Statins are widely prescribed for reducing LDL-cholesterol (C) and risk for cardiovascular disease (CVD), but there is considerable variation in therapeutic response. We used a gas chromatography-time-of-flight mass-spectrometry-based metabolomics platform to evaluate global effects of simvastatin on intermediary metabolism. Analyses were conducted in 148 participants in the Cholesterol and Pharmacogenetics study who were profiled pre and six weeks post treatment with 40 mg/day simvastatin: 100 randomly selected from the full range of the LDL-C response distribution and 24 each from the top and bottom 10% of this distribution . The metabolic signature of drug exposure in the full range of responders included essential amino acids, lauric acid , and alpha-tocopherol . Using the HumanCyc database and pathway enrichment analysis, we observed that the metabolites of drug exposure were enriched for the pathway class amino acid degradation (p<0.0032). Metabolites whose change correlated with LDL-C lowering response to simvastatin in the full range responders included cystine, urea cycle intermediates, and the dibasic amino acids ornithine, citrulline and lysine. These dibasic amino acids share plasma membrane transporters with arginine, the rate-limiting substrate for nitric oxide synthase (NOS), a critical mediator of cardiovascular health. Baseline metabolic profiles of the good and poor responders were analyzed by orthogonal partial least square discriminant analysis so as to determine the metabolites that best separated the two response groups and could be predictive of LDL-C response. Among these were xanthine, 2-hydroxyvaleric acid, succinic acid, stearic acid, and fructose. Together, the findings from this study indicate that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism may play a role in modulating the response to simvastatin treatment.NCT00451828ClinicalTrials.gov  Statins are HMG-CoA reductase inhibitors that are used to reduce LDL-cholesterol (LDL-C) and, thereby, to reduce CVD risk Multiple intervention trials with statin drugs have demonstrated a reduction in relative risk for both CVD and stroke. Nevertheless, residual CVD risk remains high and LDL-C response varies greatly. Variation in response to statins can be affected by genetic and environmental influences. Several genetic polymorphisms that contribute to variability in the LDL-C response to statins have been identified Inter-individual variation in response to statins, and the fact that LDL cholesterol and other biomarkers are not sufficient to predict clinical benefit or side effects, suggest that more reliable biomarkers are needed for identifying the sub-populations that may achieve the most benefit from statin use and those that might be at risk for developing side effects. Metabolomics provides powerful tools for mapping pathways implicated in disease and in response to drug treatment Previously, using a targeted lipidomics platform, we found that baseline cholesterol ester and phospholipid metabolites were correlated with LDL-C response to treatment in individuals selected from the upper and lower tails of the LDL-C response distribution in the Cholesterol and Pharmacogenetics (CAP) study In the present study, we used a non-targeted, broad spectrum pathway agnostic GC-TOF mass spectrometry platform to measure 160 metabolites in 148 CAP study participants and considered the following questions:What is the metabolic signature of exposure to simvastatin?Which elements of the drug signature correlate with LDL-C response?What metabolites at baseline define distinct metabolic profiles (metabotypes) that can distinguish between good and poor response to simvastatin?An untargeted, broad range GC-TOF metabolomics platform was used to profile plasma from 100 participants from across the full range of the distribution of LDL-C response in the CAP study . SamplesFollowing simvastatin treatment, a significant decrease was found in plasma levels of cholesterol , alpha-tocopherol , and gamma-tocopherol , (vitamin E), and lauric acid  . ChangesAnalysis of the metabolite changes that correlated with LDL-C response in the full range group identified urea cycle intermediates and a group of dibasic amino acids related by shared transporters . SpecifiWe next sought to identify metabolites whose baseline levels were predictive of LDL-C response to simvastatin in the full range group . A test In good responders we found a decrease in cholesterol  and an increase in shikimic acid  . ShikimaWhile metabolomics measurements detected no reduction in plasma cholesterol levels in poor responders  several metabolites did change significantly in the poor responder group upon treatment with simvastatin . In poorWe detected several metabolites with highly significant correlations between pre-treatment levels and LDL-C response to simvastatin in the good responder group . Lower b2 of 0.87 and a Q2 of 0.31. Furthermore, 7-fold cross validation was then used to better evaluate the prediction of drug response. Specifically, in each round of cross validation, about 6/7 of the subjects were used to build a three component OPLSDA model, which was then used to predict the drug response for the remaining 1/7 of the subjects. It was found that on average the OPLSDA models achieved a prediction accuracy of 74% with 70% sensitivity and 79% specificity. The ROC curve of true positive rate vs false positive rate yields an area under the curve (AUC) of 0.84  was used to compare baseline levels of metabolites in good and poor responders . The res of 0.84 . In geneIn this study a mass spectrometry-based metabolomics platform (GC-TOF) that detects small molecules below 550 Da in a broad spectrum of chemical classes and metabolic pathways was used to map global metabolic effects of simvastatin. An untargeted metabolomics approach can yield many unrelated findings; but the dataset reported here has some striking patterns. In broadly sampled full range responders, we have detected an increase in amino acids, specifically essential amino acids and their degradation products. An increase in dibasic amino acids was correlated with response to simvastatin, again not a random set of amino acids, but specifically those transported by the cystinuria and arginine transporters. In poor responders we detected a decrease in plasma fructose, glucose and sugar acids that was not seen in good responders. This combination of findings may suggest a distinct pattern of energy usage between poor responders and the broader spectrum of response phenotypes. Poor responders could also be separated from good responders using an OPLS-DA model of baseline metabolites. Together these findings suggest that metabolomics analysis might be developed for defining a population unlikely to benefit from statin use sufficient to warrant risk of the spectrum of side-effects.A more detailed analysis of the GC-TOF data reveals changes in a diverse set of compounds, some anticipated and some unexpected. The expected changes included decreased free cholesterol in individuals selected from both the full range of LDL-C response  and thosThe increase in creatinine and modulation of several amino acids with simvastatin treatment  was assoPseudouridine, the C-glycoside isomer of the RNA nucleotide uridine, is present in essentially all ribosomal RNAs and seems to be affected by simvastatin. In humans pseudouridine synthase converts uridine to pseudouridine, which is a dead-end metabolite and generally excreted in urine. An increased level of pseudouridine suggests a decrease in the rate of excretion, but together with a reduced level of uridine, a more likely scenario is an increase in the activity of pseudouridine synthase.The inter-correlated changes in several amino acids and their degradation products and the correlation between changes in these amino acids and LDL-C change  point toThe previously published identification of amino acid metabolism as a marker of changes in cholesterol metabolism is consistent with the development of peptide biomarkers of cardiovascular disease The metabolite most significantly different between good and poor responders at baseline was the purine metabolite xanthine, which is implicated in oxidative stress cascades . Xanthinex vivo cultures of intestinal bacteria to metabolize simvastatin have revealed a degradation of drug, producing a series of metabolites including and similar to 2-hydroxyvaleric acid (2-hydroxypentanoic acid) in vivo treatment with simvastatin  are biochemically distinct suggests that metabolomics might be a powerful tool for sub classification of individuals as an important step in streamlining clinical trials and personalizing treatment. Finally, the application of both mass spectrometry and lipidomics platforms in the CAP study has demonstrated the value of this approach in mapping global metabolic effects of simvastatin and in highlighting the pathways that may modulate variation in its clinical efficacy and in its multiple biological effects. We have undertaken this systems biology approach to define pharmacometabolomic changes to generate hypotheses of pleiotropic mechanisms mediating simvastatin effects. These results suggest that functional studies are warranted into the interaction of simvastatin with endogenous and enterobiome metabolites at bile and amino acid transporters.Plasma samples were analyzed from participants in the Cholesterol and Pharmacogenetics (CAP) study, a trial in which 944 Caucasian and African-American men and women with total cholesterol levels of 160–400 mg/dL were treated with simvastatin at 40 mg per day for six weeks . InformaTwo subgroups of participants were selected: the first from the extreme range of response, ‘good and poor responders’, consisted of 24 individuals selected from the top 10% of the LDL-C response distribution who were matched for body mass index, race, and gender to 24 individuals in the lowest 10% of responders, with response to therapy defined as the percentage change in LDL cholesterol from baseline. The second set ‘full range’ was 100 individuals randomly selected from the entire CAP study, excluding participants who had been selected for the extreme range group. These full range participants are representative of the population for age, race, gender, and BMI. On the other hand, the extreme range subset provides a means to explore the differences in metabolite profiles and predictors of the highest and lowest responses to drug therapy. Metabolomic analyses of statin-induced changes in bile acids in both groups Plasma samples (30 µl) were extracted and derivatized as reported previously -1 and 1750 V detector voltage. Result files were exported to our servers and further processed by our metabolomics BinBase database A Gerstel MPS2 automatic liner exchange system was used to inject 1 µl of sample at 50°C (ramped to 250°C) in splitless mode with 25 s splitless time. An Agilent 6890 gas chromatograph (Santa Clara CA) was used with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS column with 0.25 µm 5% diphenyl film and additional 10 m integrated guard column was used . Chromatography was performed at constant flow of 1 ml/min ramping the oven temperature from 50°C for to 330°C with 22 min total run time. Mass spectrometry was done by a Leco Pegasus IV time of flight mass spectrometer with 280°C transfer line temperature, electron ionization at −70 V and an ion source temperature of 250°C. Mass spectra were acquired from m/z 85–500 at 20 spectra sThe Wilcoxon signed rank test was used to evaluate lipid changes in metabolites pre and post treatment. For relationships between changes in metabolite and statin response of LDL-C, metabolite changes were defined as post-treatment level minus pre-treatment level. For the 100 randomly selected full range participants, metabolite changes were correlated with statin response of LDL-C by calculating their Spearman’s correlation coefficients with post-treatment level LDL-C, adjusting for pre-treatment level of LDL-C. The adjusted Spearman’s correlation is equivalent to the Pearson’s correlation between the residuals of the linear regression of the ranks of post-treatment level of LDL-C and the metabolite change on the ranks of pre-treatment level of LDL-C. To associate metabolite changes with statin response of LDL-C using good and poor responder data, the difference of metabolite changes between good responders and poor responders using the Wilcoxon rank sum test was tested. Those significant metabolites are the compounds whose change is associated with statin response of LDL-C.For predicting response of LDL-C to simvastatin, baseline metabolite levels were correlated with statin response of LDL-C by calculating their Spearman’s correlation coefficients with post-treatment level of LDL-C, adjusting for pre-treatment level of LDL-C using the same aforementioned method. In order to associate metabolites at baseline to statin response in LDL-C using extreme subjects’ data, the difference in metabolites at baseline between good responders and poor responders was tested using the Wilcoxon rank sum test. For all the above univariate tests, q-values Correlations among metabolites were obtained by deriving Spearman’s correlation coefficient between each pair of metabolites. Correlation matrixes were used to visualize the correlation between metabolites and drug responses. The modulated modularity clustering algorithm is used to cluster metabolites based on their pairwise Spearman’s correlation coefficients Orthogonal partial least square discriminate analysis (OPLSDA) was used to classify good and poor responders. OPLSDA is built on OPLS Table S1Demographics of CAP patients included in the current GC-TOF metabolomics study. The table lists the comparison of the full-range, good and poor responder subgroups used in this study based on age, gender, race, BMI, change in LDL-C and basal levels of LDL-, HDL- and total cholesterol.(DOC)Click here for additional data file.Table S2Metabolites for which baseline levels were significantly correlated with response to simvastatin in full range participants. The table shows the association of pre-treatment levels of gluconic acid, pseudouridine, maltose, leucine and uridine to the amount of change in LDL-C after simvastatin administration. Metabolites listed are significantly correlated to response to simvastatin based on p-values, but not following correction for false-discovery rate .(DOC)Click here for additional data file.Table S3Metabolites significantly altered by simvastatin in good responders among extreme range participants. The table shows the direction of change in cholesterol, shikimic acid and ethanolamine following simvastatin administration. Metabolites listed are significantly altered by simvastatin based on p-values, but not following correction for false-discovery rate .(DOC)Click here for additional data file.


The successful control of HBV infection requires an efficient expansion of distinct elements of the adaptive immune system  that, due to the hepatotropic nature of HBV, need to operate in the liver parenchyma. In this respect, we will discuss broad features of HBV immunity in patients with resolved or chronic HBV infection and analyze how the liver environment can directly modulate HBV-immunity. A network of cell types, which can play protective or pathogenic roles, forms the anti-viral adaptive immune response during HBV infection. These different components are: a) CD4 T cells, classically referred to as helper T cells. They are robust producers of cytokines and are required for the efficient development of effector cytotoxic CD8 T cells and B cell antibody production. In addition, CD4 T cells can potentially modulate inflammatory events through secretion of pro  or anti-inflammatory (IL-10) cytokines. b) CD8 T cells are able to directly recognize virus-infected cells and clear HBV-infected hepatocytes through cytolytic and non-cytolytic mechanisms , reducinThe ability to develop and preserve an efficient HBV-specific adaptive immune cell network is thought to represent the most important discriminatory factor between HBV control or chronicity , 6. SuchWhat is clear in HBV infection is that HBV-specific CD4 and CD8 T cell frequency and function is far superior in patients who resolve HBV infection than in subjects with chronic infection. The ability of patients who resolve the infection to produce anti-viral cytokines (IFN-γ and TNF-α) results in HBV clearance from infected hepatocytes without extensive direct killing –20. The A level of coordination between the different components of adaptive immunity seems necessary to achieve sustained HBV control. For example, woodchucks infected with WHV (woodchuck hepatitis B virus) demonstrate the importance of a coordinated helper and cytotoxic T cell response in controlling hepadnavirus infections. It was observed that a reduced early expansion of virus-specific T cells during WHBV infection was associated with virus persistence . In patiInterestingly, the development of functional HBV-specific memory CD8+ T cells is linked to down-regulation of PD-1 expression (and up-regulation of the IL-7 receptor CD127) on HBV-specific CD8 cells , 25. IncSince most of these studies have been performed in humans, their observational nature does not allow us to firmly assert that T-cell defects observed in chronic HBV patients are the cause or the consequence of HBV persistence. However, studies in animal models and clinical observations are filling this gap. CD8 T cell deletion experiments performed in HBV-infected chimpanzees have provided strong support for the concept that CD8 T cells are responsible for viral clearance . FurtherAdditional evidence that CD4 and CD8 T-cell responses are accountable for the immunological control of HBV are represented by the association of particular HLA class I and class II genetic profiles with resolution . InteresChronic viral infection alters the quantitative and functional profile of virus-specific immunity. Detailed functional studies have mainly focused on the analysis of CD8 T cells, which during persistent viral infection lose their ability to lyse, proliferate and produce different cytokines , and only partially maintain the ability to produce IFN-gamma . This dy6 HBV-DNA IU/mL) and HBV-specific T cells are often undetectable in the majority of patients with higher levels of HBV replication . T cells specific for HBV antigens that are more expressed in the liver are deleted in HBV transgenic mice [7 copies/mL [HBV-specific CD8+ T cells detected during chronic infection often display similar defects. They produce low levels of antiviral cytokines, proliferate poorly in vitro, express the inhibitory receptor PD-1, and are prone to apoptosis , 35. Hownic mice , and theopies/mL . Importaopies/mL .3–106 fold excess over whole virions and can reach 1–10 ug/mL in the serum [We believe that the prolonged exposure to large quantities of soluble HBV antigens (HBsAg and HBeAg) and the tolerogenic features of the liver are the principal causes of the functional alteration and deletion of virus-specific B and T cells present in chronic HBV patients. Antigen persistence alters T-cell function and causes deletion in different viral infections . In HBV he serum .Such a high and persistent load of viral antigens allows them to be cross-presented by liver professional antigen-presenting cells , which can have a tolerizing effect , 41. ThiAdditional factors might contribute to the maintenance, if not directly induce, HBV-specific immune dysfunction. Some could exert their function mainly in the intrahepatic environment, while others are not topologically restricted to the liver . Regardiin vitro, but such modulation was HBV-specific for some [The impact of T regulatory cells in the modulation of HBV-specific T cell immunity is also controversial . Trfor some , but notfor some .We think it is important to consider that these studies were limited to the analysis of the CD4+ CD25+ cells present in the blood, while a detailed analysis of the intrahepatic frequency and function of these cells has not been performed. Furthermore, it is possible that a population of HBV-specific regulatory cells, different from the CD4+ CD25+ T cell subset, analogous to the presence of IL-10-producing HCV-specific T cells , might bWe have discussed the features of adaptive immunity in patients with chronic and resolved HBV infection and the potential mechanisms that are likely to affect their quantitative and qualitative profile. A final issue that we would like to briefly discuss is the modulation of effector T cell function caused by the liver environment. This aspect is different from the tolerogenic features of the liver which act on the induction phase, and instead deals with the potential direct influence of liver environment and hepatocyte antigen presentation on T cell effector function.The fact that virally infected hepatocytes are largely resistant to perforin/granzyme-mediated killing , but higet al. have shown in an in vivo model of HBV infection that effector T cells can change their cytolytic or non-cytolytic function after recognition of antigen presented by hepatocytes [Isogawa atocytes . Their datocytes  or be a atocytes . Both hyatocytes . On the atocytes .7 copies/mL serum), while chronic patients that control HBV replication (< 106 copies/mL serum) can do so without signs of liver inflammation. In both situations, the quantity of intrahepatic HBV-specific CD8+ T cells are similar and the discriminatory factor is the level of HBV replication, suggesting that the availability of antigen modulates T cell function [The possibility that increased availability of MHC-viral peptide complexes on hepatocytes, as a consequence of different levels of HBV replication, could trigger differential function in HBV-specific T cells is consistent with clinical and new experimental observations. Hepatic flares are associated with increased levels of HBV replication (> 10function .The concept that effector T cells can modulate their function in relation to different levels of hepatocyte antigen presentation is further supported by recent observations that classical Th1/Tc1 HBV-specific T cells can produce the neutrophil chemotactic factor CXCL-8 when stimulated by high antigen dose. Parenchymal recruitment of granulocytes is a common step of immunopathological processes triggered by CTLs during infection with non-cytopathic viruses , and seeThe cross-talk between different components of immunity, like the ability of HBV-specific T cells to produce chemokines involved in the recruitment of granulocytes, opens a further chapter; the influence of other cell types on the function of adaptive immunity in the liver. Work on HBV transgenic mice has clearly shown that activated platelets influence the recruitment of virus-specific CTLs into the liver . Plateleet al. has shown that depletion of arginine in the inflamed liver can directly interfere with intrahepatic T cell function, particularly with their ability to produce IL-2 and proliferate [Thus, protective or pathogenic effects of HBV-specific T cells in the liver are not only dependent on their quantity and intrinsic function, but can be modulated by external factors like platelets and granulocytes. In addition to these data, a recent report by Das liferate . This adBeyond the well characterized differences in HBV adaptive immunity of resolved and chronic patients lie a number of issues that have been difficult to determine. We know that the host response to HBV infection is a complex coordinated process and that the liver environment might tune effector T cell function. Studies have begun to shed light on the peculiarity of the intrahepatic environment and how this can modulate virus-specific immunity. In fact, modification of the liver environment may already be the basis of the therapeutic effect of IFN-alpha that, through activation of the immunoproteasomes in hepatocytes, can change the quantity of HLA-class I/peptide complexes available for CD8+ T cell recognition. These types of questions are exceedingly difficult to study in humans but it is necessary to understand how the immune system operates in the liver if immunotherapeutic strategies under development are to have any success in achieving sustained viral control.In addition to the liver environment, a better understanding of how the network of immune cells, parenchymal/non-parenchymal cells, and platelets interact and communicate to achieve HBV clearance or liver damage is critical. Multiple components are involved in each process and being able to separate protective from inflammatory processes could increase the efficacy and safety of HBV treatment. As a further and final note, the study of HBV-specific adaptive immunity in patients is heavily skewed towards analysis of T cell function. Although there is no doubt that T cells play an essential role during HBV infection, perhaps new exciting revelations of HBV pathogenesis will derive from new accurate studies of HBV-specific B cell function.


Reperfusion hemorrhage is an independent predictor of adverse left-ventricular remodeling following acute myocardial infarction. Iron chelation may potentially alleviate the toxic and pro-inflammatory effects of iron degradation products. CMR can provide insights into the interaction between hemorrhage and iron chelator.It has been speculated that iron chelation may be beneficial in acute myocardial infarction (AMI) and that early treatment can limit ischemia-reperfusion injury and also reduce infarct size. However, the role of iron chelation in hemorrhagic myocardial infarction, where it would be most suited, has not yet been explored. Reperfusion hemorrhage results in accumulation of iron degradation products of hemoglobin that may be pro-inflammatory as free iron is toxic in nature. The purpose of the study was to investigate the interaction between iron chelating agent deferiprone (DFP) and hemorrhage in a porcine model of myocardial infarction and monitor remodeling by cardiovascular magnetic resonance (CMR).The study involved two groups of animals that were subjected to a 90 min balloon occlusion of the LAD followed by reperfusion - untreated (N=2) and DFP treated (N=2). DFP  was administered  a few hours before the procedure (pre-loading) and treatment was continued with a daily dose of 100 mg/kg. Imaging was performed on a 3T MRI scanner  pre-AMI  and day 2-week 4 post-AMI. Edema was evaluated by T2 quantification using a T2-prepared spiral sequence and hemorrhage was identified by T2* determined using a multi-echo gradient-echo acquisition. Infarct assessment was performed by delayed hyperenhancement (DHE) using an IR-GRE sequence.Figure Hemorrhage may be a source of iron toxicity and a mediator of inflammation, directly contributing to adverse remodeling in the setting of AMI. DFP was able to penetrate the infarct zone and was also effective in neutralizing hemorrhagic byproducts. Elimination of hemorrhage resulted in faster resolution of edema and normal ventricular volumes, representing a beneficial remodeling process. Iron chelation could potentially serve as an adjunctive therapy in hemorrhagic AMI.We would like to acknowledge funding support from the Ontario Research Fund, the Canadian Institutes of Health Research and GE Healthcare.


INTRODUCTION: The purpose of this study was to evaluate the morphologic surface of guttapercha cones after cutting with different methods.MATERIALS AND METHODS: The apical 3 millimeters of forty standardized, gutta-percha cones size 40 were cut off using scissors or a scalpel against a glass slab. The samples were then examined under scanning electron microscopy (SEM) for topographic deformity.RESULTS: According to results, cutting with scissors produced significant topographic deformity in the standardized gutta-percha cone surface but cutting with sharp surgical instrument against a glass slab allowed the development of a smooth and unmodified guttapercha cone surface.CONCLUSION: Results of this study recommended that cutting the tip of a guttapercha point with a sharp scalpel against a glass slab would produce more reasonable surface morphology than using scissor for the same procedure. The objectives of modem endodontic therapy are to clean and shape the root canal system to create a fluid tight seal along the entire length of the cavity . In otheThe fitting of the master cone in the apical portion of the root canal plays a very important role in the attainment of an adequate apical seal. In addition, fitting is important in preventing the extrusion of the filling material through the apical constriction .There have been many studies on the gutta percha cones used in endodontic therapy. The sizing of gutta-percha cones is based on having a similar size and taper as the endodontic files. However, studies have shown that there are variations existing between different brands of cones and discrepancies in the diameters of instruments and gutta-percha points of the same size -6. Thus;The aim of this study was to investigate the surface morphology of standardized gutta percha cones after cutting them with two different methods.Forty gutta-percha standardized cones size 40  were used in this experiment. Cones were divided in 2 groups of 20 samples each. In group 1, the cones were clipped at 2-3 mm from the tip by means of a sharp surgical scissors . In group 2, cones were cut with a sharp scalpel blade No.15  against a glass slab . Gutta-percha cones were then investigated without any coating process using a conventional and low vacuum scanning electron microscope (SEM) . Scanning electron micrographs of the gutta-percha cones were evaluated by three endodontists, according to the following criteria. Morphological event was evaluated by scores of 1 to 2, with 1 being the supenor.Cutting surface:1- Presence of one plane2- Presence of two planesFlange:1- Absent2- Presence of one or two flangesBurs:1- Absent2- PresentInvestigation of the standardized gutta-percha cones after two different cutting procedures revealed different features. In group 1, the cut surfaces of all samples showed two plane, one or two convergent flanges and burs . In grouCutting of gutta-percha cones with sharp surgical instrument or scissors is commonly necessary during the master cone -fitting procedure. Discrepancies induced in the cone tip after cutting may theoretically impede a good adaptation in the apical portion of the root canal. In consequence, inadequate master cone fitting may compromise the attainment of the apical seal provided by the root canal filling. The cut of the gutta-percha cones occurs by the application of a shear stress in the transverse section of the cone. The cutting procedure induces an elastic compression of the material.Close to the cut surface, cone fibers bend in a similar direction to the movement applied to the cutting instrument. When the applied pressure overcomes the elastic resistance of the material, the cone is separated in two parts. The cone fibers bending during the cut show a plastic deformation in the whole cross-section of the material . The resThe results of this research showed that the tips of standardized gutta-percha cones can be regularly cut off using sharp surgical instrument against a glass slab. Cuts using scissors produced irregular surfaces that might impede adequate master cone fitness.


Transient or long-term DNA self-assembly participates in essential genetic functions. The present review focuses on tight DNA-DNA interactions that have recently been found to play important roles in both controlling DNA higher-order structures and their topology. Due to their chirality, double helices are tightly packed into stable right-handed crossovers. Simple packing rules that are imposed by DNA geometry and sequence dictate the overall architecture of higher order DNA structures. Close DNA-DNA interactions also provide the missing link between local interactions and DNA topology, thus explaining how type II DNA topoisomerases may sense locally the global topology. Finally this paper proposes that through its influence on DNA self-assembled structures, DNA chirality played a critical role during the early steps of evolution. Transient or long-term DNA self-assembly participates in essential genetic functions. For example, the compaction and the three-dimensional organisation of DNA are crucial for cellular processes such as transcription, DNA replication and segregation of daughter chromosomes during cell division. Also, bringing DNA sites into proximity is required for DNA recombination, chromatin packaging and building architectural complexes that control transcription and replication –3. AlthoIn contrast, the closely packed DNA helices observed in DNA crystals are profoundly influenced by the DNA geometry, chirality and sequence –13. The right-handed crossovers since they are characterized by positive values of their crossing angle. Since the sugar-phosphate backbone is fitted into the major groove, the crossing angle is dictated by the chirality and geometry of the double helix—mainly the angle between the major groove and the helical axis. Thus, the self-fitting of double helices into a crossed structure both avoids electrostatic repulsion between double helices and optimizes energetically favourable intermolecular contacts . Th. Th22]. contacts ,24. Remacontacts . Interescontacts . Inter-hcontacts ,28. Morecontacts ,30. The contacts . ProbablLeft-handed B-DNA crossovers that are characterized by negative values (−40° and −80°) of the crossing angles have been also observed in many RNA or DNA crystals. However, this mode of assembly in which the helices are juxtaposed by groove-groove interactions, prevents the tight self-fitting of the double helices and is neither stabilized by direct sequence-specific contacts between DNA segments, nor by intermolecular divalent cation bridges  Figure .Right-handed crossovers of B-form DNA double helices are therefore unique in that they are assembled by a sequence-dependent interaction. Crystallographic studies have shown that while the B-DNA double helix dictates the geometry of inter-helical assembly, cytosines play a key role in controlling the interaction through specific interaction of their N4 amino groups with phosphate groups –24. ThesThus, the formation of inter or intramolecular H-bonds between the N4 amino group of cytosine and a phosphate group plays a key role in controlling DNA-DNA interactions in a sequence dependent manner.Consistent with these findings, a recent survey of the Nucleic Acids Database shows that, without exception, cytosine-phosphate interactions are strictly required for stabilising right-handed DNA crossovers . ProbablIn addition, crystallographic studies of methylated DNA duplexes showed that C5-methyl cytosines also promote and stabilize the formation of DNA crossovers at the modified C5-mpG sequences . The twoet al. (2009)  of 600 mM) but increasing attraction above [Mg2+] of 50 mM [2+[2+ ions are also required for the folding of both DNA and RNA molecules. They mediate the folding of Holliday junctions from a planar open structure into a compact stacked conformation [2+[The strict requirement for Mgof 50 mM . Also, rof 50 mM . These fof 50 mM . In addi50 mM [2+. Divalenormation . Indeed,ation [2+,34. The ation [2+–43. A coation [2+.Like a directing piece for a “supramolecular construction set”, the B-DNA double helix dictates the overall geometry of DNA self-fitted assemblies. As exemplified in In this context, the recent discovery that right-handed crossovers are stable at close to physiological conditions  supportsOur model proposes that the cell may dispose of a collection of DNA-DNA interactions with varying degree of stability that can be exploited for tuning chromatin compaction. In addition, the finding that 5-methyl cytosine promotes close DNA-DNA interactions  may alsoin vivo[Transient DNA-DNA close interactions also play a critical role in the control of DNA topology. Indeed, within the interwound plectonemic supercoiled DNA, tight intersegmental contacts occur in the presence of divalent cations, under physiological conditions ,50. The in vivo–56. Typein vivo.Although acting locally, a remarkable property of type II topoisomerases is their sense of the global DNA topology. For example, when a negatively supercoiled ring is singly linked to a nicked ring, these enzymes preferentially unlink the ring rather than remove the supercoils . TopoisoApplying our crystallographic lessons to DNA topology brought two useful insights to solve this question. First, it has been demonstrated that the various topological states of the cell are associated with different local inter-segmental interactions . KnowingStable right-handed crossovers constitute therefore the local signature of an unusual topological state in the cell, such as positively supercoiled or relaxed DNA. Local information such as the differential stability and geometry of crossovers may be therefore exploited for sensing the global topology of DNA . This suSecond, a recent study has demonstrated how binding right-handed crossovers specifically across their large angle imposes a different topological link between the type II topoisomerases and the plectonemes of opposite sign , top 2121. WithiDNA topology and topoisomerases have been the subject of adaptive pressure in organisms that live at different temperatures for maintaining the balance between the melting potential and functional stability . The dynThus, among other physical properties of DNA, such as its anisotropic flexibility ,76, or tIndeed, in contrast to life at high temperature that can tolerate various topological states of DNA—from negative supercoiled DNA to slightly positively supercoiled DNA –81, adapOn the other hand, emerging from the RNA world, the DNA has been selected to support the genetic information of complex organisms. It could be speculated that this early “choice” of the double helix has been influenced by its ability to self-assemble into a simple hierarchic organization. Indeed, RNA and DNA not only differ by their respective chemical stabilities, but also by their packing modes and consequently the overall architecture of their supramolecular assembly. Although similar electrostatic rules govern the self-contacts between RNA and DNA molecules, their helical interactions differ as a consequence of their distinct secondary structures. In addition, to adopt a regular A-conformation, the RNA molecules most often fold into more complex structural motifs due to the presence of the 2′-hydroxyl group and extended base pairing rules . RNA strin vivo molecular interactions and bring precious insights about how a molecule is influenced by a defined intermolecular environment. Many crystal packings of nucleic acid double helices are indeed very similar to the dense packing of double helices observed in large nucleic acid assemblies such as the recently solved ribosomal particles or nucleosomal DNA. In the present review, an analysis of the reciprocal influence of the double helix structure on its crystal packing shows how the interplay of DNA sequence and electrostatic environment controls DNA-self assembly. This study reveals how tight DNA-DNA interactions greatly enhance the effect of chirality on the overall architecture of DNA higher-order structures thus providing the missing link for the local sensing of the global DNA topology by type II DNA topoisomerases. This study also suggests that the structural simplification of the genetic material in passing from the RNA to the DNA world has probably contributed to the storing and packing of larger genomes, in favouring the simple and codified hierarchical assembly of double helices. The detailed knowledge of the structure and energetics of close DNA-DNA interactions is therefore indispensable for a complete understanding of the genetic functions.Crystal packing effects and crystal intermolecular contacts are most often considered as disturbing artefacts in crystallography whose main purpose is to solve the structure of a single molecule in a “structure-function” perspective. An alternative point of view is that crystal packing may mimic However, many questions remain to be solved as for example how do the B-A transitions affect the energetic and sequence dependence of tight DNA interactions? It has been proposed that the A-form of DNA protects the geometry of the active site of DNA-polymerases against deleterious structural variations of sequence hotspots, thus directly contributing to DNA-polymerase fidelity . More re


Nonsense Mediated Decay (NMD) degrades transcripts that contain a premature STOP codon resulting from mistranscription or missplicing. However NMD's surveillance of gene expression varies in efficiency both among and within human genes. Previous work has shown that the intron content of human genes is influenced by missplicing events invisible to NMD. Given the high rate of transcriptional errors in eukaryotes, we hypothesized that natural selection has promoted a dual strategy of “prevention and cure” to alleviate the problem of nonsense transcriptional errors. A prediction of this hypothesis is that NMD's inefficiency should leave a signature of “transcriptional robustness” in human gene sequences that reduces the frequency of nonsense transcriptional errors. For human genes we determined the usage of “fragile” codons, prone to mistranscription into STOP codons, relative to the usage of “robust” codons that do not generate nonsense errors. We observe that single-exon genes have evolved to become robust to mistranscription, because they show a significant tendency to avoid fragile codons relative to robust codons when compared to multi-exon genes. A similar depletion is evident in last exons of multi-exon genes. Histone genes are particularly depleted of fragile codons and thus highly robust to transcriptional errors. Finally, the protein products of single-exon genes show a strong tendency to avoid those amino acids that can only be encoded using fragile codons. Each of these observations can be attributed to NMD deficiency. Thus, in the human genome, wherever the “cure” for nonsense (i.e. NMD) is inefficient, there is increased reliance on the strategy of nonsense “prevention” . This study shows that human genes are exposed to the deleterious influence of transcriptional errors. Moreover, it suggests that gene expression errors are an underestimated phenomenon, in molecular evolution in general and in selection for genomic robustness in particular. In biological systems mistakes are made constantly because the cellular machinery is complex and error-prone. Mistakes are made in copying DNA for transmission to offspring (“genetic mutations”) but are much more frequent in the day-to-day task of gene expression. Genetic mutations are heritable and therefore have long been the almost exclusive focus of evolutionary genetics research. In contrast, gene expression errors are not inherited and have tended to be disregarded in evolutionary studies. Here we show how human genes have evolved a mechanism to reduce the occurrence of a specific type of gene expression error—transcriptional errors that create premature STOP codons . Nonsense errors are potentially highly toxic for the cell, so natural selection has evolved a strategy called Nonsense Mediated Decay (NMD) to “cure” such errors. However this cure is inefficient. Here we describe how a preventative strategy of “transcriptional robustness” has evolved to decrease the frequency of nonsense errors. Moreover, these “prevention and cure” strategies are used interchangeably—the most transcriptionally robust genes are those for which NMD is most inefficient. Our work implies that gene expression errors play an important role as supporting actors to genetic mutations in molecular evolution, particularly in the evolution of robustness. Drosophila melanogaster, PABP-dependent NMD is the primary pathway in operation since only a minority of spliced transcripts are under the surveillance of EJC-dependent NMD Saccharomyces cerevisiae only the intron-independent NMD pathway is known to operate. Finally, in Schizosaccharomyces pombe, although intron-dependent NMD is active it is believed to be EJC-independent In mammalian transcripts, premature termination codons (PTCs) are normally detected by one of two NMD pathways −5 errors per nucleotide −3 errors per nucleotide PTCs may arise from heritable nonsense mutations in the germline or they can be created by transient errors in transcription and splicing. Previous attention has been paid to splicing errors as a source of nonsense errors since introns retained in the mature mRNA can either introduce an intron-encoded PTC or induce a frameshift leading to the formation of an exon-derived PTC The possible consequences of translating PTC-containing transcripts include loss of functional molecules, dominant negative interactions and gain-of-function activities. The deleterious effects of these outcomes are highlighted by the fact that, in mammals, knockouts of the core effectors of NMD have lethal effects NMD mitigates the negative effects of PTC-containing transcripts and hence can be considered as providing a partial “cure” for transcriptional errors. In mammals, the efficiency of NMD in detecting PTCs varies both among and within genes. Single-exon genes appear insensitive to NMD In this study we propose that natural selection has promoted a dual–strategy of prevention and cure to deal with nonsense errors in gene expression. Specifically we predict that selection compensates for the partial efficiency of NMD through a preventative approach of “transcriptional robustness” that minimizes the consequences of transcriptional error. We investigated whether human genes eschew codons that can be changed into a stop codon by one mutation (‘fragile codons’ Schizosaccharomyces pombe) in which a mechanistically distinct form of intron-dependent Nonsense Mediated Decay appears to be active.Using comparisons between single and multi-exon genes (intergenic) and between different exons of multi-exons genes (intragenic), we show how each of these mechanisms is used to increase transcriptional robustness of mammalian genes. Single-exon genes should provide the clearest signal of transcriptional robustness and thus provide a “litmus test” of this hypothesis. Therefore, our first, intergenic, analysis contrasts fragile codon usage and fragile amino-acid usage in single-exon genes and multi-exon genes. We performed a second, intragenic, analysis at the exon-level by taking advantage of the fact that, in mammals, last exons of multi-exon genes, like single-exon genes, are invisible to EJC-dependent NMD. Finally, we demonstrate that the two hallmarks of transcriptional robustness detectable in mammals (synonymous codon usage and amino-acid usage) can also be detected in the genes of fission yeast  metric. Being mutational neighbours of STOP codons, the 18 fragile codons are relatively AT-rich. It is known that GC-content varies along chromosomes and that these variations in base composition affect both synonymous codon usage and amino-acid usage. These variations are particularly strong within mammalian genomes . To account for both this compositional bias and the influence of amino acid content, we used codon usage among facultative amino acids as the basis of the NFCU metric. NFCU measures the relative usage of fragile and robust codons among 5 groups of codons where all codons in a given group (i) have the same GC-content and (ii) are synonymous.p<10−15, Wilcoxon rank sum test; D.melanogaster in which both gene sets should on average be equally well served by NMD since the intron-dependent NMD pathway shows much reduced activity in fly We measured NFCU in 2422 single-exon genes and in 20563 multi-exon genes in the human genome. We observe a 8% depletion of fragile codons in single-exon genes that is highly significant  shows that the depletion of fragile codons in human and mouse single-exon genes is not a consequence of amino acid usage. Therefore, we can ask whether there is evidence for usage of the second mechanism of transcriptional robustness by testing whether the amino acid usage of single-exon gene products reduces the frequency of nonsense transcriptional errors. Accordingly, we observed that the usage of fragile amino acids (FAU) among proteins encoded by single-exon genes is 17% lower than those of multi-exon genes in human and 21% lower in mouse whereas no difference is observed in fly . TherefoWe repeated this analysis to control for GC content differences between single-exon and multi-exon genes. We used a normalized fragile amino-acid usage metric (NFAU) that considers the relative usage of fragile amino-acids among two groups of amino-acids that are encoded by codons having the same GC-content. Once GC content is controlled for we observe that the fragile amino acid content of single-exon gene products is 12% lower than those of multi-exon genes in human and 16% lower in mouse whereas a 2% enrichment is seen in fly .Thus far our analysis has demonstrated that, in human and mouse, single exon genes bear two hallmarks of transcriptional robustness relative to multi-exon genes (i.e. depletion of fragile codons and amino acids). These hallmarks highlight two mechanisms by which natural selection can prevent nonsense transcriptional errors: at the level of gene sequence through synonymous codon choice and at the level of protein sequence through amino-acid choice. Provisionally, we can attribute this observation to the inactivity of EJC-dependent NMD in single-exon genes. However, since all biological processes are inherently inefficient, EJC-dependent NMD is inevitably also suboptimal. This raises the question whether there is a genome-wide requirement for transcriptional robustness. One approach to answering this question is to ask whether, among all human genes, there is correlated usage of the two mechanisms (codon-level and protein-level) to achieving transcriptional robustness.a/Ks (the ratio of non-synonymous to synonymous substitution rates). For the vast majority of human genes, estimates of Ka/Ks lie in the range 0-1. Broadly, we might expect two different patterns: (i) For genes with Ka/Ks close to 1, synonymous and non-synonymous sites are equally modifiable. Therefore, where nonsense errors are deleterious, natural selection can use both mechanisms to increase transcriptional robustness (i.e. both fragile codons and fragile amino-acids can be depleted) leading us to expect an overall positive correlation between fragile codon usage and fragile amino-acid usage. (ii) For genes with Ka/Ks close to 0 there is strong selective constraint on the protein sequence and non-synonymous sites are much less modifiable than synonymous sites. Here natural selection can use only one mechanism to increase transcriptional robustness: that of synonymous codon choice since transcriptional robustness can be less readily increased through amino-acid choice. Notably, the protein products of many such genes might be enriched in fragile amino acids due to functional requirements e.g. proteins involved in signal-transduction tend to be tyrosine rich and zinc-finger proteins and proteins enriched in disulfide bonds are cysteine rich. If nonsense errors are deleterious in such genes then there should be strong counterselection of fragile codons to compensate for functionally-determined fragile amino-acid content. For genes under strong selective constraint at the amino-acid level we would therefore expect an overall negative correlation between fragile codon usage and fragile amino-acid usage. Among genes with strong selective constraint on protein sequence, the magnitude of this negative correlation is likely to depend on the abundance of genes with functions requiring high fragile amino-acid content.However, for a given gene the nature of the correlation between these two mechanisms is likely to depend on the relative level of constraint on synonymous and non-synonymous sites as measured by Kp<10−15). More specifically, for human genes having mouse orthologs we can determine how this correlation depends on the strength of selective constraint. We observe that the sign and magnitude of the correlation between both transcriptional robustness mechanisms (codon-level and protein-level) is dependent on the strength of selective constraint on protein sequence in agreement with our prediction  we see a positive correlation between both transcriptional robustness mechanisms , we observe a positive correlation between normalized fragile codon usage (NFCU) and normalized fragile amino-acid usage (NFAU)  for NFCU versus NFAU in Ka/Ks quartiles 2-4: 0.050 (p = 0.001); 0.068 (p<10−5); 0.076 (p<10−6)). Therefore with increasing ‘flexibility’ of the protein sequence there is an increasing tendency for transcriptional robustness to be realized not only through synonymous codon choice but also through amino-acid choice.First, for genes towards the upper end of the Ke (NFAU) . In othea/Ks for the first quartile of selective constraint  =  0.03) the correlation between transcriptional robustness mechanisms is negative (Spearman's rho for NFCU versus NFAU in Ka/Ks quartile 1: -0.025 (p = 0.09); However, for genes under strong selective constraint at the protein level . Since these hexamer sequences overlap codons, their presence imposes additional constraints on the coding sequence of multi-exon genes We found that ESEs indeed tend to encode fragile codons and that ESEs are depleted in single-exon genes. However the difference in fragile codon usage between single and multi-exon genes persists when this is controlled for showing that our observation is not a side-effect of differences in splicing related constraints .Drosophila genome as a negative control.The patterns of fragile codon usage in single-exon and multi-exon genes could be due to a more familiar source of codon usage bias that has nothing to do with their propensity for nonsense errors. In fly, selection for translational efficiency or accuracy leads to the preferential usage of optimal codons in highly expressed genes op) Among codons we see no association in either human or fly between fragility with respect to nonsense errors and translational optimality  are thought to be detected by the EJC-dependent NMD pathway suggests that, similarly to single-exon genes, last exons of multi-exon genes constitute a blind-spot for EJC-dependent NMD and are served only by PABP-dependent NMD. Although a mechanism for detection of PTCs downstream of the last EJC has been described (“fail-safe” NMD), its activity is believed to be restricted to exceptional targets p = 0.0002, Wilcoxon rank-sum test) 7% depletion among last exons  compared to upstream exons   among last exons  compared to upstream exons . As a control we performed the same analysis on Drosophila multi-exon genes in which NMD should be equally efficient in upstream and last exons. In accordance with our expectation we saw no depletion of fragile codons among last exons  compared to upstream exons  of Drosophila multi-exon genes.For human multi-exon genes whose coding sequence is completely contained within the last exon we see a significant 8% depletion of fragile codons compared to multi-exon genes having at least two coding exons (see  = 0.50) . We repep<10−16, binomial test). Considering NFCU for all 12324 pairs of upstream and last exons showed that their fragile codon content is significantly different .These results were confirmed by an analysis based on matching the last exon of each human multi-exon gene with the upstream sequence of the same gene. There are 12324 human multi-exon genes with reliably annotated last exons and for which NFCU is defined in both the last exon and in the upstream sequence. Among the 12265 genes for which NFCU differs between these two regions, for 6599 (54%) fragile codon content is lower in the last exon than in the upstream sequence whereas in 5666 genes NFCU in the last exon is greater than in the upstream sequence . If these PTCs remain undetected by NMD, the resultant short peptide truncations should have trivial fitness consequences compared to longer peptide truncations caused by undetected PTCs in upstream exons. Consistent with this, the fragile codon content of last exons of human multi-exon genes (median NFCU = 0.47) is greater than that of human single-exon genes (median NFCU = 0.43) despite the fact that both are invisible to EJC-mediated NMD.We attempted to control for the effect of trivial peptide truncation by analyzing NFCU in regions on either side of the boundary of EJC-dependent NMD but located at least 50 codons away from the normal termination codon. We focused on multi-exon genes having reliably annotated last exons longer than 100 codons and having at least 50 codons in the NMD-competent region of upstream exons . Specifip = 0.016, Wilcoxon rank-sum test). As a negative control we repeated this analysis on Drosophila multi-exon genes in which EJC-dependent NMD shows reduced activity implying that surveillance of nonsense errors should be equally efficient in the 5′ and 3′ windows. As expected we saw no difference in fragile codon density between these window sets .In this analysis we saw a 10% reduction in fragile codon density in 3′ (NMD-compromised) windows compared to 5′ (NMD-competent) windows .In summary, we see a consistent pattern of depletion of fragile codons in the last exons of human multi-exon genes when compared with upstream exons. This accords with their status as NMD-compromised regions that are invisible to EJC-dependent NMD and visible only to the PABP-dependent NMD pathway. The selective pressure to avoid fragile codons in last exons imposed by the requirement for transcriptional robustness is likely to be partly offset by increased tolerance of PTCs lying close to the NTC. Nevertheless, transcriptional robustness appears to be an important determinant of codon choice in the last exons of human multi-exon genes as well as in single-exon genes.Since transcriptional robustness is likely to be a consequence of reduced NMD potency we can attempt to dissect the relative contributions to this phenomenon of each of the two mammalian NMD pathways. The large depletion of fragile codons in histone genes relative to other single-exon genes  suggestsp<10−15), 0.90 (p<10−15), 0.97 (p = 0.03), 0.99 (p = 0.91), respectively. Thus only the longest single-exon transcripts have fragile codon content equal to that of multi-exon transcripts of similar length. This result suggests that the increased transcriptional robustness of single-exon genes is primarily due to the lack of supplementary nonsense surveillance from EJC-dependent NMD. However, the longest single-exon transcripts also benefit from an increase in the efficiency of PABP-dependent NMD.We investigated whether decreased efficiency of PABP-dependent NMD alone is responsible for the greater transcriptional robustness of single-exon genes due to the shorter length of their transcripts. We compared NFCU between single and multi-exon genes binned by CDS length  and founRepeating this procedure in our analysis of fly genes revealed no difference in NFCU between single-exon and multi-exon genes when length differences are controlled for (data not shown). Thus the modest 2% depletion of fragile codons seen in fly single-exon genes compared to multi-exon genes is entirely due to the inefficiency of PABP-dependent NMD of shorter transcripts .Schizosaccharomyces pombe in a manner apparently independent of the EJC S.pombe genes and found that single-exon genes (median NFCU = 0.43) show a highly significant  6% depletion of fragile codons relative to multi-exon genes (median NFCU = 0.46) echoing our observations in human and mouse. However, in contrast to the situation in mammals and fly, the CDS of single-exon genes is longer than that of multi-exon genes in S.pombe (the coding sequence (CDS) of S.pombe single-exon genes and multi-exon genes have a median length of 1220 nts and 1053 nts, respectively). Therefore we tested for the possibility that the signal of robustness in single-exon genes in S.pombe is muted by the reduced efficiency of PABP-dependent NMD in the shorter mRNAs of multi-exon genes. We compared single and multi-exon genes in bins of equal CDS length and found that fragile codons are significantly depleted in single-exon genes for all length bins except the shortest: the ratios  of median NFCU in single-exon genes relative to median NFCU in multi-exon genes for length bins Q1-Q4 are 0.99 (p = 0.83), 0.92 (p<10−7), 0.93 (p<10−8), 0.95 (p<10−8), respectively).It has recently been demonstrated that splicing enhances NMD in S.pombe. Among fission yeast proteins we observe a 3% depletion  of fragile amino-acids in the protein products of single-exon genes (median NFAU = 0.397) compared to multi-exon gene products (median NFAU = 0.410).The signal associated with the second mechanism of transcriptional robustness (amino-acid choice) is less strong in In this study we set out to investigate whether human genes have evolved “transcriptional robustness” to reduce the frequency of mistranscription events leading to nonsense errors. More specifically we hypothesized that such a strategy of nonsense prevention should complement the cure for nonsense errors provided by Nonsense Mediated Decay (NMD). Indeed by comparing single-exon genes (in which NMD is inefficient) and multi-exon genes (in which NMD is much more efficient) we show that intergenic variation in transcriptional robustness reflects intergenic differences in NMD efficiency. Specifically, the primary hallmark of this robustness is a depletion of “fragile” codons that are susceptible to mistranscription into a STOP codon. We took account of differences between single-exon and multi-exon genes that, although unrelated to transcriptional robustness, might nevertheless covary with fragile codon usage. Our result is not explained by biologically confounding variables such as differences in splicing constraints or transcription associated mutational biases  equal transcriptional error rates and (ii) similar selective costs associated with the toxic effects of truncated proteins. However, if either of these quantities was, on average, greater in single-exon genes than in multi-exon genes then this would promote increased transcriptional robustness of single-exon genes even if NMD were equally potent in both groups.Our analysis of intragenic patterns of fragile codon use addresses both assumptions, by isolating the influence on transcriptional robustness of variable NMD efficiency from the influences of variable transcriptional fidelity and selective costs of nonsense errors. First, although transcriptional fidelity might vary between genes, it is not likely to vary within genes. In contrast, the efficiency of NMD varies not only between genes but also within genes since, like single-exon genes, last exons of multi-exon genes are invisible to EJC-dependent NMD in mammals. Therefore if selection for transcriptional robustness was mediated by transcriptional fidelity then this could explain the intergenic but not the intragenic patterns of fragile codon use that we see (i.e. depletion of fragile codons in last exons). Second, although the selective costs of nonsense errors might vary within genes this should result in an enrichment of fragile codons in last exons rather than the depletion observed.Therefore the intragenic pattern of fragile codon usage in mammalian genes, and in particular the depletion of fragile codons in last exons, suggests that variable NMD-efficiency underlies transcriptional robustness. Although these patterns do not implicate variation in transcriptional fidelity or in the selective costs of undetected nonsense errors as unique explanations of transcriptional robustness these factors might also contribute to intergenic differences in robustness. Importantly, the result of our intragenic analysis strongly suggests that constraints on codon usage imposed by the requirement for transcriptional robustness are common to all mammalian genes and are not peculiar to the minority of genes that are intronless in mammals .cis and trans strategies in limiting protein misfolding We conclude therefore, that natural selection has promoted a dual strategy of “prevention and cure” to deal with the problem of nonsense transcriptional errors and that these strategies are used interchangeably in mammals. This example of complementarity between strategies for error-prevention and error-mitigation in mammals echoes the recent demonstration in bacteria of complementarity between There is evidence that both the intron content Drosophila annotations We used Ensembl release 49 gene annotations for human and mouse and Flybase release 5.4 for We defined two categories of codons using a classification introduced by Normalized fragile codon usage (NFCU) was calculated by considering only groups of codons that are synonymous and have equal GC content but differ with respect to their fragility. These groups were chosen from among codons encoding “facultative amino acids” . Four suFor each gene we considered its encoded peptide  and calculated the fraction of all amino acids that are fragile   considerNormalized fragile amino-acid usage (NFAU) was calculated by considering only groups of amino-acids that are encoded by codons of equal GC content. For each gene we considered its longest encoded peptide and, for two separate groups of amino-acids, calculated the fraction of amino-acids that are fragile. The first group consists of four amino-acids encoded by codons for which 1/6 nts are G or C: Tyr (fragile), Lys (fragile), Asn (robust) and Phe (robust). The second group consists of eight amino-acids encoded by codons for which 1/2 nts are G or C: Gln (fragile), Glu (fragile), Cys (fragile), Ser (facultative), His (robust), Thr (robust), Val (robust), Asp (robust). For each protein we computed the fractional fragile amino-acid usage for each of these two groups . Finally, for each protein we expressed the normalized fragile amino-acid usage (NFAU) as the average of defined fractions.a/Ks i.e. the ratio of non-synonymous substitutions per non-synonymous site (Ka) to synonymous substitutions per synonymous site (Ks). Four groups of human genes of similar selective constraint were constructed based on quartiles of Ka/Ks for all human-mouse orthologs. For each group we calculated the Spearman correlation between normalized fragile codons usage (NFCU) and fragile amino-acid usage (NFAU) metrics.Human-mouse orthologs were retrieved using the BioMart tool. In the case of human genes having multiple co-orthologs we retained the longest mouse ortholog (choosing a random protein in the case of length ties). We aligned human-mouse orthologous pairs using CLUSTALW We performed an exon-based analysis to compare NFCU in last exons and upstream exons. We considered only genes for which we could be sure that the annotated last exon is the true last exon. Last exons were considered as reliably annotated if they (i) have a CDS sequence that terminates with a STOP codon, (ii) do not have a downstream non-coding exon (and therefore do not have a downstream EJC) and (iii) have an annotated 3′ UTR of at least 100 nts. For each gene having a reliably annotated last exon we assigned the last exon to one group and each remaining exon to a second group (“upstream exons”). We calculated NFCU for each exon using only those codons completely encoded by the exon and compared NFCU in the “last exon” and “upstream exon” groups.s).Using the dataset of histone genes retrieved from Ensembl as described above we constructed sequence alignments between all pairs of histone proteins using CLUSTALW Figure S1The absence of splicing constraints cannot explain the lower fragile codon content of single-exon genes. Normalized fragile codon usage (NFCU) of human single- (S) and multi-exon (M) genes binned by ESE density within the CDS. For each quartile of ESE density , NFCU for single and multi-exon genes is plotted separately. The width of each bin is proportional to the square root of the number of genes it contains.(TIF)Click here for additional data file.Figure S2opGC). For each quartile of FopGC , NFCU for single and multi-exon genes is plotted separately. The width of each bin is proportional to the square root of the number of genes it contains.Fragile codon usage patterns among human genes are not due to selection for translational accuracy. Normalized fragile codon usage (NFCU) of human single- (S) and multi-exon (M) genes binned by the fraction of translationally optimal codons per gene controlling for GC content (F(TIF)Click here for additional data file.Figure S3Drosophila single- (S) and multi-exon (M) genes binned by the fraction of translationally optimal codons per gene (Fop). For each quartile of Fop , NFCU for single and multi-exon genes is plotted separately. The width of each bin is proportional to the square root of the number of genes it contains.Fragile codon usage patterns among fly genes are not due to selection for translational accuracy. Normalized fragile codon usage (NFCU) of (TIF)Click here for additional data file.Table S1Fragile codon usage (FCU) of multi-exon genes and single-exon genes in the human, mouse, and fly genomes.(DOC)Click here for additional data file.Text S1Supporting results.(DOC)Click here for additional data file.


Based on the results of recent randomized, controlled clinical trials and analyses of their follow-up periods the concept of metabolic memory cannot be restricted to antihyperglycaemic treatment only, rather it can be extended to lipid-lowering and antihypertensive treatment and even life-style modification. This broadened concept can be designated as vascular memory. According to this new concept, not only immediate and short-term but long-term effects of the metabolic and cardiovascular risk milieu are of great importance. Consequently, early and intensive lifestyle interventions, treatment of hyperglycaemia, lipid abnormalities and hypertension can result in beneficial effects on cardiovascular outcomes even in the long run. On the contrary, failing in target-oriented treatment from early detection of abnormalities can be associated with life-threatening cardiovascular events subsequently. Additional experimental studies are needed to characterize the exact pathomechanism of vascular memory and further clinical trials are also essential to explore its real clinical significance. The concept of metabolic memory was first described among patients with type 1 diabetes in 2005, based on the results of the follow-up observation of the original cohort in the DCCT . The pheIn this paper, clinical evidence concerning the late effect of antihyperglycaemic treatment is summarized. Additionally, the late effects of lipid-lowering and antihypertensive treatment as well as life-style modification are also reviewed. Taken together, results from recent clinical trials suggest that the original concept of metabolic memory can be defined in a much broader context.versus conventional insulin therapy during the DCCT) on the late development and progression of microvascular and macrovascular complications [The DCCT was a multicenter, randomized, controlled clinical trial which compared intensive insulin therapy with conventional insulin regimens in patients with type 1 diabetes . Originaications . AlthougAs for microvascular complications, the risk of retinopathy remained significantly reduced in the former group with intensive insulin treatment 4 years after completion of the original trial . In addiAs for cardiovascular complications, a beneficial effect of early intensive glycaemic control on surrogate endpoints such as progression of carotid intima-media thickness ,15 or coTaken together, the DCCT/EDIC study demonstrated that an average period of 6.5 years of intensive insulin treatment had a long-term, sustained effect on the subsequent risk of late micro- and macrovascular complications Figure . This phversus conventional treatment (i.e. better glycaemic control) was associated with a reduced risk of clinically evident microvascular complications and a nonsignificant reduction in the relative risk of myocardial infarction in patients with newly diagnosed type 2 diabetes mellitus [The UKPDS, one of the largest randomised clinical trials in diabetology, embracing 20 years of study 1977‐1997) followed by a further 10 years of post-trial monitoring, has radically altered our knowledge about the natural course and treatment of type 2 diabetes. The original trial with 4209 randomized patients documented that intensive glucose therapy mellitus . In the 977‐1997 mellitus .The legacy effect of lipid lowering treatment was documented by extended follow-up investigations of randomized controlled trials with statins  supporting the idea of vascular memory.The 4 S was one of the earliest randomized trials for assessing the effect of simvastatin in the secondary prevention of myocardial infarction . The benIn the HPS, the efficacy and safety of lowering LDL-cholesterol with daily 40 mg simvastatin (versus placebo) were investigated . During The WOSCOPS was designed to determine whether pravastatin in men with hypercholesterolaemia and no history of myocardial infarction reduced the combined incidence of nonfatal myocardial infarction and death from coronary heart disease . After rversus control arms despite conversion of curves representing the changes of LDL-cholesterol values over time  versus placebo in 9014 patients (age 31‐75 years) with a history of myocardial infarction or hospitalization for unstable angina and initial plasma total cholesterol levels of 4.0 – 7.0 mmol/l . The meaThe ASCOT-LLA was a placebo-controlled randomized trial for evaluating the effects of atorvastatin 10 mg daily in the primary prevention of coronary heart disease in hypertensive subjects who had a total cholesterol level of ≤6.5 mmol/l. The trial was stopped prematurely after a median 3.3-year follow-up due to substantial benefits of atorvastatin (36 % relative risk reduction) on composite primary endpoint of cardiovascular events . The resThe legacy effect of treatment with antihypertensive drugs was observed in some randomized, controlled trials with post-trial observational follow-up. Nevertheless, the beneficial late effect of tighter antihypertensive control was not observed in the UKPDS 10-year follow-up.versus placebo was assessed in patients who were at high risk for cardiovascular events but who did not have left ventricular dysfunction or heart failure [In the HOPE study, the effect of ramipril  failure . A total failure . During versus placebo group was 22 % (p = 0.001). The long-term benefit of trandolapril was evaluated over 10‐12 years of follow-up [In the TRACE study, patients with myocardial infarction and left ventricular systolic dysfunction (ejection fraction ≤35 %) were randomly assigned to receive oral trandolapril or placebo . The folollow-up . In the versus placebo was added to conventional therapy in patients with reduced (≤35 %) ejection fraction and a significant reduction in the incidence of heart failure and the rate of related hospitalizations was observed in a median duration of 37.4 months. In addition, there was a trend toward fewer deaths due to cardiovascular causes among the patients who received enalapril [In the SOLVD trial, enalapril nalapril . In ordereduced ≤ % ejectiIn the SYST-EUR study, elderly patients with isolated systolic hypertension after 2 years of randomized treatment with nitrendipine or placebo were followed for an additional 4 years with open-label antihypertensive treatment . PatientIn the UKPDS with newly diagnosed type 2 diabetic patients, the effect of tight blood pressure control was also evaluated and, therefore, over a 4-year period 1148 patients with hypertension were randomized to tight or less-tight blood-pressure control regimens. Tight control was achieved by using ACE-inhibitor (captopril) or beta-blocker (atenolol) while less-tight control was maintained by antihypertensive treatment that excluded these agents. In the randomized trial, for tight as compared with the less-tight control of blood pressure, there were relative risk reductions of 24 % for any diabetes-related endpoint, 32 % for diabetes-related death, 44 % for stroke, and 37 % for microvascular disease .In the FDPS, 522 middle-aged, overweight subjects with impaired glucose tolerance were randomly assigned to either the intervention or the control group . Each suIn the China Da Qing Diabetes Prevention Study, 577 adult subjects with impaired glucose tolerance were randomly assigned to either the control group or to lifestyle intervention groups . Active intervention took place between 1986 and 1992 . In 1992In the 2.8 years of the DPP randomised clinical trial, diabetes incidence in high-risk adults was reduced by 58 % with intensive lifestyle intervention and by 31 % with metformin, compared with placebo . During The STENO-2 study, conducted over 7.8 years, recognized that the risk of cardiovascular events and death could be halved among patients with longstanding diabetes and microalbuminuria by intensive multifactorial treatment . PatientAlthough the exact pathomechanism of vascular memory is not clearly understood some elements of the pathophysiologic process have already been highlighted by experimental studies. Furthermore, explanations of the late effect of an earlier treatment have become available from clinical studies.versus conventional treatment were compared [As for antihyperglycaemic treatment, increased formation of AGE could be a plausible cause of structural and functional changes occurring in the early metabolic environment but carrying implications for developing and progressing micro- and macrovascular complications in the long run -50. In acompared . Interescompared . Recentlcompared .Although late beneficial effect of early therapy with statins was documented in several lipid trials, the patomechanism remains obscure. Although the time of initiating treatment with statins and the dose of the respective statin are of great importance, the non-lipid lowering benefits of statins should also be considered. Generally, statins are linked to the inhibition of HMG-CoA reductase and a subsequent reduction in synthesis of isoprenoid intermediates by which a number of critical intracellular signalling processes are prevented leading to beneficial effect on subclinical inflammation and endothelial function. All these factors may have a role in vascular protection with statin therapy even long term ,63.versus immediate start of antihypertensive treatment may have a deleterious effect on cardiovascular outcome [As for treatment with antihypertensive drugs, it should be emphasized that no legacy effect in the UKPDS follow-up was observed . Neverth outcome .Regarding life-style modification it is of great importance that weight reduction can result in improvement in both insulin resistance and beta-cell function. Accordingly, the incidence rate of diabetes mellitus in subjects originally recruited with impaired glucose tolerance proved to be lower in the intervention versus control groups during the randomized trial and even the post-trial period . In thisIt is very likely that the term of metabolic memory observed in type 1 and type 2 diabetic patients with different antihyperglycaemic treatment ,71 can bAccording to the concept of the vascular memory, not only immediate and short-term but even long-term effects of the metabolic and cardiovascular risk milieu could be expected. Consequently, early and intensive treatment of hyperglycaemia, lipid abnormalities, and hypertension can result in beneficial effects on cardiovascular outcomes even in the long run (“good memory”). On the contrary, failing in target-oriented treatment from early detection of abnormalities can be associated with subsequent life-threatening cardiovascular events (“bad memory”) . SimilarTaken together, the “memory effect” is a new challenge for treatment aiming to reduce cardiometabolic risks and events. It is obvious that not only experimental studies but further clinical trials are needed to explore the exact pathomechanism and the particular clinical significance of the vascular memory.ACE = Angiotensin-convertase enzyme; ADDITION-Europe = Anglo-Danish-Dutch Study of Intensive Treatment In People with Screen Detected Diabetes in Primary Care; AGE = Advanced glycation end products; ASCOT-LLA = Anglo-Scandinavian Cardiac Outcomes Trial Lipid Lowering Arm; DCCT = Diabetes Control and Complications Trial; DPP = Diabetes Prevention Program; EDIC = Epidemiology of Diabetes Interventions and Complications; FDPS = Finnish Diabetes Prevention Study; HOPE = Heart Outcomes Prevention Evaluation; HPS = Heart Protection Study; LIPID = Long-term Intervention with Pravastatin in Ischaemic Disease; RAAS = Renin-angiotensin-aldosterone system; SYST-EUR = Systolic Hypertension in Europe Trial; SOLVD = Studies of Left Ventricular Dysfunction; TRACE = Trandolapril Cardiac Evaluation; UKPDS = United Kingdom Prospective Diabetes Study; WOSCOPS = West of Scotland Coronary Prevention Study; 4 S = Scandinavian Simvastatin Survival Study.The author declares that he has no competing interests.GJ conceived, wrote and approved the final manuscript.


G protein-coupled receptors (GPCRs) constitute a large family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses. GPCR are involved in a wide variety of physiological processes, including in the neuroendocrine system. GPCR are also involved in many diseases and are the target of 30% of marketed medicinal drugs. Whereas the majority of the GPCR-targeting drugs have proved their therapeutic benefit, some of them were associated with undesired effects. We develop two examples of used drugs whose therapeutic benefits are tarnished by carcinogenesis risks. The chronic administration of glucagon-like peptide-1 (GLP-1) analogs widely used to treat type-2 diabetes was associated with an increased risk of pancreatic or thyroid cancers. The long-term treatment with the estrogen antagonist tamoxifen, developed to target breast cancer overexpressing estrogen receptors ER, presents agonist activity on the G protein-coupled estrogen receptor which is associated with an increased incidence of endometrial cancer and breast cancer resistance to hormonotherapy. We point out and discuss the need of pharmacological studies to understand and overcome the undesired effects associated with the chronic administration of GPCR ligands. In fact, biological effects triggered by GPCR often result from the activation of multiple intracellular signaling pathways. Deciphering which signaling networks are engaged following GPCR activation appears to be primordial to unveil their contribution in the physiological and physiopathological processes. The development of biased agonists to elucidate the role of the different signaling mechanisms mediated by GPCR activation will allow the generation of new therapeutic agents with improved efficacy and reduced side effects. In this regard, the identification of GLP-1R biased ligands promoting insulin secretion without inducing pro-tumoral effects would offer therapeutic benefit. Seven transmembrane receptors, also termed G protein-coupled receptors (GPCR), form the largest class of the cell surface membrane receptors, involving 850 members in the human genome. GPCR are generally expressed in several different tissues in the same individual and involved in numerous physiological processes, including in the neuroendocrine system by playing a pivotal role in the control of feeding behavior, reproduction, growth, hydromineral homeostasis and stress response. At the cellular level, biological effects triggered by GPCR often result from the activation of multiple intracellular signaling pathways which are dependent or independent of G protein coupling  and the transactivation of the EGF-R  leading to the activation of phosphatidylinositol-3 kinase (PI3K), Protein Kinase Cζ (PKCζ), Akt-protein kinase B, Extracellular Regulated Kinase (ERK1/2) signaling pathways and to the up-regulation of the expression of the cell cycle regulator cyclin D1 , LiVolsi,  and calcLiVolsi, . NeoplasLiVolsi, .There are very few data on GLP-1R-induced proliferative signaling in thyroid C-cells. Chronic administration of liraglutide did not modify ERK phosphorylation, but increased ribosomal S6 phosphorylation, a downstream target of mTor and PI3K activation which plays a role in regulating cell proliferation and survival by growth factors  incretin could be also another alternative in type-2 diabetes treatment, especially as the GIP receptor (GIP-R) was not expressed in the normal thyroid and the exocrine pancreas unlike GLP-1R Ahren, , 2012. Bcis-acting estrogen response elements in the promoter and enhancer regions of hormonally regulated genes (genomic effects of estrogen)  is the main mechanism responsible for the diverse biological effects of the hormone  Figure  (Ring an) Figure . Estroge) Figure .Estrogen is the one of the risk factors for breast tumors, which accounts for 40% of cancer among the women and approximately 50% of all breast cancers demonstrated elevated levels of ER expression (Pike et al., in vivo and of human endometrial carcinoma cell lines and these effects were mediated by GPER (Gottardis et al., An increased incidence of uterine malignancies in association with tamoxifen treatment has been reported. The incidence and severity of endometrial cancer increased by 4- to 6.9-fold in women with 5 years of exposure to tamoxifen (van Leeuwen et al., in vivo (He et al., G protein-coupled estrogen receptor promotes carcinogenesis by endometrial cancer cells as down-regulation of GPER led to reduce growth and invasion by RL95 endometrial cancer cells treated with 17β-estradiol and to decrease tumorigenesis The majority of breast cancers is ER-positive and depends on estrogen for growth. Therefore, blocking estrogen signaling remains the strategy of choice for the treatment and the prevention of breast cancer. Tamoxifen is the prototypical drug that targets ER. It presents potent anti-estrogenic properties and has been used extensively for the past 40 years to treat and prevent breast cancer (Jordan and Morrow, In vitro prolonged tamoxifen treatment leads to an increased cell surface expression of GPER and also to clonal selection of GPER-positive MCF-7 breast cancer cells (Ignatov et al., The tumor resistance to tamoxifen treatment is associated to a decrease or a loss of ER expression and to an increase of GPER expression. GPER protein is expressed in ∼50% of all breast cancers including half of ER-negative tumors and correlates with increased tumor size and metastasis (Filardo et al., G protein-coupled estrogen receptor mediates the proliferative and tamoxifen-resistance effects through EGF-R transactivation leading to the phosphorylation of ERK and Akt (Filardo et al., In conclusion, tamoxifen has been the only available hormonal option for the systemic treatment for breast cancer from 1973 to 2000. Despite the clinical success of tamoxifen, the development of drug resistance and endometrial cancers leads to the requirement of alternative hormonal therapy. In this regard, the knowledge of the contribution of GPER-mediated signaling in the undesired effects of estrogenic antagonist uses for breast cancer treatment should allow the future development of new molecules. Moreover, further researches are required to define the role of GPER signaling in estrogen undesired physiological effects and to elucidate the role of non-selective estrogen receptor ligands in health and disease.G protein-coupled receptors are generally expressed in several different tissues and involved in numerous physiological processes. Many natural ligands can bind and activate several subtypes of GPCR. This is illustrated, for example, with cholecystokinin and somatostatin receptors (Guillermet-Guibert et al., One strategy to overcome these limitations would be to examine the initial steps following receptor activation. The release of X-ray structures of agonist/GPCR complexes (Chung et al., Although GPCRs can modulate a large variety of distinct signaling pathways, classification of biased ligands are actually restricted to two groups depending on their ability to activate two main transduction pathways (Whalen et al., The vast majority of biased ligands identified so far exhibits exclusive β-arrestin activity for a number of receptors (Rajagopal et al., Few ligands have been yet identified as perfect G protein-biased ligands, namely inducing G protein signal transduction without any β-arrestin recruitment (Whalen et al., Interestingly, some ligands are biased in regard to the different G protein families and can trigger opposite cellular responses (Reversi et al., Biased signaling can also exist with respect to other signaling proteins than G proteins and arrestins. The internalization of apelin receptor takes different signaling pathways depending of the apelin isoforms (Lee et al., Results obtained from preclinical and clinical studies tend to support a pro-tumoral action of GLP-1 in the pancreas and the thyroid. Deciphering the signaling networks engaged following GLP-1R agonist administration in pancreatic ductal cells and thyroid C-cells comparatively to pancreatic β-cells is critical to unveil their contribution in the different cellular processes. The identification of GLP-1 analogs that promote insulin secretion to treat type-2 diabetes without inducing pro-tumoral effects is therefore a timely challenging issue. Like most GPCRs, the GLP-1R couples to different classes of heterotrimeric G proteins, including Gαs, Gαq, and Gαi, regulatory proteins such as the β-arrestins, and activates multiple signaling pathways such as cAMP production, intracellular calcium mobilization, phosphorylation of ERK1/2. While GLP-1 can activate all of these signaling pathways, some compounds were shown to present biased activity on GLP-1R. Oxyntomodulin and glucagon biased the GLP-1R toward cAMP accumulation over the recruitment of β-arrestins, BMS21 compound toward ERK1/2 activation and cAMP production over β-arrestins recruitment, BETP compound toward calcium mobilization and β-arrestins recruitment over cAMP production and ERK1/2 activation (Jorgensen et al., The most crucial GLP-1R signaling pathway for enhancing glucose-dependent insulin secretion involved the receptor coupling to Gαs proteins and the activation of cAMP production (Baggio and Drucker, in vivo and open the door to the generation of diagnostics and therapeutics directed at individual or both estrogen receptors. Such compounds might represent an important new approach for cancer therapy, thus increasing the armamentarium of drugs used to treat estrogen-sensitive and resistant cancers. On the other hand, aromatase inhibitors which act by preventing the enzyme aromatase to convert androgens into estrogen have been also brought forward as a potential alternative (Josefsson and Leinster, Despite the clinical success of tamoxifen in breast cancer treatment, the development of drug resistance and endometrial cancers involving the GPER leads to the requirement of alternative hormonal therapy. In this regard, the contribution of GPER-mediated responses estrogen antagonists must be considered in the future development of anti-estrogenic molecules. Recent studies based on pharmacological structure/function relationship on ER and/or GPER have identified selective GPER antagonists which completely block uterine epithelial cell proliferation mediated by GPER and which are poorly active or inactive on ER (Dennis et al., In conclusion, GPCRs provide huge therapeutic opportunities; some are already in use. The progress in the knowledges of signaling pathways downstream of these receptors and the effects arising, their regulation by pharmacological agents, and the data from the receptor structure provide new opportunities which should lead to new generation of ligands with minimized side effects.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.