. Commonly, imaging methods in current usage include ultrasound imaging, PET/SPET, magnetic resonance imaging (MRI), optical imaging and CT imaging. Among them, cardiac MRI is undoubtedly the most promising molecular imaging means in clinical transformation as it can provide anatomical information with relative safety and superior resolution and sensitivity without radiation.Stem cell transplantation treatment: Stem cell transplantation methods using adult stem cells, embryonic stem cells（ESC）or induced pluripotent stem cells (iPSC) have become a major research focus in the field of treatments for ischemic heart disease. For example, using an MRI detection method, Grajek and colleagues have reported that patients with acute anterior wall myocardial infarction show improved myocardial perfusion 12 months after injection of bone marrow stem cells (BMCs), but that the left ventricular ejection fraction (LVEF) does not increase.5 Kraehenbuehl and colleagues have also reported that embryonic stem cell transplantation in a rat myocardial ischemia model can reduce the left ventricular expansion and the area of myocardial infarction after 3-6 weeks.6 Furthermore, Nelson and colleagues have shown that treatment with human stem cell factor, OCT3/4, SOX2, KLF4 and c-MYC is able to induce embryonic stem cells into iPSCs, form regenerative myocardial, smooth muscle or endothelial vascular cells in situ, repair myocardial infarction and increase ventricular wall thickness and electrical stability.7 In addition, a meta-analysis of randomised controlled clinical trials demonstrated that intracoronary adult bone marrow stem cell therapy in the setting of acute myocardial infarction (AMI) could improve left ventricular function and reduce the incidence of heart failure.8Direct labelling of stem cells: It is difficult to produce ideal stem cell MRI contrast effects. Hence, a great deal of research into the MRI-tracing of transplanted stem cells has focused on the development and application of novel contrast agents. Commonly used stem cell MRI contrast agents are divided into two major categories. The first involves contrast agents based on gadolinium (Gd) and manganese (Mn), such as gadolinium chelating agents (Gd-DTPA)9 and manganese chloride (MnCl2).10 These mainly provide T1 positive contrast effects. The second involves paramagnetic/super paramagnetic contrast agents based on iron oxide nanoparticles that produce strong T2/T2* negative contrast effects.11,12Strategies of iron oxide particle labelling: Iron oxide nanoparticles are a family of paramagnetic/superparamagnetic contrast agents, consisting of a ferrite (maghemite or magnetite) core and a polymeric coating. Depending on the diameter sizes , the nanoparticles can be divided into the SPIO (diameter size 60nm-150nm), USPIO (diameter size 10nm-40nm) and MION (diameter size 10nm-30nm) categories.13 Ferucarbotran (Resovist) and Ferumoxides (Endorem or Feridex) are MRI enhancement contrast agents approved by FDA, that have been widely applied in the clinical diagnosis of liver tumours and metastatic lymph nodes. High concentrations of ferromagnetic material can shorten both the T1/T2 values as well as the effect of T2*, resulting in a significant reduction in MR relaxation and higher biological safety.14-1617-19 Due to the negatively charged membrane surface, the unmodified ferric oxide particles are unable to attach to the stem cells. Polycation TAs are macromolecular substances with positively charged surfaces that may include polylysine20 or protamine sulfate.18 With a strong positive/negative interaction, SPIO/TA composites are able to adhere to the surface of cell membranes, improve the phagocytosis of iron oxide particles and avoid aggregation of SPIO particles.21 For example, Frank and colleagues mixed Ferumoxides (Feridex) with USPIO (MION-46 L) and added cationic TAs, successfully raising the concentration of intracellular SPIO particles. After 4-48 h incubation with 25 µg Fe/ml TA-(USPIO), target cells demonstrated a significant reduction in T2 values.22,23 Subsequent work has optimized this method by mixing Ferumoxides with protamine sulphate (50:3) µg/ml. Following incubation with the human mesenchymal stem cells, hematopoietic CD34+ stem cells and other mammalian cells overnight, the iron content of the cells was found to be 1.47 pg/cell-17.31 pg/cell.24Most labelled cells do not spontaneously internalise SPIOs and effective strategies are therefore needed to promote endocytosis. For example, positively charged polymer transfection agents(TAs) can be coated on the surface of magnetic iron oxide particles resulting in negatively charged cells nonspecifically uptaking the particles through the membrane surface. At present, composites of SPIO and polycation TAs are the most commonly used method to enhance the endocytosis of iron oxide particles.25 In addition, iron oxide particle surfaces can be modified by receptors such as vascular cell adhesion molecule-126 and membrane mucin A527 resulting in the nanoparticle targeting of specific tissues or organs. These methods require specific target receptors in the membrane, which greatly limits their application in stem cell tracking. Another method for increasing the efficiency of nanoparticle endocytosis is known as magnetoelectric perforation. Toxicity testing of mesenchymal stem cells, neural stem cells and adipose cells in vitro have all produced encouraging results with this method. Magnetoelectric perforation does not require a prolonged cell incubation time and the contrast agent for the target cells is safe and effective and approved by the FDA.28,29 However, from the perspective of biological safety considerations further research is still needed.Besides cationic polymerization material coating, other iron oxide particle surface modifications can also enhance cellular endocytosis. Koyama and colleagues fused monoclonal antibodies of pancreatic cancer specific antigen (PAP2a) with dextran modified SPIOs. Due to the nature of antigen-antibody reactions, this represented a novel approach for targeting the iron oxide particles to pancreatic cancer cells and promoting receptor-mediated SPIO endocytosis.Biological safety of iron oxide particle labelling: Cell labelling with iron oxide provides the potential for a rapid translation from preclinical medicine to a clinical setting. However, intensive toxicity tests are necessary for every protocol and cell type before clinical application. SPIO agents approved by the FDA  are mainly cleared by the reticuloendothelial system. Richards and colleagues isolated peripheral blood mononuclear cells from volunteers, labelled them with Ferumoxides in vitro and administrated these cells through intravenous injection. In T2* weighted images and R2* maps they observed that the labelled cells could efficiently migrate to the inflammation damage areas in tuberculin skin test.15 In spite of this result, many additional pre-clinical experiments will be required to verify the bio-safety of paramagnetic contrast agents. Sensitivity of MRI detection in vivo: The MRI detection of labelled stem cells is affected by intracellular iron distribution, MRI sequence, spatial resolution, magnetic field intensity and surrounding tissue heterogeneity. A higher intracellular iron content results in a more obvious relaxation time shorten. The T2* weighted image is highly sensitive for iron oxide particle labelling and its sensitivity can reach 3000 times that of T1 WI or 60 times that of T2 WI. 30 The most commonly used T2* sequence is the steady-state free precession (SSFP) and it is now the preferred sequence for detecting SPIO labelled cells. The T2* sequence is vulnerable to the influence of intracellular magnetic field inhomogeneity and the interference of the surrounding normal tissues, especially in high field MRI. However, conventional fast 3D gradient echo (GE) sequences are able to balance T2* sensitivity, spatial resolution and imaging time. In 2006, Fayad and colleagues reported an appealing MR sequence named ‘Gradient echo Acquisition for Superparamagnetic particles with Positive contrast’ (GRASP), which creates a positive rather than negative contrast of SPIO. The main advantage of this method is that it overcomes the interference of other sources of T2* effects and that the hyperintense signal may increase the sensitivity and specificity of cell tracking.31 Theoretically, when the quantity of the target cells is small, a smaller imaging voxel size should be chosen in the high field MRI. Limitations of SPIO direct labelling: Although the SPIO particle direct tracing method is the most commonly used cell labelling technique for MRI, it still has some shortcomings. For example, signal is usually found in areas of noninterest (e.g. the cardiopulmonary junction).32 Also, as a paramagnetic material accumulating in haemorrhagic infarction disease, haemoglobin shows low signal on T2* weighted imaging.33 It is important to note that with the death and rupture of transplanted cells, targeted SPIO nanoparticles can be phagocytosed by surrounding tissue cells or reticuloendothelial cells and subsequently redistributed, deposited or differentiated. Considering these facts, direct iron oxide labelling is more appropriate for short-term cell tracing in vivo or in vitro. The reason for false positive signals may be due to phagocytosis by surrounding cells, such as macrophages, or simply iron oxide distributed extracellularly. Moreover, partial volume effects or low concentrations of cells in one imaging voxel can lead to false negative results and with every subsequent cell division intracellular iron content will be halved, thus leading to a gradual reduction in cell detection sensitivity. For example, it has been reported that after 6 weeks MRI is unable to detect the differences between visible and invisible cells after transplant stem cell were administrated to the heart.34 Despite these limitations and shortcomings, paramagnetic/superparamagnetic iron oxide particles are still highly popular in the field of stem cell tracing largely because of their high sensitivity.Reporter gene labelling: Reporter gene labelling is achieved through the fusion of an MRI reporter gene to a target gene. Transfection of a target cell can then produce reporter gene expression for indirect MRI detection in vivo. The products of reporter genes are only expressed in living cells because genes are incorporated into the cellular DNA via transgenic methods. Thus, with these important advantages, transgenic gene labelling strategies are highly valuable in long-term studies of labelled cell survival, proliferation and differentiation in vivo. 35 including β-galactosidase, cytosine deaminase, creatinine kinase, tyrosinase and arginine kinase; 2) Ferritin or transferrin receptors.36 Among MRI reporter genes, the ferritin receptor has attracted a great deal of attention. Ferritin is a metalloprotein composed of 24 subunits that can bind up to 4500 Fe3+ ions. Excessive expression of ferritin can increase the intake of iron and the redistribution of intracellular iron which leads to the accumulation of transverse relaxation rates and a reduction of T2 values. Genove and colleagues reported that following adenovirus-ferritin reporter gene injection into murine corpus striatum, a robust contrast could be observed on T2 and T2* weighted imaging from 5 to 39 days.37The MRI reporter gene can be divided into two categories based on the product of its expression: 1) Intracellular enzymes Despite the advantages of MRI reporter gene imaging, there still remains a difficulty in avoiding potential damage to cell proliferation and differentiation. Additionally, apart from concerns regarding the sources and safety of cells, issues relating to gene mutation and sensitivity still need to be solved.The rapid development of in vivo imaging techniques has benefited the dynamic monitoring of stem cell therapies following myocardial infarction. CMR, which is safe, sensitive, lacks radiation and provides good resolution can produce accurate information regarding anatomy and changes in cardiac function. Currently, CMR is undoubtedly the most likely methodology regarding the clinical prospects of molecular imaging technologies. For researchers who study MRI, a major aim of future work is to develop new molecular probes and sequences in order to improve its sensitivity and specificity. At the same time the potential biological damage caused by reporter genes and immune responses needs to be limited. With the help of imaging technology, stem cell therapy will have a major part to play in the therapy of myocardial infarction.Xi Lu, Rui Xia, and Bing Zhang: Carried out the literature search and wrote the manuscript.Fabao Gao: Reviewed and finalized the article.


Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene  Magnaporthe oryzae, is one of the most devastating rice diseases worldwide. The rice blast infection can damage almost all parts of the plant at various growth stages11Rice is a staple food for more than half of the world’s population and therefore important for global food security. Rice blast, which is caused by the fungus R) genes and over 350 quantitative trait loci (QTLs) have been identified4567R genes include the introgression of Piz5 and Pi54 into an elite basmati rice restorer line ‘PRR78’Pib and PishPi1, Piz-5 and Pita combinationsR genes, rice blast remains problematic because resistance is often lost in new varieties after a few years of intensive agricultural use. The rapidly evolving blast fungus can overcome the resistance conferred by major blast resistance genes. Because of the importance of the disease, M. oryzae was the first phytopathogenic fungus whose genome was sequenced. The M. oryzae genome contains retrotransposons and abundant repetitive elements in regions carrying the avirulence (Avr) genesM. oryzae strains, genomes of three additional M. oryzae strains were re-sequenced13Avr genes revealed high levels of non-synonymous variations as well as frequent loss and gain of Avr genes, which explains the rapid evolving nature of the blast fungus13Deployment of host plant resistance is the most effective approach for the control of rice blast. To date nearly 100 blast resistance , lead to major differences in their function or resistance spectrum. For instance, only few amino acid changes among the predicted proteins of blast resistance genes Pi2, Pi9 and Piz-t determine their resistance specificitiesR genes also often have varied specificities. For example, a single amino acid change differentiates the resistant and susceptible alleles of rice blast resistance genes Pita and Pid221Pm3 gene in wheat exhibit different resistance patterns against a range of powdery mildew isolates2324R gene in a single genetic background or through multiline approach is a possible strategy to improve resistance in the field26Pm3 where the specificities of Pm3d and Pm3e were combined into a single allele. Moreover, a detailed molecular analysis of such allelic R gene variants facilitated understanding of host-pathogen interactions and evolution of disease resistance genes. Therefore, exploring naturally available genetic diversity for new alleles of resistance genes is an efficient approach for broadening the resistance sources against rice blast.Pertaining to the genetic variability and pathogenicity of the rice blast fungus, genes involved in host plant resistance also co-evolved with high levels of allelic and copy number variationsXa21 from O. longistaminataPi9 from O. minutaPid3-A4 from O. rufipogonPi-hk1 from a landrace HeikezijingSeed banks are rich sources of such natural genetic diversity considering their large collections of cultivars, traditional varieties and wild relatives of many crops. This genetic potential can be accessed for identification of novel genes and/or their functional alleles. Several agriculturally relevant genes were obtained from wild relatives and landraces, such as the bacterial blight resistance gene Pi54 in Indian germplasm. Pi54 (formerly known as Pi-kh) was first identified in the Indian rice cultivar HR22 and was cloned from the indica type rice cultivar Tetep. The gene confers broad-spectrum resistance against Indian rice blast isolatesPi54 belongs to the ‘Coiled Coil - Nucleotide Binding Site - Leucine Rich Repeats’ (CC-NBS-LRR) class of R genes and the protein activates several downstream defence related pathways upon pathogen attackPi54 alleles that we have identified expand the known Pi54 allelic series and could potentially serve as a genetic resource for breeding or engineering resistance to rice blast.Here we report the allelic diversity of a major rice blast resistance gene Pi54 gene (M. oryzae strains when screened in the uniform blast nursery (UBN). Under controlled conditions, the selected accessions were found resistant against at least one of the five tested individual blast strainsPi54 using the ‘Pi54 MAS’ markerPi54 allele is present or a 359 bp fragment if a Pi54 allele is interrupted by a 144 bp insert (often associated with the susceptible phenotype)Pi54 Pi54 .Pi54 MAS’, the full length coding region of the gene was amplified from all 329 accessions. The primers flanking the start and stop codons as annotated for Pi54 allele from the cultivar TetepPi54_Tetep) were used for the sequence based amplification of Pi54 alleles. Detailed analysis of the sequences obtained from these 329 accessions identified eleven alleles of Pi54, based on the comparison with Pi54_TetepPi54 allelic sequences43839Pi54_Tetep. The allele Pi54_3708 and the previously reported Pi54 alleles from a landrace Satti and the wild rice O. nivara were found identical. Similarly Pi54_40636 was found identical to alleles in the haplogroup-H2 from recently reported Pi54 allelesPi54 alleles to the reference allele Pi54_Tetep. Except for the three alleles Pi54_42439, Pi54_22419 and Pi54_3708 that were exclusively identified in single accessions, each of the remaining eight alleles was identified in several rice accessions  have a complete open reading frame (ORF) similar to that of Pi54_Tetep. The remaining 5 alleles  appear to have shorter ORFs of variable sizes due to predicted premature stop codon(s) introduced by deletions of either 2 bp  or 8 bp (Pi54_42439 and Pi54_40996)  sites such as 13 casein kinase II (CK2) phosphorylation sites, four N-glycosylation sites, four N-myristoylation sites and three protein kinase C (PKC) phosphorylation sites were predicted in all Pi54 allelesPi54_3708, which differs from Pi54_Tetep by only 2 SNPs at positions 591 and 795. One of the two SNPs in Pi54_3708 is a synonymous mutation at position 591 that is shared with all other identified Pi54 alleles . Seven alleles have at least one unique SNP (four of them non-synonymous) or deletions specific to that allele but absent in the other Pi54 alleles  , Table 1 and 429 . Five SN and 429 . In case and 429 . All Pi5Pi54 alleles were performed in DnaSPPi54 alleles was calculated as 0.01551. The sliding window analysis of nucleotide diversity of Pi54 alleles showed a high rate of diversity within the SNP-rich region described above  does not indicate a strong positive selection.Nucleotide polymorphism analyses for the eleven ed above . The numPi54_Tetep reference allele were subjected to protein prediction and analyses. Pi54_Tetep has a NBS domain of 21 amino acids (AA) and two non-synonymous mutations in the NBS domain were found among the studied Pi54 alleles. One of these non-synonymous mutations replaces tyrosine with phenylalanine at position 155 in all predicted proteins except Pi54_3708. The second non-synonymous mutation changes a candidate serine casein kinase II phosphorylation site at position 158 to threonine in Pi54_41818, Pi54_22126, Pi54_40996 and Pi54_42439. Pi54_Tetep has two LRR domains, each 23 AA in length. The only non-synonymous mutation in LRR1 that replaces leucine with phenylalanine at position 265 is observed in Pi54_3708 exclusively  results in a predicted imperfect leucine zipper-like pattern (LxxxxxxLxxxxxxLxxxxxxL) but the predicted protein structures of various alleles retain a typical horse shoe-shaped conformation  are distributed among all three subspecies  compared to Pid3 orthologues from other cultivated and wild rice varietiesR genes revealed a unique spectrum of resistance among these alleles, including alleles of Pi35Pi54 from O. rhizomatis (Pi54rh) and O. officinalis (Pi54of)4AC134922 locusPi35 that confers quantitative broad-spectrum resistance is an allele of Pish, which confers race-specific resistance against rice blastPi54, a major blast resistance gene conferring resistance against rice blast in India. We identified nine new alleles of Pi54 that significantly extend the known Pi54 allelic series. Among the investigated Indian accessions, the distribution of new Pi54 alleles appeared to be enriched in indica as compared to japonica and javanica accessions.Considering the narrow genetic base of agriculturally grown varieties, exploration of natural genetic diversity is critically important to broaden and diversify our resistance sources against detrimental crop diseases, including rice blast. Allelic variants of an Pi54  interact with AVR-Pi54 using various domain as well as non-domain regionsPi54 alleles, while the majority of the sequence polymorphisms are in the region between the tyrosine kinase phosphorylation site and the NBS domain, which we refer to as the ‘SNP-rich region’. The SNP-rich region as well as the overall nucleotide polymorphism of the alleles identified in this study is shared among the fifty Pi54 haplotypes that were recently reported from Indian rice varietiesPi54 alleles by gene conversion and recombination.Pi54 has a NBS-LRR structure with a CC domain at the N terminus5051Pi54 are responsible for the resistance phenotype in TetepPi54 alleles to different rice blast strains further indicate a possibility of novel and/or altered resistance spectrum as compared to the known Pi54 alleles (R gene(s) and/or QTLs cannot be ruled out. Once functionally validated, these alleles could be used in various combinations with previously reported allelic forms of Pi54 or with other rice blast resistance genes in order to enhance the durability of blast resistance. Furthermore, detailed understanding of polymorphic sites controlling resistance will facilitate intragenic allele pyramiding to combine resistance specificities of different allelesWe found that 67% of the amino acid changes among the minor motifs/patterns and PTM sites are also in the SNP-rich region of the gene. The majority of the polymorphisms at the PTM sites does not result in their loss but could influence the accessibility of acceptor residue(s) within a PTM site for the modifying enzymes. More than half of the polymorphic AA residues predicted as exposed are located within a PTM site. It has been suggested that the difference in number and distribution of phosphorylation motifs between the resistant Tetep and susceptible Nipponbare alleles of  alleles . HoweverPi54 using a functional co-dominant marker ‘Pi54 MAS’Pi54 alleles with and without an insertion of 144 bp, the former being correlated with the susceptible allele. Genomic DNA extracted from Pi54 monogenic line was used as the positive control and that from susceptible rice cultivar LTH was used as the negative control for the Polymerase chain reaction (PCR), in addition to a water control.The rice germplasm material for this study was obtained from the International Rice Genebank (IRG) of the International Rice Research Institute (IRRI), Philippines. The accessions were screened for rice blast resistance against field mix-inoculum and five pure blast isolatesPi54 alleles as annotated for Pi54 allele from cultivar TetepForward primer 5′-TACCTGATGGTTCTTTAAAATTGGG (designed for this study) and reverse primer 5′-CATAAGCTAGACCTTGAAGGATGTCPi54 alleles were designed in DNASTAR-SeqBuilder, using Pi54_Tetep as reference (Accession Nr. AY914077). DNA sequencing was performed using Applied Biosystems Capillary Sequencer 3730. Sequence assembly, consensus development and alignments were done using ‘CLC Genomics Workbench, version-7.5′. Multiple sequence alignment was performed to identify the Single Nucleotide Polymorphisms (SNPs). Sequence polymorphism analyses such as, number of segregation sites and mutations, sliding window analysis of nucleotide diversity, ka/ks ratio and Tajima’s D test were performed using DnaSP-v5Forward primer 5′-GTTAGGCCTTCAGGAATGGAGTGC was used as internal sequence primer in addition to the standard forward and reverse primers from the pJET1.2 cloning vector for the complete sequence coverage. The primers for isolation and sequencing of Pi54 alleles together with previously reported Pi54 alleles from cultivated rice, landraces and wild rice species to analyse their genetic relatedness43839Pi54 alleles i.e., from start to stop codon  corresponding to that of Pi54_Tetep were obtained from NCBI. The sequence alignments were performed in CLC genomics workbench. The tree was constructed using ‘Neighbor-joining’ method and ‘Jukes-Cantor’ for nucleotide distance measure and bootstrap performed with 1000 replications.Phylogenetic analysis was performed using DNA sequences of the newly identified Pi54 alleles were obtained using the EXPASY translation tool (http://web.expasy.org/translate/). The major domains such as NBS and LRR regions were assigned as previously reported for Pi5437http://www.ch.embnet.org/software/COILS_form.html). The patterns and post-translational modification sites were predicted using ScanProsite serverPi54_Tetep reference allele. The prediction parameter was set to ‘normal’ mode for all the proteins analysed. PyMOL-version 1.3 was used to visualize, analyse and edit the protein structures for their secondary structure elements and orientation.The protein sequences of Accession codes: The newly identified Pi54 sequences have been submitted to the GenBank database with accession numbers, Pi54_40996 = KR052922, Pi54_13758 = KR052923, Pi54_22126 = KR052924, Pi54_22597 = KR052925, Pi54_40636 = KR052926, Pi54_10202 = KR052927, Pi54_41818 = KR052928, Pi54_52694 = KR052929, Pi54_42439 = KR052930, Pi54_22419 = KR052931, and Pi54_3708 = KR052932.How to cite this article: Vasudevan, K. et al. Identification of novel alleles of the rice blast resistance gene Pi54. Sci. Rep.5, 15678; doi: 10.1038/srep15678 (2015).


Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting.Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed.A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation and reverse cleavage amongst the embryos observed was 25% and 7%, respectively. Over 40% of embryos exhibiting these characteristics did however form blastocysts meeting our criteria for freezing.These data provide us with a platform with which to potentially enhance embryo selection for transfer. In vitro fertilization (IVF) success rates have increased since 1978 and the birth of the first IVF baby. The implantation rate per embryo transferred in patients under 35 in 2011 was still however only 36%, according to the SART (Society of Assisted Reproduction) national IVF registry. In spite of ASRM guidelines to reduce the number of embryos transferred, many centers still transfer 2-3 embryos despite the increased risk of multiple pregnancy, with its associated neonatal and maternal complications, in order to maximize the chance for pregnancy (SART.org).The most critical step during IVF is embryo selection for transfer. For the last two decades, the conventional method of embryo selection for transfer has been based on critical assessment of morphologic parameters during embryonic development. Currently, these morphological assessments are limited to once a day at set time points, since repeated removal of embryos from the incubator environment for observation may result in undesired temperature and pH shifts in the embryo culture dish.Embryo development is a dynamic event and static observations of embryonic growth can therefore be limiting in their ability to discern differences between embryos at similar cell stages. The introduction of time-lapse imaging and monitoring systems in the clinical IVF laboratory has allowed more detailed observations on embryo developmental kinetics. Numerous data suggest that the precise timing of specific events such as pronuclear formation, syngamy, early cleavage events, cell cycle intervals, synchronicity of cell division and initiation of blastulation are indicators of an embryo’s developmental potential-7. The aThe specific aims of this study were: (1) to present our initial clinical outcome data with continuous time-lapse imaging in the Embryoscope, (2) to determine if there are kinetic differences between embryos with limited developmental potential and those forming blastocysts of a quality suitable for transfer or freezing , (3) to determine if there are differences in kinetics between implanting and non-implanting blastocysts and (4) to compare these observations to embryo selection markers proposed by other laboratories.This was a retrospective study of prospectively acquired data of time-lapse imaging of human embryos during in vitro growth. This research was carried out at the Cleveland Clinic Fertility Center between April and November 2012. The study was approved by our Institutional Review Board and was carried out following ethical guidelines set forth by our institution. Patients with 10 or more mature oocytes and at least 7 fertilized oocytes were offered the opportunity to participate in this investigation. The study was restricted to this subset of patients to only include those who had enough embryos that selection parameters could potentially aid in embryo selection for transfer. The exclusion criteria for this study were maternal age over 39 and cases involving surgically retrieved sperm. A total of 81 patients were enrolled. The number of patients included in the study was determined by the total number of patients recruited during six months of data collection. The data set analyzed included 648 embryos, derived from 60 patients, cultured until the blastocyst stage (day 5-6). The remaining 21 patients had a day 3 transfer and their morphokinetic data was excluded. Time-lapse images of human embryo development were critically assessed by one of three observers, the lab director and two senior embryologists. At the outset of the study all three observers viewed videos as a group and established annotation guidelines. Consistency of grading and annotation were thereafter reviewed by the lab director and discussed at weekly lab meetings.2 and 21% atmospheric oxygen in a Forma incubator.Women were treated with either a GnRH-agonist (gonadotropin releasing hormone) or a GnRH-antagonist to suppress ovulation until follicle maturity was attained. Ovarian stimulation was initiated using daily injections of follicle stimulating hormone (FSH) with starting doses based on serum anti-Mullerian hormone levels, antral follicle counts and previous responses to ovarian stimulation. The subsequent doses were adjusted according to follicle growth and serum estradiol levels. Final follicular maturation was triggered with human chorionic gonadotrophin (hCG) when at least two lead follicles measured 18 mm in mean diameter. Oocytes were collected 36 hours later by transvaginal needle aspiration of follicles under ultrasound guidance. Oocytes were placed in a 35 mm dish containing 25 μl drops of Global fertilization medium  supplemented with 10% human serum albumin  under an oil overlay. Dishes were incubated at 37˚C with 6% CO2 and 21% atmospheric oxygen.Oocytes were denuded of cumulus cells 2-3 hours after the retrieval using hyaluronidase (Sage). Intracytoplasmic sperm injection (ICSI) was then performed on mature oocytes. Injected oocytes were moved to pre-equilibrated 25 μl drops of Global medium with 10% Synthetic Protein Supplement  under an oil overlay and cultured overnight at 37˚C with 6% COAn EmbryoSlide™  was prepared on the day of egg retrieval for zygote culture after the fertilization check. The EmbryoSlide™ containing 12 individual wells was prepared by filling each well with 25 μl of Global medium with 10% SPS and overlaying with 1.2 ml of washed oil. The slide was equilibrated overnight.2 with 5.5% oxygen. Medium was refreshed on day 3 by performing a half-change with freshly prepared, pre-equilibrated culture medium. An Eppendorf pipette was used to withdraw 12 μl of fluid from each well and this was replaced with fresh medium, taking care not to disturb the embryo. The image acquisition software collected images of each embryo as it developed during the six day culture interval. The time-lapse video created from these images was used to monitor cell cleavage anomalies as well as the timing of specific cell cycle events.Oocytes were examined 16-18 hours after insemination for signs of fertilization and the presence of two pronuclei. Zygotes were moved to the wells of the pre-equilibrated EmbryoSlide™. Care was taken to remove any bubbles using a finely drawn glass micropipette before placing the zygotes in the wells. Slides containing zygotes were placed in the Embryoscope chamber immediately following fertilization check and cultured for up to 6 days. Embryos were cultured at 37°C at 6% COThe Embryoscope, recently FDA cleared in the U.S. for clinical embryology, was used for time lapse imaging of the embryos. It is a tri-gas incubation chamber equipped with a built in microscope (Hoffman Modulation contrast objective) and a high definition camera , allowing continuous monitoring of embryonic growth. The chamber design and camera software is capable of imaging up to 72 embryos (6 patient slides with 12 embryos). The image acquisition system was set to capture high contrast 200× images from 5-7 focal planes for each embryo, every 15 minutes.Observations were made daily in the morning using the Embryoscope viewer. The time of ICSI was designated as time zero (t0). All timings were thereafter expressed as hours post-insemination (hpi) and zygotes were first placed in the Embryoscope at 17-18 hpi. For day 2 embryos, observations were made 42-44 hpi, day 3 at 66-68 hpi, day 4 at 90-92 hpi, day 5 at 114-116 hpi and day 6 at 138-140 hpi. Embryos were first graded at these set time points without viewing the accumulated time-lapse images. Information was recorded in the patient’s laboratory chart.Cleavage stage embryos were assessed for cell stage, percent fragmentation, multinucleation and blastomere symmetry,12. IncrBlastocysts were selected for transfer based on conventional grading criteria without the use of kinetic data. Good quality blastocysts arising from embryos displaying an optimal growth pattern  were given preference. Selection was based on blastocyst grade in the morning at 114-116 hpi even though transfers were generally scheduled for late afternoon. In patients presenting with only a single suitable quality blastocyst (n = 8), morula were transferred along with the blastocyst. Morula observed with beginnings of fluid were often early blastocysts by the time of transfer. Supernumerary blastocysts Grade B-E with an inner cell mass and a trophectoderm of good cell number and quality were cryopreserved. Time-lapse videos were reviewed in detail once conventional grading had been completed.Time lapse microscopy (TLM) allowed the visualization of nuclear and cytoplasmic events during the entire in vitro culture interval. Using the Embryo Viewer software, video records were annotated for the timing of specific developmental stages, cleavage anomalies and blastomere nuclearity from the point of ICSI. The following early kinetic markers were assessed: time to pronuclear fading or syngamy (tPNf), time to 2 cells (c) (t2), 3c (t3), 4c (t4), 5c (t5), 8c (t8) and 9c + or partially compacting (t9). We also looked at late kinetic parameters coinciding with genomic activation, specifically, morula (tMor), start of blastulation (tSB), blastocyst (tBL), and expanded blastocyst (tEBL). Embryos were labeled as morula when greater than 90% of the embryo was compacted and individual blastomeres could no longer be identified. Start of blastulation was defined as the earliest frame in which fluid could be distinctly seen amongst blastomeres in the morula stage embryo. Embryos were annotated as blastocysts when a crescent shaped region with fluid could be visualized. Expansion of the blastocyst in diameter was the point identified as tEB. Durations of the second cycle , third cell cycle  as well as intervals between 4 and 5 cells  and t5-t2 were also calculated. The final kinetic parameters looked at were the time to complete synchronous divisions s1 (t2-tPNf), s2 (t4-t3) and s3 (t8-t5).Two cleavage anomalies specifically monitored were: reverse cleavage (RCLV), where a blastomere was re-absorbed after cleavage and direct cleavage (DC), where a single blastomere divided directly from 1 to 3 cells in less than 5 hours, as described by Rubio et al. PresencEmbryo transfer was performed on day 5 under trans abdominal ultrasound guidance using a Wallace Sure-View catheter. Patients used either intravaginal progesterone  or intramuscular injections of progesterone in oil (100mg) for luteal support starting the day of oocyte retrieval. Supernumerary blastocysts of good morphology, displaying an inner cell mass and adequate trophectodermal cells were cryopreserved.Serum beta-hCG levels were measured 15 days after transfer. A clinical pregnancy was defined as visualization of an intrauterine gestational sac with fetal heart activity on ultrasound 5 weeks after the embryo transfer. The implantation rate (IR) was calculated by dividing the number of embryos with cardiac activity by the number of embryos transferred.For the purpose of data analysis cultured embryos were divided in to three groups: (A) Blastocysts transferred (BL-T) (B) Blastocysts frozen (BL-F) and (C) Poor quality blastocysts and non-blastulating embryos ultimately discarded (PQ-D). Kinetic data was compared between the three groups. We also looked specifically at transfers with known implantation data (KID). Timing of developmental endpoints were compared between transfers where all embryos implanted (KID+) and those in which all embryos failed to implant (KID-). The mean timing of cell division and cell cycle intervals were compared using the ANOVA parametric test and the Student t-test. The chi square test was used for analysis and comparison of proportions. P-values of <0.05 were considered statistically significant.Patient demographics and cycle characteristics are shown in Table The 648 cultured zygotes were divided in to three groups (A-C) according to quality and final disposition as described earlier. A total of 335 blastocysts (BL-F) were frozen and 208 embryos (PQ-D) were discarded. Group A designated as BL-T, represents each patient’s best quality blastocysts selected for transfer. Amongst the 105 blastocysts transferred 69% were expanded blastocysts (Grade C or D), 31% were early blastocysts (Grade A or B). The timings of cleavage and specific early developmental endpoints amongst embryos from the three groups are shown in Table To better understand the impact of different embryo kinetic parameters on ability to implant we looked at transfer cycles with “known implantation data” (KID). We had 38 KID patients in which all transferred blastocysts either implanted (KID+) or failed to implant (KID-). Table IVF outcome data from national registries like SART give evidence to the wide variation in clinical outcomes amongst clinics. Certainly differences in laboratory protocols and expertise factor in to these data and the ultimate implantation potential of embryos being generated. It is therefore of paramount importance that laboratories collect time-lapse image information and first establish baseline kinetics for embryo development within their own clinical and laboratory setting.This study represents our first experience with the use of the Embryoscope and time-lapse imaging for embryo culture in our laboratory. The controlled environment provided within the time lapse-chamber proved to be beneficial for cultivation of human embryos resulting in high day 5 pregnancy outcomes. Our initial findings suggest that early morphokinetic parameters do in fact differ between embryos making good quality useable blastocysts as compared to those embryos failing to blastulate or showing poor blastocyst morphology. Furthermore even amongst good quality transferred blastocysts, we were able to detect differences amongst implanting versus non-implanting blastocysts using time-lapse data. This latter observation may be especially valuable as our laboratory moves towards elective single embryo transfer (SET).To date, there have only been a few studies looking at the relationship between early kinetics, blastulation and implantation potential,7,15-17.Embryo kinetics for predicting blastocyst formation as opposed to implantation may potentially differ. Kirkegaard et al. reported that duration of the first cytokinesis, duration of the 3 cell stage and direct cleavage to 3 cell were useful parameters in predicting blastocyst formation. At the In the present study with 648 embryos from an infertile patient population, we did not find cc3 or t5 to be helpful in discriminating between embryos making blastocysts versus those of limited viability either failing to blastulate or developing in to poor quality, unusable blastocysts. At the same time, It should also be pointed out that the t5 values  in our transferred blastocysts were quite in line with those observed by Herrero et al in their grouping of blastocysts with optimal morphology. In the Our initial KID data albeit limited are in agreement with other studies suggesting that kinetic markers associated with blastocyst formation may not necessarily be the same as those defining an embryo’s competence to implant. Clearly, ability to implant may be influenced by both patient-specific factors such as age, oocyte quality, endometrial receptivity, and sperm factor, as well as laboratory culture conditions influencing blastocyst quality and ploidy,10,18-21The combining of morphokinetics with preimplantation genetic screening (PGS) technology to develop a non-invasive model to identify euploid embryos has garnered a lot of interest. Hong et al observed a reduced prevalence of aneuploidy in blastocysts with early time to cavitation. In a reCulture media and specifically the use of single step versus sequential media may also contribute to the observed kinetic pattern within a cohort of embryos. Ciray et al observed that embryos cultured in a single step medium were advanced from the first mitosis thru to the 5-cell stage over sibling embryos cultured in sequential media but the cell cycle intervals  and s2 did not differ. Pregnancy and implantation rate were also not affected. In contrast Basile et al comparing single step medium  to a sequential medium (Quinn’s Cleavage) observed no significant differences in timings or outcome parameters. The protocol in this initial TL study used Global as a single step medium with a medium refresh on Day 3. We have since shifted over to uninterrupted culture in Global medium from day 1 thru 5 with equally good clinical results. In deptIn addition to kinetic parameters, there are other events witnessed with time-lapse monitoring that may benefit embryo selection. The visualization of anomalous events such as direct cleavage and reverse cleavage previously not possible with conventional static microscopy may be useful as deselection criteria. Direct cleavage first studied by Rubio et al., was shown to be associated with lower implantation rates. These iIncreased detection of multinucleation with time-lapse morphology may be another tool for embryo deselection. Multinucleation may arise from karyokinesis without subsequent cytokinesis or alternatively from errors during mitosis in chromosome segregation and packaging, quite often resulting in chromosomally abnormal cells. Data suLaboratories acquiring new time lapse capabilities face the dilemma of how to move forward using the technology. This study’s sample size was limited to six months of data collection to allow for an expedient analysis of data to gain an understanding of the morphokinetic patterns within our own laboratory environment. A limitation of this study was its retrospective nature and restriction to a study population of patients with a larger number of embryos. A prospective randomized trial to see if conventional morphology combined with kinetics can help to “refine” selection of blastocysts on day 5 and increase implantation rates is currently underway .The present work illustrates the importance of sequential observation of embryo developmental patterns and the wealth of additional information that can potentially be incorporated in to an embryo classification model.The authors declare that they have no competing interests.ND designed the study, critically assessed data and drafted manuscript. SP analyzed data and performed statistical analysis. LG helped revise manuscript. JG, CA and TF reviewed manuscript. All authors read and approved the final manuscript.


Genome-wide data is accumulating in an unprecedented way in the public domain. Re-mining this data shows great potential to generate novel hypotheses. However this approach is dependent on the quality  of the underlying data. Here we performed a systematic analysis of chromatin immunoprecipitation (ChIP) sequencing data of transcription and epigenetic factors from the encyclopaedia of DNA elements (ENCODE) resource to demonstrate that about one third of conditions with replicates show low concordance between replicate peak lists. This serves as a case study to demonstrate a caveat concerning genome-wide analyses and highlights a need to validate the quality of each sample before performing further associative analyses. The easy accessibility of these data, including processed files, has resulted into many researchers world-wide using the ENCODE resource to learn new biology, either by integrating existing data The ENCODE consortium at UCSC provides 690 ChIP sequencing data sets for transcription factors and transcription regulators, performed using 189 different antibodies in 91 cell lines, sometimes under different cell treatments. It contains 57 conditions where ChIP-seq for the same factor in the same cell line with the same treatment (or absence of treatment) were done multiple times  and not merged. These experiments were not merged because they differ in one or more of the following: antibody used for the ChIP, laboratory that performed the experiment, or library preparation protocol for the sequencing. It is not mentioned whether these replicates give redundant information, complementary information, or if one of the replicates is of higher quality than the other(s), leaving the end user unarmed to decide which peak list should be used. Notably, these peak lists have also not been merged in other ENCODE data repositories such as encodeproject.org or factorbook.org 2http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeAwgTfbsUniform/ (Uniform SPP peaks). Common and sample specific peaks were identified using bedtools http://ftp.ebi.ac.uk/pub/databases/ensembl/encode/integration_data_jan2011/byDataType/peaks/jan2011/peakSeq/optimal/hub/. Bam files for the corresponding experiments were downloaded from https://www.encodeproject.org/, and merged using Samtools De novo motif analysis was performed using HOMER https://github.com/gdevailly/ENCODE-TFBS-replicate-quality).ChIP-seq experiments with replicates in the ENCODE TFBS dataset were identified and uniform TFBS peaks files for the corresponding samples were downloaded from the website 33.1PCR1x” and the “V0422111” protocols). Two corresponding peak lists are provided by the ENCODE (“wgEncodeAwgTfbsHaibA549Usf1V0422111Etoh02UniPk.narrowPeak” and “wgEncodeAwgTfbsHaibA549Usf1Pcr1xEtoh02UniPk.narrowPeak”) and both experiments are in different lanes in the UCSC track .We identified 57 conditions within the ENCODE transcription and epigenetic factors ChIP sequencing data where the same experiment was done multiple times (between 2 and 5) for the same factor in the same cell line with the same treatment (or absence of treatment), and the replicates were provided without being merged. For example, the USF1 ChIP-seq in A549 cells treated with 0.02% of ethanol was performed two times by the HudsonAlpha laboratory with the same antibody but with two different library preparation protocols . If the overlap between all replicate peak lists and the common peak list was 50% or more (i.e. if at least 50% of the peaks were reproducible in every replicate), then the condition was labelled as “similar” A. Withinnsitive” B where tsimilar” C. Of thesimilar” . Five ofcommon peaks” , at peaks detected in a given experiment but not in any other replicate (“sample specific peaks”), and at peaks detected in any of the other replicates, but missed in a given experiment  procedure instead of representing true ChIP enrichment differences, we obtained the raw read counts for each sample from the ENCODE resource. For each of the 135 experiments (from 57 conditions), we computed mean coverage at “Peak lists generated by another peak caller (peakSeq) for 6 replicates  classified the samples in the same groups  showed a discrepancy between the de novo motifs identified in the replicate experiments , whereas the REST-NRSF motif is the top enriched motif in the Broad sample . As GATA1 is a master regulator of erythropoiesis and K562 is a leukaemia derived cell line, the HudsonAlpha HDAC2 experiment (and antibody) appears more reliable than the Broad HDAC2 in K562.HDAC2 experiments in K562 cell line – histone deacetylase HDAC2 experiments in K562 cell line were generated by the Broad and HudsonAlpha laboratories using different antibodies. When comparing to other ENCODE ChIP-seq experiments, HudsonAlpha HDAC2 ChIP-seq clusters with P300 (as identified by the Sydh laboratory) while Broad HDAC2 clusters with HDAC6 . In H1-h2.CMYC experiments in H1-hESC cell line – CMYC replicate experiments in H1-hESC cell line were generated by the Stanford and UTA laboratories. Amongst H1-hESC samples, the Stanford CMYC replicate clusters together with MAX and is part of a larger cluster including RNA polymerase II and other promoter associated factors, such as TAF1 and TBP. This is in accordance with the proven role of CMYC in RNA polymerase II elongation The promoter proximity of binding and its preference for specific sequence motifs are characteristics of many transcription factors and can be used as a proxy to establish biological validity of ChIP-seq samples. We annotated peaks with their distance to the nearest transcription start sites (TSS). This revealed differences between replicates, especially for dissimilar conditions . We alsoeriments . This waDe novo motif discovery detected different primary (Although further analysis is indeed a way forward as demonstrated above, it does not always provide sufficient evidence to prefer one experiment over other for the same condition. For instance, the two CHD1 ChIP-seq experiments in H1-hESC are dissimilar . Interes primary  and seco4The ENCODE ChIP-seq data is of great value to computational and non-computational biologists alike and is widely used by the scientific community de novo motif discovery (The practical question still remains which replicate to use or what is the best way to merge replicates. Intersecting peak lists is the most conservative approach, and retains a small number of high quality peaks. Although the most reasonable approach in most situations, it suffers a drawback: replicating a ChIP-seq experiment will effectively reduce the number of peaks detected. We therefore suggest other strategies, on a case-by-case basis . For mosiscovery . For RNAiscovery .Taken together, this study demonstrates that the high standards set for technical quality control achieved by the ENCODE consortium does not guarantee the robustness of the sample. This highlights a potential caveat concerning mining of the vast amounts of data deposited in the public domain in general, where even high technical quality of the data is not always documented.The authors declare no competing interests.


Apelin and its G protein-coupled receptor (GPCR) have emerged as a key signalling pathway in the cardiovascular system. The peptide is a potent inotropic agent and vasodilator. Remarkably, a peptide, Elabela/Toddler, that has little sequence similarity to apelin, has been proposed as a second endogenous apelin receptor ligand and is encoded by a gene from a region of the genome previously classified as ‘non-coding’. Apelin is downregulated in pulmonary arterial hypertension and heart failure. To replace the missing endogenous peptide, ‘biased’ apelin agonists have been designed that preferentially activate G protein pathways, resulting in reduced β-arrestin recruitment and receptor internalisation, with the additional benefit of attenuating detrimental β-arrestin signalling. Proof-of-concept studies support the clinical potential for apelin receptor biased agonists. Since its discovery in 1993, there is increasing evidence of the importance of the apelin receptor in the mammalian cardiovascular system. This review provides an update of apelin receptor pharmacology, cardiovascular physiology and pathophysiology, and discusses the role of a second receptor ligand, Elabela/Toddler. Finally, there is increasing impetus to develop biased apelin receptor agonists for clinical use.1 receptor Homo sapiens) apelin receptor consists of 380 amino acids and is identified and conserved in many species, including mouse (Mus musculus), rat (Rattus norvegicus), Western clawed frog , and zebrafish (Danio rerio). To date only one apelin receptor has been shown to exist in mammals, although two subtypes are present in amphibians and fish The apelin receptor  is a class A GPCR discovered in 1993 based on its sequence similarity with the angiotensin ATαiαq-linked activation of phospholipase C (PLC) and protein kinase C (PKC) α13-activated myocyte enhancer factor 2 has been proposed 1]apelin-13 via an interaction with the C-terminal phenylalanine residue in the peptide ligands in vivo has not yet been obtained in native tissue; however, in vitro studies suggest that such receptor complexes may confer novel modes of receptor activation and signalling Apelin responses are in part sensitive to pertussis toxin, consistent with coupling via GThere is widespread distribution of apelin receptor in peripheral organs including heart, lung, kidney, and placenta 1]apelin-13  retains comparable biological activity to the full-length peptide and its endogenous expression can be localised to endothelial cells Evidence indicates that apelin acts as a paracrine/autocrine mediator because peptide expression patterns follow that reported for the receptor pelin-13 , may be (1–16)) or [Pyr1]apelin-13 is reported to favour G protein over β-arrestin signalling (1–16), a linear unmodified peptide, also reported to be G protein biased, lacked vasodilator capacity in an in vivo rat renal afferent arteriolar preparation preconstricted with angiotensin II Development of synthetic apelin agonists has included cyclisation 1 receptor MM54, designed using a bivalent ligand approach, was the first competitive apelin receptor antagonist reported with nanomolar affinity for cloned human apelin receptor but with no affinity for the ATA diverse range of physiological functions of apelin signalling have emerged since its discovery. This review focuses on the regulation of the cardiovascular system by apelin and its receptor. For reviews of other important functions of apelin peptides including fluid homeostasis and metabolism see 1 receptor or infusion of angiotensin 1–7 Important insights into the cardiovascular roles of apelin receptor signalling have been obtained from the generation of apelin 1 receptor double-knockout mouse that exhibits elevated baseline blood pressure Homozygous apelin receptor knockout mice are born in sub-Mendelian ratio apela, was identified in a conserved but previously designated non-coding region of the genome Apela mutant zebrafish exhibit severe developmental defects including rudimentary or absent heart formation, reminiscent of the apelin receptor phenotypes. In addition, the Elabela/Toddler and apelin receptor spatiotemporal expression pattern, mutant rescue, and receptor internalisation experiments also suggest that Elabela/Toddler may be a ligand of the apelin receptor. These studies predicted the existence of three mature Elabela/Toddler peptides with 32, 21, and 11 amino acids , and Na+/Ca2+ exchanger (NCX), together with inhibition of MLC phosphatase 2+-activated K+ channels via phosphatidylinositol 3-kinase (PI3K) in vivo in rats In addition to an effect on the vasculature, apelin may be an important regulator of cardiac contractility via activation of the receptor present on cardiomyocytes 2+ sensitivity 2+]i transients and Ca2+ availability independently of PKC In the heart, apelin signals via phospholipase C, PKCɛ, NHE, NCX apela knockout is anticipated and will confirm whether this is also the case for mammals. However, appropriate expression of apelin may also be required for cardiac progenitor cells The apelin receptor appears to be essential for normal cardiac development, as shown by the severe cardiovascular phenotype of the receptor knockout mice Much of the current interest in apelin and the cardiovascular system has focussed on the potential therapeutic benefit of targeting the apelin receptor in pulmonary arterial hypertension (PAH) and HF. By contrast, there is currently less evidence supporting the contribution of the apelin pathway in other cardiovascular conditions (but see reviews 1]apelin-13 to prevent the development of monocrotaline-induced PAH PAH is a fatal disease characterised by complex remodelling of the pulmonary vasculature, that results in increased pulmonary arterial pressure and vascular resistance, leading to right ventricular hypertrophy and eventually death from right ventricular failure Apelin may be beneficial in PAH by influencing multiple aspects of the disease. First, enhancing apelin signalling lowered indices of pulmonary arterial pressure in animal models in vitro and in vivo, whereas suppression of CD39 gave rise to apoptosis-resistant PAECs which may be responsible for advanced vascular lesions in PAH in vivoSecond, apelin may resolve the imbalance between proliferation and apoptosis in PAH, thus limiting the extent of vascular remodelling. Apelin promotes proliferation in normal PAECs in vivo studies using PAH models consistently reported reduced right ventricular hypertrophy in response to administered apelin Finally, in addition to effects on the pulmonary vasculature, apelin may also beneficially augment right ventricular performance in PAH. Apelin increased contractility in trabeculae from failing right ventricle caused by hypoxia-induced pulmonary hypertension In addition to right ventricular failure in PAH, enhancing apelin receptor signalling may also be beneficial in left ventricular HF. In humans, plasma apelin levels are unaltered or increased in early stages of HF αi signalling is protective αi-biased agonists in this condition The effect of HF on apelin receptor expression is less clear, and levels were reportedly unchanged or reduced in patients with cardiomyopathies Therefore, as predicted, exogenous apelin is beneficial in a range of animal HF models Emerging evidence suggests that apelin receptor signalling is beneficial beyond established HF and confers cardioprotection during myocardial injury. Cardiac apelin expression was upregulated in response to hypoxia/ischaemia apela within a region of the genome hitherto classified as ‘non-coding’ may hold the key to the identification of endogenous ligands for the remaining 130 orphan GPCRs The discovery of a second peptide, Elabela/Toddler, provides an elegant explanation of why apelin peptide knockout does not recapitulate the dramatic failure of the cardiovascular system to develop following deletion of the apelin receptor gene. The identification of


Pain is undoubtedly unpleasant sensation that affects the life style of a large population around the world. Several therapeutic approaches are used for the effective management of pain including use of drugs and alternate measures. This special issue describes the various events in traditional/alternative systems treatment for the management of painful condition.The article entitled “Laser Acupuncture for Postoperative Pain Management in Cats” demonstrated that laser acupuncture significantly reduces postoperative analgesic requirements in cats undergoing ovariohysterectomy. Low back pain (LBP) is one of the most common complaints in the emergency department (ED) but the available therapies are having serious side effects. The clinical study in an article “Efficacy and Safety of Acupuncture for Acute Low Back Pain in Emergency Department: A Pilot Cohort Study” showed that acupuncture offered significant pain reduction in low back pain in patients treated in emergency department without any side effect.µ receptor partial agonist/antagonist widely used because of its potency and safety. The clinical studies in the article “Dezocine Prevents Postoperative Hyperalgesia in Patients Undergoing Open Abdominal Surgery” showed that dezocine offers a significant antihyperalgesic and analgesic effect in patients undergoing elective open gastrectomy for up to 48 h postoperatively.Postoperative pain is very frequent and hard to treat. Dezocine is an opioid The review article “Complementary and Alternative Medicine for the Management of Cervical Radiculopathy: An Overview of Systematic Reviews” concluded that current systematic reviews showed potential advantages to CAM for CR in alleviating neck pain or related symptoms. Due to the frequently poor methodological quality of primary studies, the authors suggested that the conclusions should be treated with caution for future clinical practice.Approximately 75–90% of cancer patients experienced pain while 50% of patients are not satisfied with pain treatment that could affect the cortisol level. The article of this issue “Decreased Cortisol and Pain in Breast Cancer: Biofield Therapy Potential” exhibited marked reduction in cortisol concentration treated with bioenergy, suggesting reduction in stress due to pain.The review article of Lo et al. “The Effects of Acupuncture on Cerebral and Muscular Microcirculation: A Systematic Review of Near-Infrared Spectroscopy Studies” based on available literature believed that research utilizing Near-Infrared Spectroscopy Studies to investigate the hemodynamics of acupuncture presently lacks in scope and quality. Improved designs, for example, placebo-controlled, randomized trials, and standardized intervention reporting could enhance study quality.γ-Terpinene-Mediated Antinociception” the gamma terpinene (γ-TPN), a monoterpene, when studied for antinociceptive effect showed significant antinociceptive activity in various pain assessment models. The results suggest that the γ-TPN (p.o.) produced antinociceptive effect in models of chemical nociception through the cholinergic and opioid systems involvement.In this research study entitled “Involvement of Cholinergic and Opioid System in A research article “Transcutaneous Electrical Acupoint Stimulation Improves the Postoperative Quality of Recovery and Analgesia after Gynecological Laparoscopic Surgery: A Randomized Controlled Trial” based on prospective, randomized, double-blind, placebo-controlled clinical study displayed that preoperative transcutaneous electric acupoint stimulation enhances quality of recovery, improves postoperative analgesia and patient's satisfaction, alleviates postoperative side effects, and accelerates discharge after general anesthesia for gynecological laparoscopic surgery.Auricular point acupressure (APA) is a promising treatment for pain management. The pilot prospective randomized clinical trial entitled “The Anti-Inflammatory Actions of Auricular Point Acupressure for Chronic Low Back Pain” demonstrated that APA treatment affects pain perception in CLBP patients through modulation of the immune system, as reflected by APA-induced changes in serum inflammatory cytokine and neuropeptide levels.β1-receptor intervention.The article of this special issue “Study on the Antinociceptive Effects of Herba Epimedium in Mice” when studied in various pain models evoked marked antinociceptive effect possibly mediated through 5-HT receptors (1A and 2A receptors) and adrenaline The article “Efficacy of Pulsed Radiofrequency on Cervical 2-3 Posterior Medial Branches in Treating Chronic Migraine: A Randomized, Controlled, and Double-Blind Trial” showed in a randomized, double-blind, and controlled clinical trial that the pulsed radiofrequency on the cervical 2-3 posterior medial branches could provide satisfactory efficacy in the treatment of chronic migraine without obvious adverse effects.The research article of the issue “Peripheral Neuropathic Facial/Trigeminal Pain and RANTES/CCL5 in Jawbone Cavitation” exhibited regulated on activation, normal T-cell expressed and secreted (RANTES) overexpression in silent inflamed jawbones as a possible cause for atypical facial pain/trigeminal neuralgia (AFP/TRN). Thus, the surgical clearing of fatty degenerated jawbone might diminish RANTES signaling pathways in neurons and contribute to resolving chronic neurological pain in AFP/TRN patients. Artemisia dracunculus L. Aqueous Extract on Fructose Fed Male Rats” reported that the extract of A. dracunculus elicited significant antinociceptive and anti-inflammatory effects in fructose fed male rats.The research article entitled “The Nociceptive and Anti-Inflammatory Effects ofThe research article entitled “Chinese Herbal Therapy for Chronic Tension-Type Headache” provided the evidence that Chinese herbal therapy can be clinically useful for the treatment of chronic tension-type headache.In short, the various articles of this special issue explored the importance of each traditional/alternative way of treatment and clinical significance thus signifying that pain is best controlled using coordinated efforts using different traditional/alternate measures.Haroon KhanHaroon KhanBruno EtoBruno EtoVincenzo De FeoVincenzo De FeoAnwar-Ul-Hassan GilaniAnwar-Ul-Hassan Gilani


NPM1) mutations are frequently found in patients with acute myeloid leukemia (AML) and the newly generated sequences were suggested to induce immune response contributing to the relatively favorable outcome of patients in this AML subset. We hypothesized that if an efficient immune response against mutated nucleophosmin can be induced in vivo, the individuals expressing HLA alleles suitable for presenting NPM-derived peptides should be less prone to developing AML associated with NPM1 mutation. We thus compared HLA class I frequencies in a cohort of patients with mutated NPM1 , a cohort of patients with wild-type NPM1  and in normal individuals (large datasets available from Allele Frequency Net Database). Several HLA allelic groups were found to be depleted in NPMc+ patients, but not in NPMwt compared to the normal distribution. The decrease was statistically significant for HLA B*07, B*18, and B*40. Furthermore, statistically significant advantage in the overall survival was found for patients with mutated NPM1 expressing at least one of the depleted allelic groups. The majority of the depleted alleles were predicted to bind potent NPM-derived immunopeptides and, importantly, these peptides were often located in the unmutated part of the protein. Our analysis suggests that individuals expressing specific HLA allelic groups are disposed to develop an efficient anti-AML immune response thanks to aberrant cytoplasmic localization of the mutated NPM protein.Nucleophosmin 1 ( NPM1) mutations are the most common single gene abnormality detected in adult acute myeloid leukemia (AML) and might represent the initiating event for AML development in about 60% of AML patients without causative cytogenetic abnormalities [NPM1 mutations [Nucleophosmin 1  since the year 2001. Of those, 152 samples were negative (wild-type), mutation type A was found in 114 samples  and mutation type D in 10 samples. Mutations of other types were found in the remaining 26 samples.A total of 302 samples from AML patients were tested for All patients signed written informed consent to the use of their biological material for research purposes in agreement with the Declaration of Helsinki. The study has been approved by the Ethics Committee of the Institute of Hematology and Blood Transfusion of the Czech Republic.Induction therapy 7+3  is followed by 3–4 cycles of consolidation therapy (HIDAC) in responding patients. The induction cycle 7+3 is repeated in case of persisting residual disease or when 5–10% blasts are detected in the bone marrow aspirates on day 14 or 21. Reinduction/salvage therapy (FLA/Ida) is given to patients with refractory disease. Allogeneic stem cell transplantation is indicated in the following cases: disease refractory to 7+3 therapy, achievement of second complete remission after disease relaps, higher-risk disease, Flt3-ITD positivity.NPM1 cDNA comprising exons 11 and 12 were the following: forward 5´-GGTGGTTCTCTTCCCAAAGT-3´ and reverse 5´-AACATTTATCAAACAC GGTA-3´[Mononuclear cells were isolated from the bone marrow or from the peripheral blood of AML patients. Total RNA was prepared with RNA-Bee—RNA isolation reagent  according to the manufacturer’s instruction. Complementary DNA PCR templates were generated from total RNA (2 μg per reaction) by SuperScript II RNase H reverse transcriptase (Invitrogen). Primers for amplification of a segment of C GGTA-3´. PCR proPatients with AML indicated for related or unrelated hematopoietic stem cell transplantation are routinely typed in class I loci  and in class II loci  on high-resolution level. We use combination of sequence-based typing (SBT) and polymerase chain reaction with sequence-specific primers (PCR-SSP) to solve ambiguous results and all null alleles are excluded –15.The MHC I binding predictions were made on 6/19/2014 using the IEDB analysis resource Consensus tool  which coThe frequencies of the individual class I allelic groups in patients with mutated NPM and with wild-type NPM were compared with normal frequencies of these allelic groups using contingency tables. A p-value of less than 0.05 was considered indicative of a statistically significant difference between groups. Survival curves were created and statistically evaluated using GraphPad Prism software. This software was also used to evaluate the association between changes in HLA-B frequency distribution and the predicted ability of HLA-B alleles to bind NPM-derived immunopeptides (Spearman non-parametric test).Results of HLA typing were available for 63 AML patients bearing type A or type D nucleophosmin mutations (group NPMc+) and for 94 AML patients with wild-type nucleophosmin (NPMwt). HLA I allele frequencies for the two AML patient cohorts were calculated and compared with allele distribution in two merged large datasets obtained from neighbouring European populations: 2907 healthy individuals from Poland and 1698 normal samples from Germany-Austria minority, both of them accessible at Allele Frequency Net Database . As showWe also examined the frequence distribution of HLA class II and noted statistically significant decrease of DRB1*04 in NPMc+ compared to NPMwt patient group . InteresThe depletion of certain HLA class I alleles in NPMc+ AML group indicated that individuals with specific HLA types may develop an efficient anti-NPM immune response and be more resistant to AML development. Subsequently, we searched for possible effect of HLA type on AML patient outcome. In a retrospective analysis, NPMc+ AML patients with complete data (N = 60) were separated into two groups: those bearing at least one of the depleted allelic groups  and those not having any of these alleles. Group characteristics are specified in The analysis of the overall survival revealed significantly better prognosis for patients expressing the selected HLA alleles . On the Immunoepitopes derived from mutated nucleophosmin and restricted to HLA class I molecules were predicted using the Immune Epitope Database (IEDB). We first searched for leukemia-specific peptides involving the mutated C-terminal sequence (beginning at position 288). The cut-off value for percentile rank was set to 1% as recommended by the authors of the site and peptides of 8–11 aminoacids were screened for the probability to be exposed on HLA-A, -B and -C alleles. The aberrant cytoplasmic localization of the mutated protein may, however, favor NPM processing and presentation of all NPM-derived peptides. We thus performed similar search for high-affinity binding peptides  from the whole NPM sequence. As it follows from ex vivo experiments [in vivo conditions which are more complex and can involve a variety of immunosuppresive mechanisms. We hypothesized that if an efficient immune response against mutated nucleophosmin can be induced in vivo, the individuals expressing HLA alleles suitable for presenting NPM-derived peptides should be less prone to developing AML due to NPM1 mutation. Indeed, a few HLA class I alleles had strikingly reduced incidence in NPMc+ patient group compared to healthy controls as well as to the patients with NPMwt  and with high binding affinity (IC50 less than 50 nM). Significant association of changes in HLA frequency distribution due to NPM1 mutation with predicted immunoepitopes was found for HLA-B alleles . While sin vitro responses to stimulation with two peptides derived from mutated C-terminal NPM sequences [in vitro assay used in these studies rather reflects the general ability of patient T-cells to be stimulated by a foreign peptide. This may actually be important for the development of specific T-cell response which would explain the observed difference in the outcome of responding vs non-responding patients. However, any other NPM-derived peptide may have been responsible for the induction of in vivo anti-NPM immune reaction in responding patients.Greiner et al. have recently found significant differences in survival of AML patients between small groups  with different equences . The resequences . We thusex vivo experiments [in vivo [The search for immunopeptides suitable for immunotherapy often focuses on ligands of the most frequent allelic group, HLA-A*02. The peptide AIQDLCLAV (derived from the mutated C-terminus) that had been tested in the above cited eriments  has a pr[in vivo . Thus, HNPM1 mutations are associated with the induction of efficient spontaneous immune response in vivo. This response may rescue a large part of individuals expressing appropriate HLA alleles from developing AML initiated by nucleophosmin mutations and helps achieve durable response to treatment in the remaining cases. The immunogenicity of the mutated protein is probably due to its aberrant cytoplasmic localization and involves mainly non-mutated aminoacid sequences. Although our results need to be validated on larger patient cohorts, they indicate that patients bearing suitable alleles may benefit from immunomodulatory treatment in combination with standard chemotherapy. As our analysis provided useful hints as to suitable NPM-derived immunopeptides, our future project will include attempts to detect specific cytotoxic T-lymphocytes recognizing these peptides in AML patients.In conclusion, although our evidences are indirect, they strongly suggest that S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.